79 BioTek Ebook FINAL - Compressed

Long-Term 3D Spheroid
Culture & Analysis

Gain a greater understanding of disease biology with validated
methods for long-term spheroid culture and analysis
BROUGHT TO YOU BY INDEPENDENT SCIENCE PUBLISHER IN PARTNERSHIP WITH

Long-Term 3D Spheroid Culture & Analysis
Introduction
From basic cell science through to
translational research and pre-clinical
investigations, 2D culture systems have
Contents
remained the gold standard for many
decades. Yet, it is widely recognized that • Introduction
cells cultivated in 2D are poor predictors
•M
odeling Tumoroid Invasion
of physiology and pathophysiology in vivo,
Long Term
owed largely to the lack of tissue-specific
architecture and extra-cellular matrices. •M
onitoring Chronic Hepatotoxicity
Nevertheless, the popularity and persistence
•S
tem Cell Differentiation
of 2D models can be credited to their ease of
cultivation, compliance with high-throughput •E
xtended Analysis of Immune
screening campaigns, and amenability to long Mediated Killing
term experimentation.
Recently, there has been an intensified • Featured Products
interest in the use of 3D models in
life sciences research, to improve the
understanding of basic cellular processes
and reduce drug attrition rate. This has been cell differentiation.
compounded by the emergence of more This application-based eBook will focus on
advanced technology systems that overcome the use of spheroids as a powerful tool for
many of the cumbersome complications translational research in 3D – demonstrating
of 3D cultivation and analysis. In the past, robust workflows for high-throughput
3D models have proven difficult to reliably spheroid assembly and long-term quantitative
cultivate long enough to perform multiple analysis. With a focus on four key applications,
dosing studies as well as to monitor chronic we provide examples of varied assembly
toxicity, long-term kinetic analyses, and stem systems (including scaffold and scaffold-
2
free) and advanced microscopy analyses Monitoring Toxicity in Multiple-Dosing Studies

to effectively discern disease biology in 3D. A significant proportion of candidate drugs fail
Find out how methods such as magnetic in early-stage clinical trials due to unforeseen
bio-printing, specialized media exchange hepatotoxic effects, also known as ‘drug-
technology and ultra-low attachment culture induced liver injury’, or DILI. The main reason
plates enable uniform culture and prevent for this is the incongruity between animal
accidental loss of spheroids during extended models and the human hepatic response,
experimental analyses. preventing accurate prediction of drug
metabolism in the liver. To overcome this
Modeling Tumoroid Invasion Long Term roadblock in the discovery pathway, primary
Multicellular spheroids are an excellent tool human hepatocyte-derived 3D models have
for modeling tumor biology due to their emerged as a means of more thoroughly
structural and functional heterogeneity, investigating toxicity before transitioning
that more closely mimics the collective to the clinical stage. However, many of the
“
behavior of cancer cells in the body. One chronically damaging activities of some
such behavior is the invasion of cells through compounds do not surface in these short-
biological matrices that enables a tumor to term pharmacokinetic studies in vitro.
disseminate. It is estimated Primary cells are also
that nearly 90% of cancer inherently variable and have
deaths are due to metastases;
for example, the hepatocellular
Nearly 90% of limited availability – making
them far from ideal for high-
carcinoma five-year survival rate
falls to just 3% if the tumor has
disseminated. Hence, a great
deal of research is invested
in developing compounds to
prevent the processes that
are due to
metastases
“
cancer deaths
throughput, automated testing
systems.
Induced pluripotent stem
cells (iPSCs) are a promising
alternative, therefore. They
offer the potential for studying
initiate or perpetuate cellular full pharmacology in longer-
invasion. There is mounting term studies in 3D. Here, we
evidence, for instance, to suggest that matrix present a comparative study of iPSC-derived
metalloproteinases (MMPs) play a significant hepatocyte spheroids exposed to multiple
part in penetrating biological membranes, but concentrations of three drug compounds.
more accurate model systems are required Using the Cytation 5, researchers were able
to elucidate the mechanisms of the invasive to simultaneously determine the presence
phenotype. of reactive oxygen species and the integrity
In this guide, we present a method for of plasma membranes, as well as to quantify
long-term kinetic analysis of embedded mitochondrial membrane potential. We also
spheroid invasion, using glioblastoma cell provide a method for hepatotoxicity testing
lines co-cultured with fibroblasts, to derive using cytochrome P450 as a measure for
uniform spheroids. Spheroids were monitored hepatic function, and the CellTiter-Glo® assay
over several days with the automated to quantify viability.
environmental control and image capture and
analysis capabilities of the Cytation 5 Cell Stem Cell Differentiation
Imaging Multi-Mode Reader. Differences in Understanding the role of stem cells
their invasive phenotype were easily quantified in normal physiology and in disease is
and compared in 3D. In a separate study, the essential to appreciating the processes
same microscopy system was used in tandem involved in development, growth, and repair
with protease activity detection technology or regeneration, as well as advancing the
to simultaneously deduce phenotypic and applications of tissue engineering. Human
molecular response to MT(membrane- mesenchymal stem cells (hMSCs) are an ideal
tethered)1-MMP and MMP14 inhibitors. tool for this type of study: they are easily
3
isolated and can differentiate into multiple including cancer and neurodegeneration. It
“
tissue types. The biggest problem with has become clear that the immune system
conventional 2D cultivation formats, however, often transitions from disease-fighting to
is their loss of replicative and differentiation disease-favoring, as dysregulated cells
ability over serial passages. develop methods to
3D cultivation formats counteract the
have been proposed in the
past but are not suited to
The immune cytotoxic effects of
the immune system.
high-throughput screening
campaigns due to the high
cost and large volume of
sample required. More
recent methods, such as
those proposed in this
system is a major
“
contributor to many
types of disease
Many paradigm-
changing therapies
have emerged from
these discoveries, most
notably CAR-T cell
therapy for leukemias
guide, are able to rapidly and lymphomas. Now,
generate hMSC spheroids in scientists are working
an automated fashion. In this eBook, discover to refine these cell-based therapies even
a step-by-step process for the generation of further, but this requires more representative
biomimetic hMSC spheroids, using magnetic 3D models of the complex tumor
bioprinting. Find out how robust, long- environment. In this guide, discover a method
term cultivation could be achieved for full for T cell-target cell interaction analysis using
chondrocyte differentiation in vitro. 3D bio-printed multicellular breast cancer
spheroids. T cells were activated using a
Extended Analysis of T-Cell Mediated Killing direct or general approach, and the method
Recently, the immune system has emerged as of cytotoxicity effectively determined with
a major contributor to many types of disease, quantitative microscopy.
References
1. L
i L, Qian M, Finkelstein D, Johnson M, H D, Lopez-Terrada, et al. Model liver cancer
metastasis using 3D spheroids derived from primary tumors in liver cancer genetic mouse
models. Cancer Research. 2018;78(10).
2. V
orrink S.U, Zhou Y, Ingelman-Sundberg M, Lauschke V.M. Prediction of Drug-Induced
Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically
Defined Conditions. Toxicological Sciences. 2018;163(2):655-65.
4
A p p l i c a t i o n N o t e
3D Cell Culture, Automation
Automated Media Exchange for Spheroid Cultures Using a Novel

MultiFlo™ FX Accessory
Brad Larson, Principal Scientist, Applications Department, BioTek Instruments, Inc., Winooski, VT
Abstract
Three dimensional (3D) spheroidal cell models have become a mainstay in life science
research due to the ability to mimic in vivo-like environments. Performing media
exchanges and washes with spheroids in cell repellent microplates can be problematic due
to the risk of accidental spheroid removal. By incorporating a novel peristaltic pump-based
tool, these procedures can be carried out in a controlled manner that eliminates spheroid
disruption and removal, enabling long-term 3D experimental procedures requiring multiple
media exchanges.
Introduction
Spheroidal 3D cellular structures are a mainstay Experimental Components

in many research areas, including oncology and The known topoisomerase I inhibitor,
toxicology1. Culturing cells in 3D provides a more camptothecin (Catalog No. 208925) was
in vivo-like environment, allowing cells to maintain purchased from EMD Millipore (Billerica, MA).
high viability when cultured for extended time DMEM, low glucose, pyruvate, HEPES (Catalog
periods. To maintain the highest levels of viability No. 12320-032), advanced DMEM (Catalog No.
within untreated cells and ensure that observed 12491-015), fetal bovine serum, 10% (Catalog No.
effects are solely from treatment, media exchanges 10437-036), and penicillin-streptomycin (10,000
Key Words: and re-dosing are necessary throughout the U/ml) (Catalog No. 15140-122), and penicillin-
experiment, particularly in vitro tests lasting streptomycin-glutamine (100x) (Catalog No.
weeks. Media exchanges with cell models that 10378-016) were purchased from ThermoFisher
AMX
do not rely on adherance to labware can be Scientific (Waltham, MA).
MultiFlo AMX daunting if performed by hand, even when
performed on a single plate. Multichannel Microplate Consumables
Spheroid pipettes must remove and dispense media at an
96-well, cell-repellent, polystyrene, round bottom,
extremely slow rate, and care must be taken to
3D clear, sterile, microplates with lid (Catalog No.
keep pipette tips away from the actual spheroids.
650979) and 384-well, cell-repellent, polystyrene,
Spheroid Proliferation
Through incorporation of the AMX Media round bottom, clear, sterile microplates with lid
Spheroid Media Exchange Exchange Module on the MultiFlo FX, risk (Catalog No. 787979) were donated by Greiner Bio-
of accidental spheroid removal from wells is One (Monroe, NC). 96-well, clear round bottom,
eliminated. Spent media is automatically removed sterile, ultra low attachment microplates with lid
and replaced with fresh media only, or fresh media (Catalog No. 7007) and 384-well, black/clear round
containing treatment concentrations. bottom, sterile, ultra low attachment microplates
with lid (Catalog No. 3830) were donated by
Corning, Inc. (Corning, NY). PrimeSurface® 96U
Materials and Methods clear round bottom 96-well microplates (Catalog
No. MS-9096UZ) and PrimeSurface 384U clear
Materials round bottom 384-well microplates (Catalog No.
MS-9384UZ) were donated by S-BIO (Hudson, NH).
Cells
U-87 glioblastoma cells were generously
BioTek Instruments, Inc. donated by Dr. Sachin Katyal (University of
P.O. Box 998, Highland Park,
Manitoba, Winnipeg, Manitoba, Canada). HT-
Winooski, Vermont 05404-0998 USA
Phone: 888-451-5171 1080 fibrosarcoma cells (Catalog No. CCL-121)
Outside the USA: 802-655-4740 and PANC-1 carcinoma cells (Catalog No. CRL-
Email: customercare@biotek.com 1469) were obtained from ATCC (Manassas, VA).
www.biotek.com
Copyright © 2018
5
Application Note 3D Cell Culture, Automation
Instrumentation Methods
Cytation™ 5 Cell Imaging Multi-Mode Reader Cell Preparation and Tumoroid Formation
Cytation 5 is a modular multi-mode microplate Prepared U-87, HT-1080, or PANC-1 cells were harvested
reader combined with an automated digital and diluted in complete media and dispensed into
microscope. Filter- and monochromator-based all microplate wells in a volume of 100 μL for 96-well
microplate reading are available, and the microscopy microplates, and 50 μL for 384-well microplates. A total of
module provides up to 60x magnification in 1000 cells was dispensed to all wells of each test spheroid
fluorescence, brightfield, color brightfield and plate. The microplates were incubated at 37 ºC/5% CO2
phase contrast. The instrument can perform for forty-eight hours to allow cells to aggregate into single
fluorescence imaging in up to four channels in a spheroids.
single step. With special emphasis on live-cell assays,
Media Exchange Method
Cytation 5 features shaking, temperature control to
65 ºC, CO2/O2 gas control and dual injectors for The AMX Media Exchange Module aspirate tips were
kinetic assays and is controlled by integrated Gen5™ positioned at the back, right corner of each well in
Microplate Reader and Imager Software, which also 96- and 384-well format, and the bottom of the tubes
automates image capture, analysis and processing. were elevated from the bottom of each well to avoid
The instrument was used to kinetically monitor disturbing the spheroid (Figure 2). Media was removed
3D tumoroid activity over the incubation period. from each well using a slow aspiration speed; with 15-
20 µL of residual media volume in 96-well plate wells,
MultiFlo™ FX Multi-Mode Dispenser and 10-15 µL of residual media volume in 384-well plate
The MultiFlo FX is a modular, upgradable reagent wells. When dispensing fresh media into 96-well spheroid
dispenser that can have as many as two peri-pump (8 plates, dispense tubes were positioned at the back, right
tube dispensers), two syringe pump dispensers and corner of the well, away from the spheroid; whereas
a strip washer. The syringe and washer manifolds can when dispensing into 384-well plates, the tubes were
be configured for plate densities from 6- to 384-well. positioned directly above the spheroid to prevent
The MultiFlo FX was equipped with the AMX Media disruption. In both microplate well densities, the bottom
Exchange Module. of the dispense tubes were elevated from the bottom
of the well in a manner such that the media droplets
AMX Media Exchange Module contacted the existing well liquid to ensure equal
volumes were dispensed to each well (Figure 3). The
Media exchange of spheroid cultures is accomplished
media dispense rate was optimized so that spheroids
through use of BioTek’s patent-pending AMX Media
were not displaced from each well center.
Exchange Module, which consists of two unique,
modified peristaltic pump cassettes with eight stainless A.
steel tube aspirate (Figure 1, right arrow) and dispense
(Figure 1, left arrow) heads. The cassette tubing is
fed through the peristaltic pumps of the MultiFlo FX
and into media bottles or tubes. Software allows the
pumps to run slowly and gently so as to not disturb the
spheroids during aspirate or dispense procedures. Each
cassette is fully autoclavable, enabling sterile processing.
B. C.
Figure 2. (A.) AMX Media Exchange Module aspirate tip

positioning; illustrating intact spheroids in residual media using
(B.) 96-well plates; and (C.) 384-well plates.
Figure 1. AMX Media Exchange Module with

separate stainless steel tube aspirate (right
arrow) and dispense (left arrow) heads.
2 6
Figure 3. AMX Media Exchange

Module 96-well plate dispense
tip positioning.
Qualitative Validation of AMX Media Exchange Module
After U-87 spheroid formation, 96- and 384-well spheroid microplates were transferred to the MultiFlo™ FX with AMX
module, and five media exchange cycles were performed concurrently to simulate a rigorous washing protocol. Once
the media exchanges were complete, microplates were transferred to Cytation™ 5 for brightfield imaging of all wells.
A 4x objective was used for all image capture. Due to the conical shape of the bottom of the well in 384-well microplates,
a black ring is seen at the outer edges of each image (Figure 4B).
Quantitative Validation of AMX Media Exchange Module
Camptothecin was diluted in media to create an eight-point titration (10 µM - 2.4 nM) including a negative control
without compound. Following aggregation, 96-well Greiner microplates containing U-87 spheroids, 96-well Corning
microplates containing HT-1080 spheroids, and 384-well S-Bio microplates containing PANC-1 spheroids were
placed into the BioSpa™ 8 Automated Incubator. At regular intervals, the plates were automatically transferred
to the MultiFlo FX, where media was removed using the aspirate cassette and replaced with the various inhibitor
concentrations using the dispense cassette. After each dosed media exchange, the plates were then transferred to
Cytation 5 for kinetic brightfield imaging to monitor spheroid growth. Multiple images were captured in a z-stack using a
4x objective to ensure accurate calculation of spheroid volume. The “Object Size” value, which is the average diameter
of the spheroid, was incorporated into a custom Gen5 metric using the mathematical volume of a sphere formula.
Spheroid Volume = (4/3) * π * (Object Size/2)3
The process was repeated over the one or two-week spheroid proliferation incubation period.
Results
Qualitative Validation of AMX Media Exchange Module
After five cycles of 85% media exchange, brightfield imaging of the 96- and 384-well microplates
(Figure 4) confirm that spheroids were not disturbed during the automated aspirate and dispense steps.
A. B.
Figure 4. Brightfield images of (A.) 96-well, and (B.) 384-well Greiner spheroid microplates following 5x media exchange. Images from
Corning and S-Bio microplates not shown.
3 7
Quantitative Validation of AMX Media Exchange Module
During the spheroid proliferation inhibitor dosing period, z-stacked brightfield images were captured kinetically using
Cytation 5. Spheroid volume was then automatically calculated using Gen5™ Microplate Reader and Imager Software.
A. B.
C.
Figure 5. Automated kinetic spheroid proliferation results using AMX media exchanges. Calculated spheroid volumes following chronic
exposure to varying camptothecin concentrations. (A.) U-87 glioblastoma spheroids in Greiner 96-well spheroid plates; (B.) HT-1080
spheroids in Corning 96-well spheroid plates; (C.) PANC-1 spheroids in 384-well spheroid plates.
Figure 5 demonstrates expected results with suitable experimental error where spheroids continue to propagate
and increase in volume over time, in all cell models and microplate densities. Futhermore, the toxin camptothecin
correspondingly interferes with spheroid propagation.
Conclusions
The MultiFlo™ FX with AMX Media Exchange Module effectively performs single and multiple media
exchanges without disturbing unattached 3D spheroids in 96- and 384-well formats. When coupled with BioTek
automation, the media exchange tool provides a walk away solution for long-term 3D experimental procedures.
References
1. Knight, E.; Przyborski, S. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.
J Anat. 2015, 227, 746-756.
4 AN072718_09, Rev. 07/27/18 8

3D Cell Culture
Brightfield and Fluorescence Imaging using 3D PrimeSurface®

Ultra-Low Attachment Microplates
Brad Larson, BioTek Instruments, Inc., Winooski, VT USA
Anju Dang, S-BIO, Hudson, NH USA
Abstract
Three-dimensional (3D) cell culture has become a well established in vitro experimental
approach as it provides an improved in vivo-like environment. The use of clear U bottom
ultra low attachment microplates that minimize cell adherance has become a standard
for applications such as spheroid proliferation. Results shown here demonstrate the ability
to generate quality results using both brightfield and fluorescence microscopy.
Introduction Materials and Methods
Culturing cells in three-dimensions (3D) has Materials

become a well established approach as it is
more representative of the in vivo environment Cells and Media
than traditional two-dimensional (2D) cultures.
HT-1080 fibrosarcoma cells (Catalog No. CCL-
Allowing cells to interact with each other in a
121) were purchased from ATCC (Manassas,
spheroid creates a micro-environment which
VA). Advanced DMEM (Catalog No. 12491-
Key Words: mimics in vivo tissue and is a better model
015), fetal bovine serum, (Catalog No. 10437-
for examining the effect of drugs in cancer.
036), and penicillin-streptomycin-glutamine
3D Cell Culture Developing uniform spheroids becomes
(100X) (Catalog No. 10378-016) were purchased
especially important as it forms the basis for
Spheroids from ThermoFisher Scientific (Waltham, MA).
robust and reliable assays. S-BIO PrimeSurface®
3D Spheroids cultureware are ultra low attachment (ULA)
Experimental Components
dishes and plates that promote scaffold free,
Spheroid Microplates
self assembly spheroid formation. The plates are
The known topoisomerase I inhibitor, camptothecin
pre-coated with a proprietary hydrophilic
(Catalog No. 208925) was purchased from EMD
polymer that enables spontaneous spheroid
Millipore (Billerica, MA). CellTox Green Cytotoxicity
formation of uniform size. PrimeSurface 96
Assay (Catalog No. G8731) was purchased from
and 384 ULA plates have good optical clarity
Promega Corporation (Madison, WI). PrimeSurface
making them highly suitable for brightfield and
96 U bottom, clear wall ULA plates (Catalog No.
fluorescent imaging. Imaging technologies such
MS-9096UZ) and PrimeSurface 384 U bottom,
as the BioTek Cytation™ 5 allows researchers to
clear wall ULA plates (Catalog No. MS-
study not only spheroid proliferation through
9384UZ) were donated by S-BIO (Hudson, NH).
brightfield imaging, but also phenotypic
events such as hypoxia, apoptosis, or necrosis
Cytation™ 5 Cell Imaging Multi-Mode Reader
induction through the use of fluorescent probes
and fluorescence imaging. Incorporation of Cytation 5 is a modular multi-mode microplate
z-stacking and projection techniques in reader combined with an automated digital
the Gen5™ Microplate Reader and Imager microscope. Filter- and monochromator-
Software create in-focus images of spheroidal based microplate reading are available, and
cells, allowing accurate, robust, and repeatable the microscopy module provides up to 60x
determination of the effect of test molecules magnification in fluorescence, brightfield,
or conditions. In this app note, we present color brightfield and phase contrast. The
BioTek Instruments, Inc.
P.O. Box 998, Highland Park, data generated with BioTek Cytation 5 using instrument can perform fluorescence imaging
Winooski, Vermont 05404-0998 USA PrimeSurface ULA plates to develop simple in up to four channels in a single step. With
Phone: 888-451-5171 robust spheroid assays for brightfield and
Outside the USA: 802-655-4740 special emphasis on live-cell assays, Cytation 5
fluorescence imaging. features shaking, temperature control to 65 ºC,
Email: customercare@biotek.com
www.biotek.com
Copyright © 2017
9
Application Note 3D Cell Culture
CO2/O2 gas control and dual injectors for kinetic 3D Image Z-Stacking Parameters
assays, and is controlled by integrated Gen5™
Microplate Reader and Imager Software, which also Focus Method Autofocus
automates image capture, processing and analysis. Number of Slices 9
The instrument was used to image spheroids in the Step Size 54.4 µm
PrimeSurface plates using brightfield and fluorescence
Images Below Focus Point 4
imaging.
Table 2. Image Z-Stacking Parameters.
Methods
3D Image Processing
3D Spheroid Formation
Following capture, a z-projection of the images in
HT-1080 cells were added to wells of the 96- and the z-stack was carried out using the focus stacking
384-well PrimeSurface U bottom microplates at algorithm to create a final image containing
concentrations of 10000, 5000, 2000, 1000, 500, only the most in-focus information (Table 3).
250, 100, and 50 cells per well in volumes of 100 or
3D Image Stitching Parameters
50 µL for the 96- or 384-well plates, respectively. The
microplates were incubated at 37 °C/5% CO2 for 48 Method Focus Stacking
hours to allow the cells to aggregate into spheroids. Channel Brightfield
Size of Max. Filter 11 pixels
Camptothecin Treatment and Dead Cell Staining Top Slice 9
Bottom Slice 1
Upon completion of the aggregation process, a subset
Table 3. 3D Z-Projection Criteria.
of spheroids seeded at 1000 cells/well were treated
with either 10,000, 500, 10, or 0 nM camptothecin by
manually removing media and replacing with an equal
volume of fresh media containing the drug. The plates Cellular Analysis of 3D Projected Images
were then incubated at 37 ºC/5% CO2 for an additional
Cellular analysis was carried out on the projected images
24 hours to induce necrosis within the treated spheroidal
of untreated variable sized spheroids using the criteria in
cells. The following day media was again removed from
Table 4.
the wells and replaced with media containing 1x CellTox
Green necrotic cell stain. The plates were incubated at Spheroid Cellular Analysis Criteria
37 ºC/5% CO2 for 5 hours to allow fluorescent probe
Channel ZProj[Brightfield]
penetration into the cells. A final media exchange was
Threshold 10000
performed to remove excess stain.
Background Light
Automated Imaging Procedure Split Touching Objects Unchecked
Fill Holes in Masks Checked
Spheroid imaging was carried out to assess the ability
Min. Object Size 75 µm
to generate quality images and accurate cellular
analysis. Brightfield and fluorescent images were Max. Object Size 1000 µm
captured using the imaging channels listed in Table 1. Include Primary Edge Objects Unchecked
Analyze Entire Image Checked
Imaging Channel Target
Advanced Detection Options
Brightfield Total Spheroids
Rolling Ball Diameter Auto
GFP Spheroidal Dead Cell Nuclei
Image Smoothing Strength 0
Table 1. Cell Imaged per Imaging Channel.
Evaluate Background On 5% of Lowest Pixels
Analysis Metric
As the cells within the spheroids exist within a range of Metric of Interest Spheroid Volume
z-planes, a z-stacking imaging procedure was setup Table 4. Spheroid Identification Criteria.
within Gen5. Following auto focusing, multiple images

were captured above and below the focal plane
to ensure cells were imaged at the proper z-height An additional cellular analysis step was performed
(Table 2). on the treated spheroids to determine the extent of
necrotic activity induced by the camptothecin treatment
(Table 5).
2 10
Spheroid Necrosis Induction Analysis Criteria A. E.
Primary Mask Criteria

Channel ZProj[Brightfield]
Threshold 25000
Background Light
Split Touching Objects Unchecked
Fill Holes in Masks Checked B. F.
Max. Object Size 1000 µm
Include Primary Edge
Unchecked
Objects
Analyze Entire Image Checked

C. G.
Rolling Ball Diameter 600
Secondary Mask Criteria
Channel ZProj[GFP]
Measure Within a Secondary
Checked D. H.
Mask
Expand Primary Mask 1 µm
Threshold 30000
Smooth 0
Method Threshold In Mask
Fill Holes in Mask Unchecked
Figure 1. 96-well U bottom brightfield spheroidal imaging. 4x
Analysis Metric z-projected brightfield images of spheroids formed from (A) 10000; (B)
5000; (C) 2000; (D) 1000; (E) 500; (F) 250; (G) 100; or (H) 50 cells per well.
Metric of Interest Object Area
Metric of Interest Area_2[ZProj[GFP]]
Custom Metric of Interest % Necrotic Cell Area
Table 5. Spheroid Necrosis Induction Analysis Criteria.
A final observation apparent from the images is the
flat, or even background signal generated from
the bottom of the microplate wells during
brightfield imaging. Using the Line Profile tool avail-
Results and Discussion able in Gen5, a line can be drawn from one side of
an image to the other, bisecting the spheroid
Brightfield Imaging of Formed Spheroids (Figure 2A). This creates a brightfield intensity graph
representative of the CCD pixel intensities along the
Visual analysis of the z-projected images reveals that line. It is evident that there is a large decrease in
HT-1080 cells are able to form tight spheroids within pixel intensity as the line passes through the spheroid
the 96-well U bottom plates. It is also evident that and that the other pixels representative of
the z-stacking and projection method used by the background are relatively bright and uniform in intensity
Gen5™ Microplate Reader and Imager Software creates (Figure 2B).
accurate, in focus spheroidal images regardless of size
(Figure 1).
3 11
A. B.
Figure 2. Image background and target object brightfield signal. (A) 4x brightfield image of 100 cell spheroid plus drawn View
Line Profile tool line. (B) Graph of brightfield signal from 1915 µm drawn line.
Figure 3 demonstrates the accurate placement of primary masks necessary for area and diameter mea-
surements using parameters in Table 4. Using the two metrics, a volume calculation can be made.
A. B. C.
Figure 3. Cellular analysis of test spheroids. Gen5 automatically drawn object masks around spheroids formed from (A) 10000; (B) 1000; and (C) 50 cells.
Spheroid analysis was performed in both 96- and 384-well PrimeSurface plates. Figure 4 illustrates the precision
and linearity of spheroid volume as a function of cell seeding density.
Figure 4. Plot and linear regression of Gen5 96- and 384-well

calculated spheroid volumes.
4 12
Fluorescent Imaging of Necrotic Cell Induction within The second step was to quantify the area covered
Spheroids solely by necrotic cells within each test spheroid. A
concern, though, with using clear walled microplates
Imaging was also carried out on treated and to perform fluorescence imaging and then ascertain
stained 1000 cell spheroids to determine the level accurate results is the autofluorescence from the
of necrotic cell induction caused by camptothecin. clear walls, contributing to high background within
Brightfield images were captured, in addition to the image. Use of the Gen5 Line Profile tool,
fluorescent images in the GFP channel to specifically however, confirms that this phenomenon was not
view necrotic cells stained by the CellTox Green witnessed when using the clear U bottom microplates
probe. An overlay was then created to visualize (Figure 6A). A consistently low background signal
cellular necrosis within each spheroid (Figure 5A). was seen from areas of the image not containing
necrotic cells, allowing for an easily distinguishable
A.
change in signal from affected cells (Figure 6B).
A.
Figure 5A. Imaging of camptothecin induced spheroidal

cell necrosis. 4x z-projected, overlaid brightfield and GFP
image of spheroid showing necrotic cell induction caused
by 24 hour 10,000 nM camptothecin.
B.
B.
Figure 5B. Primary cellular analysis to determine necrotic

cell induction. Gen5 automatically drawn primary object Figure 6. Image background and target object fluorescent
masks around 1000 cells spheroid treated with 10,000 nM signal. (A) 4x GFP image of 1000 cell spheroid treated with
camptothecin. 10,000 nM camptothecin, plus drawn View Line Profile tool
line. (B) Graph of GFP signal from 1037 µm drawn line.
To quantify the level of induced necrosis, cellular By taking advantage of the optical qualities of the U
analysis was once again performed on the bottom plates, accurate object masks were able to be
spheroidal images. To determine the area covered by placed around affected cells, regardless of whether
both live and dead cells within the captured images, high or low levels of necrotic activity were generated
object masks were placed around the spheroids by the camptothecin concentrations (Figure 7).
(Figure 5B) using the brightfield channel and
primary mask criteria described in Table 5.
13
5
A. The images in Figure 7 of object mask

placement based upon GFP signal, in addition to
the graph of camptothecin induced spheroidal cell
necrosis, confirms that accurate imaging and
analysis can be carried out when using fluorescent
channels and the U bottom microplates.
Conclusions
B.
S-BIO PrimeSurface U bottom 96- and 384-well
microplates can be used to create appropriately
sized, repeatable single spheroids within each
test well. The Cytation™ 5 and Gen5™ software
also provide the ability to capture high quality images
on multiple z-planes, suitable for spheroid imaging.
The combination of the optical quality of the
microplates and Gen5 software then allow accurate
quantification of proliferation or induced phenotypic
events within the spheroids using either brightfield
C.
or fluorescence microscopy.
Figure 7. Necrotic cell analysis of treated

spheroids. Gen5 automatically drawn object
masks around necrotic cells within spheroids
treated with (A) 10; (B) 500; or (C) 10000 nM
camptothecin.
When the necrotic cell area was divided by the total

cell area, a normalized affected cell percent coverage
area was created, which accounted for variances in
area between the treated 1000 cell spheroids. The
values were then plotted in terms of the concentration
of camptothecin used for treatment (Figure 8).
Figure 8. Plot of percent spheroid area covered by affected

necrotic cells following camptothecin treatment.
14
6 AN121917_22, Rev. 12/19/17
3D Cell Culture, Live Cell Imaging
3D Spheroid-Based Tumor Invasion Assay
Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA

Jan Seldin, Greiner Bio-One, Inc., Monroe, NC USA
Abstract
Establishment of in vitro models that mimic tumor invasion as part of the metastatic process,
are a critical part of oncology research. The need to incorporate multiple cell types, long-term
kinetic analysis, methods to allow 3D invasion into the surrounding matrix, and appropriate
detection and analysis, has made it difficult to create new models. The procedure described
here meets these needs through inclusion of advanced imaging capabilities allowing for
capture of multiple images throughout a range of z-heights, using brightfield and
fluorescence channels in a kinetic fashion. Final processed images, following stitching and
z-projection, enable accurate cellular analysis to discern the invasive capabilities of 3D cellular
structures over time.
Introduction
Oncology drug discovery has been met with with the multiple assay conditions, compounds
multiple challenges over the years. As cancers tested and kinetic images taken. This work will
develop multiple mutations during carcinogenesis, demonstrate a procedure for the generation of
Key Words: targeted approaches to individual gene mutations 3D spheroidal tumoroid structures, creation of a
common to many drug discovery campaigns suitable invasion matrix, automated kinetic image-
have mostly limited efficacy. Conversely, the based monitoring, and cellular analysis of captured
3D Cell Culture advancement of novel methods that focus on the z-stacked images of tumor invasion.
Tumoroid Invasion inhibition of the invasive phenotype of metastasis
offers greater potential for meaningful intervention, U-87 and LN-229 glioblastoma multiforme
Glioblastoma (GBM) cell lines were used in this study as they
particularly due to the fact that most cancer
U-87 patients die only after metastasis has occurred. have demonstrated phenotypic differences
For this approach to work, in vitro cell models used and metastatic ability1. Notably, the growth
Label-free Analysis
in early drug discovery should mimic as close as suppressing PTEN gene is mutated in U-87 cells,
possible the complex metastatic process. Tumors yet functions normally in LN-229 cells. Additionally,
in vivo exist as a three-dimensional (3D) mass of the human cytomegalovirus phosphotransferase
multiple cell types, including cancer and stromal protein UL-97 inhibits DNA elongation and
cells. Therefore, incorporating a 3D spheroid- replication; and is absent from U-87 cells, but
type cellular structure that includes co-cultured present in LN-229 cells2. This supports a more
cell types forming a tumoroid, provides a more aggressive growth and invasion pattern for
predictive model than the use of individual cancer U-87 cells. Both cell types were co-cultured with
cells seeded in microplates. A further constraint fibroblasts to create 3D tumoroids more closely
is the need for long term kinetic experiments representing in vivo tumor conditions and allowed
to capture the invasion process which typically to invade through a protein matrix. 17-allylamino-
require multiple days. Thus environment control 17-demethoxygeldanamycin (17-AAG), known
of the assay is needed such that the cell viability to inhibit the function of heat shock protein
of the spheroid is maintained over this long period 90 (Hsp90), a chaperone protein that stabilizes
and putative drug effects properly assessed. proteins required for tumor growth, was used
Finally, the only suitable readout for monitoring here to inhibit potential tumor invasion3.
tumor invasion is microscopy due to the small size Quantification of kinetic captured images was
BioTek Instruments, Inc. of the spheroid. To be effective in drug discovery, used to characterize the invading potential of
P.O. Box 998, Highland Park, inhibited and uninhibited tumoroid cultures.
the microscope must have automated image
Winooski, Vermont 05404-0998 USA
Phone: 888-451-5171 capture, processing and analysis to be able to cope
Outside the USA: 802-655-4740
www.biotek.com
Copyright © 2018
15
Application Note 3D Cell Culture, Live Cell Imaging
Materials and Methods MultiFlo™ FX Multi-Mode Dispenser
Materials The MultiFlo FX is a modular, upgradable reagent

dispenser that can have as many as two peri-pump (8
Cells tube dispensers), two syringe pump dispensers and
a strip washer. The syringe and washer manifolds can
U-87 GBM cells expressing GFP were generously be configured for plate densities from 6- to 384-well.
donated by Dr. Sachin Katyal (University of Manitoba,
Winnipeg, Manitoba, Canada). LN-229 GBM cells
(Catalog No. CRL-2611) were obtained from ATCC Methods
(Manassas, VA). Human neonatal dermal fibroblasts
expressing RFP (Catalog No. cAP-0008RFP) were Cell Preparation and Tumoroid Formation
purchased from Angio-Proteomie (Boston, MA).
Prepared U-87 and fibroblast cells were each harvested
and combined in a final concentration of 2.5x104 cells/mL
Experimental Components
for each cell type in complete medium. After dispensing
Matrigel® Basement Membrane Matrix, Phenol Red-Free 100 μL of cell suspension into appropriate microplate
(Catalog No. 356237) was purchased from Corning Life wells, the microplate was incubated at 37 ºC/5% CO2
Sciences (Corning, NY). The selective Hsp90 inhibitor for forty-eight hours to allow cells to aggregate into
17-AAG (Catalog No. 1515) was sourced from Tocris tumoroids. This process was repeated using LN-229 cells
Bioscience (Avonmouth, Bristol, UK). CellTracker™ stained with CellTracker Deep Red dye and fibroblast
Deep Red dye (Catalog No. C34565) was purchased cells at the same concentrations and volumes.
from ThermoFisher Scientific (Waltham, MA). 96- Invasion Matrix Preparation
well, cell-repellent, polystyrene, round bottom, clear,
sterile, microplates with lid (Catalog No. 650979) Upon completion of tumoroid formation, 70 μL of
were generously donated by Greiner Bio-One. complete medium was robotically removed from each
well, and the tumoroid plate placed on ice in a refrigerator
Cytation™ 5 Cell Imaging Multi-Mode Reader for five minutes to cool the cells. Matrigel matrix was then
thawed on ice. 17-AAG was diluted to a 2x concentration
Cytation 5 is a modular multi-mode microplate reader of 20,000 nM in invasion media and used to create an
combined with an automated digital microscope. eight-point titration from 20,000 nM to 0 nM using serial
Filter- and monochromator-based microplate reading 1:4 dilutions. The inhibitor titrations were further diluted
are available, and the microscopy module provides up to a 1x concentration by combining with either invasion
to 60x magnification in fluorescence, brightfield, color media or Matrigel matrix. With the plate still on ice,
brightfield and phase contrast. The instrument can 70 μL of Matrigel matrix plus titrated compound were
perform fluorescence imaging in up to four channels in added to each well containing tumoroids, then overlaid
a single step. With special emphasis on live-cell assays, with 100 μL invasion media containing titrated compound.
Cytation 5 features shaking, temperature control to The microplate was centrifuged at 300 × g for five minutes
65 ºC, CO2/O2 gas control and dual injectors for in a swinging bucket centrifuge that was previously set to
kinetic assays and is controlled by integrated Gen5™ 4 ºC for tumoroid positioning, then transferred to a
Microplate Reader and Imager Software, which also 37 ºC/5% CO2 incubator for one hour to initiate gel
automates image capture, analysis and processing. The formation.
instrument was used to kinetically monitor 3D tumoroid
activity over the incubation period. Tumor Invasion Assay Performance
BioSpa™ 8 Automated Incubator After gel formation was complete, the microplate
was transferred to BioSpa 8, where the software was
The BioSpa 8 Automated Incubator links BioTek readers programmed such that the microplate was automatically
or imagers together with washers and dispensers for transferred to Cytation 5 for brightfield and fluorescent
full workflow automation of up to eight microplates. imaging of the wells every twelve hours over the seven-
Temperature, CO2/O2 and humidity levels are controlled day incubation period. Table 1 lists the settings used to
and monitored through the BioSpa software to maintain perform automated image capture of each sample well.
an ideal environment for cell cultures during all
experimental stages. Test plates were incubated in the
BioSpa to maintain proper atmospheric conditions for
a period of seven days and automatically transferred
to the Cytation 5 every twelve hours for brightfield and
fluorescence imaging.
2 16
Imaging Parameters Tumoroid Invasion Analysis
Complete 3D Invading Primary mask cellular analysis criteria (Table 4) were

Brightfield Imaging Channel
Structure
applied using the brightfield channel to automatically
GFP Imaging Channel U-87 Cells Expressing GFP place object masks around the entire invading structure in
RFP Imaging Channel Fibroblasts Expressing RFP each final image. Secondary mask cellular analysis criteria
LN-229 Cells Stained with (Table 5) were also applied to place an additional mask
CY5 Imaging Channel
Deep Red CellTracker Dye around non-invasive cells remaining within the original
Objective 4x spherical tumoroid structure.
Montage 3 Row by 2 Column
Primary Cellular Analysis Parameters
Montage Overlap Auto for Stitching
Channel ZProj[Stitched[Brightfield]]
Z-Stack 20 Slices
Threshold 25,000
53.8 µm (Default for 4x
Z-Stack Step Size Background Light
Objective)
Table 1. Automated 3D tumoroid invasion imaging parameters. Split Touching Objects Unchecked
Image Processing Max. Object Size 2000 µm
Individual image tiles from each z-plane captured using Include Primary Edge Objects Checked
the three by two montage were then stitched together Analyze Entire Image Checked
(Table 2). Advanced Detection Options
Imaging Stitching Parameters Rolling Ball Diameter 2000

Registration Channel Brightfield
Fusion Method Linear Blend
Analysis Metric
Crop stitched image to
remove black rectangles on Checked Metric of Interest Object Area
the borders Table 4. Primary mask analysis parameters.
Downsize final image Checked (52.62%)
Table 2. Image stitching parameters.
Secondary Cellular Analysis Parameters
Channel ZProj[Stitched[Brightfield]]
A single image projection was then created from the 20
slice stitched z-stack (Table 3). The focus stacking method Measure Within a Secondary
Include Primary Mask
Mask
was chosen, which automatically selects the most in
Reduce Primary Mask 1 µm
focus pixel from each image in the stack for inclusion
in the final projection. This allows for the most accurate Threshold 12,000
analysis to be carried out on each invading tumoroid at Smooth 7
each timepoint. Fill Holes in the Mask Unchecked
Imaging Z-Projection Parameters Analysis Metric
Channel 1 Stitched[Brightfield] Object Area_2[ZProj[Stitched[

Metric of Interest
Brightfield]]]
Method Focus Stacking
Table 5. Secondary mask analysis parameters.
Size of Max. filter 11 px
Z-Slices Included 1-20
Stitched[GFP], Stitched[RFP],
Channels 2-4
Stitched[CY5]
Method (Channels 2-4) Use settings of channel 1
Table 3. Image Z-projection parameters.
3 17
Results and Discussion Imaging of U-87/Fibroblast Individual Co-Cultured Cell

Types
Raw Image Processing Prior to Cellular Analysis
In addition to brightfield imaging, montage tiles
During the seven-day incubation period, uninhibited and z-stacked layers were also captured using
U-87/fibroblast tumoroids continued to increase in size, the GFP and RFP channels. Overlaying images
as well as invade into the protein matrix. To ensure that tracks invasion of the entire structure (Figure 3).
the entire invading structure, including invadopodia, was
captured in the x- and y-axes, regardless of size, multiple
images were taken on a single z-plane in a montage
format (Table 1). Figure 1 illustrates four of the six images
that were captured containing portions of the invading
structure.
A. B.
Figure 3. Brightfield and fluorescent overlaid

z-projected image of U-87/fibroblast co-cultured cell
model.
C. D.
By viewing the fluorescent signal emitted by each co-

cultured cell type, in comparison to the brightfield signal
from the entire structure, individual cell migration can be
observed. In the case of the U-87/fibroblast model, the
brightfield image demonstrates extensive invadopodia
Figure 1. U-87/fibroblast 4x brightfield imaging showing (A-D) four extending out from the original propagating tumoroid
image tiles containing the invading tumoroid of interest.
(Figure 4A). From the GFP image in Figure 4B, it is evident
that GFP-expressing U-87 cells follow invadopodia
invasion into the matrix. This contrasts to the RFP
As invasion proceeds, proteins and cells invade on image in Figure 4C confirming that RFP-expressing
multiple planes within the z-axis. Therefore, images fibroblasts exhibit little to no migratory ability within this
were automatically taken across a range of z-heights so experimental model.
that all portions of the overall structure were in focus in
the final z-projected images (Figure 2 A-D). A. B.
A. B.
C. D. C.
D.
Figure 2. U-87/fibroblast 4x brightfield imaging showing (A-D) four

of the twenty images captured at multiple z-planes as part of the
z-stack and combined into the final z-projection.
Figure 4. Confirmation of invasion by individual cell types. (A) total
tumoroid invasion via overlaid GFP, RFP and brightfield channels.
Individual cellular invasion by (B) GFP-expressing U-87 cells; or (C)
RFP-expressing fibroblast cells. Digitally zoomed 4x images, 2x3
montage, 20-plane z-stack.
4 18
Kinetic Image Capture When comparing kinetic brightfield images from the two
co-cultured cell types, it is evident that LN-229/fibroblast
Finally, z-projected images are captured kinetically every tumoroids propagate over time, as seen by the increase
12 hours to observe phenotypic changes in the U-87/ in spheroid size (Figure 7), but do not exhibit the invasive
fibroblast co-cultured tumoroids following treatment properties clearly demonstrated by U-87/fibroblast
with varying concentrations of 17-AAG. Uninhibited tumoroids over the same incubation period (Figure 5).
tumoroids, as expected, demonstrate a highly invasive
nature4, exhibiting an increase in the size of the tumoroid A. B.
body as well as a dramatic increase in invadopodia
(Figure 5).
A. B.
C. D.
C. D.
Figure 7. LN-229/fibroblast tumoroid invasive potential over time, 4x

brightfield images, 2x3 montage, 20-plane z-stack. (A) 0 hours; (B) 48
hours; (C) 120 hours; (D) 168 hours.
Figure 5. U-87/fibroblast tumoroid invasive potential over time, 4x

brightfield images, 2x3 montage, 20-plane z-stack. (A) 0 hours; (B) 48
hours; (C) 120 hours; (D) 168 hours.
Gen5 Image+ Based Cellular Analysis of Tumoroid

LN-229/Fibroblast Tumoroid Analysis Invasion
LN-229/fibroblast tumoroid imaging was also conducted In addition to qualitative assessments of tumoroid
in the same manner as that for the U-87/fibroblast phenotypic changes, quantification of the extent
tumoroids. This was done to compare differences in of invasion was also carried out using the stitched,
growth and invadopodia production between GBM z-projected brightfield images. Two separate cellular
cell types known to be highly invasive (U-87) and those analyses were performed following treatment with the
with less invasive ability (LN-229)5. Changes in the 17-AAG titration. In the first, the primary cellular analysis
complete 3D structure were observed using the overlaid capabilities available in Gen5 Image+, and the criteria
brightfield and fluorescent images (Figure 6A), while listed in Table 4 used changes in brightfield signal
individual LN-229 or fibroblast invasion were monitored within the image between cellular containing pixels and
by signal captured with the individual CY5 or RFP background to place detailed object masks around the
channels, respectively (Figure 6B and C). complete invading 3D structure, despite the level of
A. B.
compound treatment (Figure 8).
A. B.
A. B.
Figure 8. Invading tumoroid object masking. Zoomed 4x brightfield

C. images of U-87/fibroblast tumoroids, 2x3 montage, 20-plane z-stack.
(A) 0 μM 17-AAG treatment; and (B) 10 μM 17-AAG treatment. Primary
masks around cells and invadopodia.
Figure 6. Imaging of LN-229/fibroblast co-cultured cell model. (A)

Total tumoroid invasion via overlaid CY5, RFP and brightfield channels.
Individual cellular invasion by (B) CellTracker Deep Red stained LN-
229 cells; or (C) RFP-expressing fibroblast cells. Digitally zoomed 4x
images, 2x3 montage, 20-plane z-stack.
5 19
The area covered by the entire tumoroid for each Gen5 Image Prime Based Cellular Analysis of Tumoroid
captured image over time was then calculated. Values Invasion
from subsequent time points were then divided by the
area value calculated at time 0 for the specific tumoroid A second cellular analysis was also performed using
to normalize results and account for variability in the primary and secondary cellular analysis capabilities
tumoroid size following cell dispensing and aggregation. available in Gen5 Image Prime, and the criteria listed
Area ratios were then plotted (y-axis) with regards to in Table 5, to measure the area solely covered by non-
time (x-axis) (Figure 9). invading cells within the tumoroid. In order to determine
the metastatic ability of 3D in vitro models, both
A. uninhibited or following treatment, it is important to be
able to distinguish between area covered by cells within
the original tumoroid and that covered by invadopodia.
As the more densely packed non-invasive, propagating
cells appear darker compared to invadopodia in a
brightfield image (Figure 10A), this additional change
in signal within the original mask allows placement
of a secondary mask to exclude the invading areas
of each tumoroid and separate the area covered by
the two portions of the entire 3D structure (Figure 10).
B. A. B.
Figure 10. Invading and main tumoroid area object masking. Zoomed
4x brightfield 2x3 montage, 20-plane z-stack images of (A) uninhibited
U-87/fibroblast tumoroids; or (B) uninhibited LN-229/fibroblast
tumoroids. Exterior primary masks around cells and invadopodia, and
interior secondary masks around non-invading cellular structure only.
Figure 9. Kinetic total tumoroid area ratios. Area ratios

plotted for (A) U-87/fibroblast tumoroids, or (B) LN-229/
fibroblast tumoroids following 0-7 day treatments with 17-
AAG concentrations ranging from 10,000 to 0 nM. Area ratio By applying the secondary mask around non-invading
calculated by the following formula: (AreaTime T/AreaTime 0).
portions of the tumoroid, differences in the invasive
qualities of the two GBM cell types then becomes clear.
From the results seen in Figure 9, it is apparent that

both U-87/fibroblast and LN-229/fibroblast tumoroids
propagate within the Matrigel matrix when unimpeded
over the seven-day incubation period. It can also be
seen that the compound 17-AAG is able to limit, or even
completely stop, tumoroid growth in a dose dependent
manner, as expected3. What is also clear from the total
area ratio graphs is the increased rate of growth for
the complete 3D structure exhibited by U-87/fibroblast
tumoroids compared to those where LN-229 cells are co-
cultured with fibroblasts. Increases in the area covered
by the entire tumoroid are approximately 2x over Figure 11. Kinetic uninhibited U-87 and LN-229 co-cultured
time for tumoroids cultured with U-87 cells (ratio: 4.4) tumoroid kinetic invadopodia areas. Calculated invadopodia
area over time for non-treated U-87/fibroblast and LN-229/
compared to those cultured with LN-229 cells (ratio: 2.4). fibroblast tumoroids. Invadopodia area calculated by the
following formula: (Total AreaTime T – Non-Invading AreaTime T).
6 20
The area covered by invadopodia for U-87/fibroblast

tumoroids increases dramatically over the seven
day incubation period. LN-229/fibroblast tumoroids,
by comparison, show little increase in invadopodia
over the same time (Figure 11). This dual analysis,
therefore, has the potential to determine not
only how rapidly tumoroid cells are propagating,
but also the invasive nature of the cell model.
Conclusions
Application of advanced cellular imaging capabilities,

such as taking multiple images on a single z-plane as
well as in a z-stack, allows all portions of an invading
3D cellular tumoroid structure to be captured in focus.
When stitched and projected to a final image, accurate
analysis can then be performed. The ability to view
entire tumoroids via brightfield imaging, as well as
individual cell types through fluorescent imaging, also
allows for determination of the invasive properties
of each co-cultured cell type within the included cell
model. Inclusion of label-free cellular analysis, carried
out on brightfield images, can then quantify not only
propagative qualities, but also the specific invasive
nature of in vitro 3D cell models when uninhibited and in
response to test molecules.
References
1. Furnari, F.B.; Lin, H.; Huang, S.H-J.; Cavenee, W.K.

Growth suppression of glioma cells by PTEN requires
a functional phosphatase catalytic domain. Proc Natl
Acad Sci USA, 1997, 94(23), 12479-84.
2. McFall, T.B. Identification of HCMV UL97 in GBM

Cell Lines and a Possible Role for Ganciclovir. Master’s
Dissertation, Northern Michigan University, Marquette,
MI, 2014.
3. Beckner, M.E.; Fellows-Mayle, W.; Zhang, Z.;

Agostino, N.R.; Kant, J.A.; Day, B.W.; Pollack, I.F.
Identification of ATP Citrate Lyase as a Positive
Regulator of Glycolytic Function in Glioblastomas. Int J
Cancer. 2010, 126(10), 2282-95.
4. Formolo, C.A.; Williams, R.; Gordish-Dressman, H.;

MacDonald, T.J.; Lee, N.H.; Hathout, Y. Secretome
Signature of Invasive Glioblastoma Multiforme. J
Proteome Res. 2011, 10(7), 3149-59.
5. Mallawaaratchy, D.M.; Buckland, M.E.; McDonald,

K.L.; Li, C.Y.; Ly, L.; Sykes, E.K.; Christopherson, R.I.;
Kaufmak, K.L. Membrane Proteome Analysis of
Glioblastoma Cell Invasion. J Neuropathol Exp Neurol.
2015, 74(5), 425-41.
21
7 AN071218_08, Rev. 07/12/18
3D Cell Culture, ADME/Tox, Cell Imaging, Cell-Based Assays
Quantification of MMP Activity and Inhibition in a 3D Tumor

Invasion Model
Using a Novel Fluorescence Detection Technology and Digital Widefield
Microscopy
Brad Larson and Leonie Rieger, BioTek Instruments, Inc., Winooski, VT

Diana Hulboy and Crystal Falco, Enzium, Philadelphia, PA
Introduction
Metastasis, the spread of cancer cells from the primary human dermal fibroblasts and MDA-
primary tumor to secondary locations within MB-231 breast adenocarcinoma cells, known
the body, is linked to approximately 90% to be invasive and metastasize to lung from
of cancer deaths1. Penetration through the primary mammary fat pad tumors7. The cells were
basement membrane is a critical step during aggregated into 3D structures using Corning
the metastatic process, and has been linked Spheroid Microplates containing an Ultra Low
to the formation of membrane superstructures Attachment surface. A novel cell imaging multi-
called invadopodia2. While being made of mode reader and cellular analysis algorithms
multiple substances, a notable component from BioTek Instruments, Inc. were incorporated
are matrix metalloproteinases (MMPs), and to provide automated, image-based detection
specifically Membrane-type 1 MMP (MT1-MMP), and quantification of invasion and enzyme
otherwise known as matrix metalloproteinase 14 activity. The combination presents an accurate,
(MMP-14). This enzyme is crucial for basement easy-to-use method to assess target-based and
membrane and interstitial matrix degradation3, phenotypic effects of new anti-metastatic drugs.
Key Words:
and has been shown to play a critical role in
conferring cells with the ability to penetrate the
3D
extracellular matrix (ECM)4. Mounting preclinical Materials and Methods
3D Cell Culture evidence linking MMPs to cancer progression5,
Spheroid combined with the issue of overlapping substrate Materials
specificity of MMP family members, has made
Tumoroid
the development of targeted MMP inhibitors an Cells
ULA attractive approach to cancer therapy. Therefore,
methods to selectively measure MMP-14 activity, MDA-MB-231 GFP cells (Catalog No. AKR-211)
Matrix Metalloproteinase
specifically within invadopodia, in a sensitive were purchased from Cell Biolabs, Inc. (San Diego,
MMP CA, USA). Human neonatal dermal fibroblasts
yet easy to perform process, are necessary.
MMP-14 (Catalog No. cAP-0008RFP) were purchased
Similarly, appropriate in vitro cell models have from Angio-Proteomie (Boston, MA, USA). Both
Tumor Invasion
been unable to accurately assess the ability cell types were propagated in Advanced DMEM
Metastasis Medium (Catalog No. 12491-015) plus Fetal Bovine
of novel therapies to inhibit tumor invasion,
Cytotoxicity including invadopodia formation. Tumors in Serum (FBS), 10% (Catalog No. 10437-028) and
vivo exist as a three-dimensional (3D) mass of Pen-Strep-Glutamine, 1x (Catalog No. 10378-016)
Imaging
multiple cell types, including cancer and stromal each from Life Technologies (Carlsbad, CA, USA).
Microscopy
cells6. Therefore, incorporating a 3D spheroid-
Cellular Analysis type cellular structure that includes co-cultured Experimental Components
Phenotypic Assay cell types forming a tumoroid, provides a more
predictive model than the use of individual EnSens® MMP-14 Activity Detection Kit (Catalog
Label-free No. 032014_03), MMP-2 Activity Detection Kit
cancer cells cultured on the bottom of a well
in traditional two-dimensional (2D) format. (Catalog No. 032014_01), and NoPro Substrate
were donated by Enzium® (Philadelphia, PA).
The EnSens MMP-14 and MMP-2 substrates
Here we demonstrate the ability to image and
are each selective for their cognate proteases.
BioTek Instruments, Inc. quantify MMP-14 activity, in addition to tumor
P.O. Box 998, Highland Park, Recombinant Human CXCL12/SDF-1 alpha
invasion, using a 3D tumoroid cell model. Enzium’s
Winooski, Vermont 05404-0998 USA (Catalog No. 350-NS-010) was purchased from
Phone: 888-451-5171 protease activity detection technology was
R&D Systems (Minneapolis, MN). DMEM/F12,
Outside the USA: 802-655-4740 incorporated into the invasion assay procedure to
HEPES, No Phenol Red Medium (Catalog No.
Email: customercare@biotek.com enable simultaneous phenotypic and mechanism
www.biotek.com 11039-021) was purchased from Life Technologies.
of action quantification. The tumoroids comprised
Copyright © 2015
22
Application Note 3D Cell Culture, ADME/Tox, Cell Imaging, Cell-Based Assays
Matrigel Basement Membrane Matrix, Phenol Red-Free (Catalog No. 356237) was purchased from
Corning Life Sciences (Corning, NY). GM 6001 (Catalog No. 2983) was purchased from R&D Systems
(Minneapolis, MN). Oridonin (Catalog No. O9639) was purchased from Sigma Aldrich (Saint Louis, MO).
Cytation 5 is a modular multi-mode microplate reader that combines automated digital microscopy and microplate
detection. Cytation 5 includes filter- and monochromator-based microplate reading; the microscopy module provides high
resolution microscopy in fluorescence, brightfield, color brightfield and phase contrast. With special emphasis on live-cell
kinetic assays, Cytation 5 features temperature control to 65 °C (37 ± 0.2 °C), CO2/O2 gas control and dual injectors for
kinetic assays. Shaking and Gen5™ software are also standard. The instrument was used to image the tumoroids as well
as the specific signal from the EnSens red fluorescent dye using the brightfield and Cy5 imaging channels, respectively.
Gen5 Data Analysis Software
Gen5 software controls the operation of the Cytation 5 for both automated digital microscopy and PMT-based microplate
reading. Image acquisition is completely automated from sample translation, focusing and exposure control. Cellular
analysis allows examination of the tumoroid as a single object to enable accurate calculations of changes in tumoroid size
and signal.
EnSens Protease Activity Detection Technology
Figure 1. EnSens® Protease Activity Detection Assay Procedure.
The EnSens protease detection technology is based on a proprietary protein substrate that can
be designed to contain a selective recognition sequence for a specific protease, in addition
to a far-red shifted fluorogenic dye. The assay procedure works in the following manner.
1. The horseshoe shaped protein substrate and dye are added to the well; 2. In the presence of the appropriate
protease, the substrate is cleaved, exposing the dye binding site; 3. Upon binding, a far-red shifted signal is
created. Therefore, little to no fluorescence is seen unless the substrate is in the presence of the correct protease.
Corning® Spheroid Microplates
Corning 96-well black, clear-bottom spheroid microplates (Catalog No. 4520) are coated with the Ultra Low
Attachment surface which is a non-cytotoxic, and biologically inert covalently bonded hydrogel that prevents cell
attachment. Novel well geometry aids spheroid formation in the center of each well. Each microplate contains an
optically clear round bottom, which is ideal for cellular imaging, as well as a black, opaque body which prevents cross talk.
2 23
Methods swinging bucket centrifuge that had been previously set

to 4 °C for spheroid positioning, and then transferred to
Cell Preparation and Spheroid Formation a 37 °C/5% CO2 incubator for one hour to promote gel
formation.
MDA-MB-231 and fibroblast cells were harvested
and diluted to a concentration of 5.0x104 cells/mL in Tumor Invasion Assay Performance
complete medium. The two volumes were combined
to create final concentrations of 2.5x104 cells/mL Using a 10x objective, and an Imaging step programmed
for each cell type. 100 µL of cell suspension was to capture a 3x3 set of images from each well, exposure
then pipetted to the appropriate wells. Following settings were optimized for the brightfield and
dispensing, the plate was placed at 37 °C/5% CO2. Cy5 (for MMP-14 protease activity monitoring) imaging
channels. Following the optimization process, automated
Image-based Spheroid Formation Monitoring day 0 imaging was performed, and continued every 24
hours pursuant, to track tumor invasion.
Spheroid formation was monitored every 24 hours. The
plate was placed into the Cytation 5, previously set to Determination of Tumor Invasion using Cellular Analysis
37 °C/5% CO2 using Gen5 as well as a gas control module.
Focusing was performed using the brightfield channel. Cellular analysis was performed with the captured
The typical cell aggregation period was 48 hours. 10x images. Multiple methods were employed using
either brightfield or Cy5 imaging channels, which
Invasion Matrix Preparation allowed the Gen5 software to examine changes in total
invading tumoroid structure, as well as MMP-14 activity.
Upon spheroid formation completion, 70 μL of complete
medium was removed from each well, washed with an
equal volume of invasion medium (serum-free, phenol
red-free medium), and the spheroid plate placed on ice Results and Discussion
in a refrigerator for 5 minutes to cool the wells. Corning®
Matrigel® Matrix, Phenol Red-Free was then thawed on Quantification of MMP-14 Activity
ice. CXCL12 ligand was diluted to a concentration of
20 ng/mL in invasion medium. Upon thawing, MMP-14 An initial experiment was performed to test the ability
substrate and dye were each added to the matrix to a of the assay components to work properly within the
concentration of 50 nM. Medium containing 20 ng/mL context of the 3D tumor invasion model. The MMP-14
CXCL12 was then diluted 1:2 in either invasion medium substrate was selected for inclusion, as this particular
or matrix containing substrate and dye. If an inhibition MMP has been shown to play a critical role in conferring
experiment was being performed, each inhibitor was cells with the ability to penetrate the extracellular matrix,
then titrated to 1x concentrations in both diluted and is directly linked to tumorigenesis and metastasis.
invasion medium containing 10 ng/mL CXCL12, and Because MMP-14 is active in invadopodia, as invasion
matrix containing 10 ng/mL CXCL12, substrate, and dye. proceeds the protease will come into contact with the
embedded EnSens MMP-14 substrate, allowing for
With the plate still on ice, 70 μL of invasion medium with cleavage to occur, binding of the dye to take place, and
CXCL12 (or CXCL12 and inhibitor) was added to each an increase in fluorescent signal to be observed in these
well. A 100 μL volume of matrix/substrate/dye, previously areas of the well.
prepared, was then added as an overlay to each well.
The plate was centrifuged at 300 x g for 5 minutes in a
Time 0 1 Day Incubation 2 Day Incubation
Time 0
4 Day Incubation 6 Day Incubation
Figure 2. Image-based Monitoring of MMP-14 Activity in 3D Tumor Invasion Assay. Final stitched images from
3x3 image montages using Cy5 channel and 10x objective.
3 24
Images were captured over a six day incubation period following initiation of the invasion process by cytokine
CXCL12 (Figure 2). A 10x objective was incorporated to allow signal capture from the dye with greater sensitivity
than would have otherwise been achieved using lower magnification. However, since the invading tumoroid covers
a large area of the well, multiple images were needed to capture the entire structure. This was accomplished by
performing a montage imaging step and then automatically stitching the final picture together using Gen5™.
The images that were captured over the incubation period (Figure 2) are as expected. Minimal signal from the dye is
initially seen, which increases dramatically over time as invasion proceeds and the MMP-14 protease is able to cleave
greater numbers of substrate molecules.
As previously mentioned, matrix metalloproteinases, such as MMP-14, have been shown to be active in the invadopodia
portions of the tumoroid structure. Therefore, a comparison of the Cy5 dye signal, to that of the complete structure as
observed through the brightfield channel, was performed.
A.
B. C.
Figure 3. Visual Confirmation of MMP-14 Activity in Invadopodia. (A.) Overlay of 10x image montages from brightfield and Cy5 channels
of signal emanating from entire invading structure. (B.) and (C.) Additional magnification of 10x images focusing on invadopodia tumoroid
structures and their MMP-14 activity.
The overlaid Cy5 and brightfield images seen in Figure 3A demonstrate that MMP-14 activity can be observed in all
portions of the tumoroid. Furthermore, when focusing solely on the areas invading furthest into the matrix (Figure 3B
and C), the increased Cy5 signal emanating from the structures identified using the brightfield channel confirms that the
protease is active within the invadopodia.
Cellular analysis can also be performed with the Cytation 5 using the Cy5 signal from the dye to quantify and correlate
the extent of invasion and MMP-14 activity. Minimum and maximum object sizes, as well as Cy5 RFU (relative fluorescent
units) threshold values are set such that a precise object mask is automatically drawn around each tumoroid in its
entirety (Figure 4A and B). The same criteria are used for all images evaluated during the experiment. This allows
for a quantitative comparison of the area covered and total Cy5 signal within each object mask to be completed.
A. B.
C. D. E.
Figure 4. Quantification of MMP-14 Activity. Images of Gen5 object masks drawn around Cy5 signal meeting minimum and maximum
object size and RFU threshold cellular analysis criteria after a (A.) 0 and (B.) 6 day incubation. Graphs generated using (C.) area and (D.)
Cy5 signal intensity within object masks. Raw µm2 and RFU values plotted on left y-axis. Ratio of values on day X compared to day 0
plotted on right y-axis. (E.) Area of invading tumoroid structure quantified using brightfield and Cy5 signals.
4 25
The results from Figure 4C and D illustrate how the area covered by the Cy5 RFU values meeting or exceeding the
set threshold increases over the total incubation period. The same trend can be seen when examining the total
Cy5 signal within the object masks drawn by Gen5. When comparing the area covered within object masks drawn using
the brightfield and Cy5 signal (Figure 4E), it can be observed that similar values are generated throughout the entire
incubation period. This further validates that MMP-14 activity can be detected in the center mass, as well as invadopodia
of the tumoroid.
Interruption of Invasion via MMP Activity Inhibition
Following confirmation that the EnSens assay components were performing correctly within the invasion matrix, and
signal was observed in the expected areas of the structure, the next step was to verify that cleavage of the substrate was
indeed from MMP activity and not from being in the presence of the components of the Matrigel matrix. Here we used the
broad spectrum MMP inhibitor, GM 6001. A titration of the inhibitor, ranging from 0 – 1000 µM, was added to the medium
and matrix surrounding the original tumoroids as previously explained, and invasion was allowed to proceed for six days.
A.
0 µM GM 6001: Day 6 31 µM GM 6001: Day 6 125 µM GM 6001: Day 6 1000 µM GM 6001: Day 6
B.
Figure 5. Demonstration of MMP Activity Detection via Inhibition. (A.) Stitched 10x images of Cy5 fluorescent signal from wells containing
various concentrations of GM 6001, following a six day incubation period. Identical exposure settings used to image all wells. (B.)
Quantification of area and Cy5 signal intensity within Gen5 drawn object masks.
A decrease in Cy5 signal emission is observed from the dye as the tumoroid is exposed to increasing concentrations
of GM 6001 (Figure 5A). This phenomenon confirms that signal generation is caused by cleavage of the substrate
due to MMP activity, and not from non-specific cleavage brought about by a component of the Matrigel matrix.
Quantification performed, once again utilizing the area covered and Cy5 signal within Gen5 drawn object masks
(Figure 5B), also confirms that inhibiting MMP activity causes a decrease in tumor invasion into the matrix as
well as MMP-14 protease activity signal generated from the included substrate and dye. The signal is never
completely eliminated, as the original tumoroid is never completely destroyed, which is similar to what has been
observed in vivo, and is most likely due to the fact that the inhibitor does not completely penetrate the tumoroid.
Substrate Selectivity and Far Red Dye Signal Confirmation
A final test was then performed to confirm that cleavage of the added substrate and subsequent signal generation could
be linked to the target MMP. Here we incorporated selective substrates for MMP-14 and MMP-2, as well as the NoPro
substrate, which is cleaved by a yeast protease not expressed in mammalian cells, making it an ideal negative control
substrate. Two different types of inhibitors were tested: the broad range MMP inhibitor GM 6001 and oridonin, which
suppresses MMP-2 and -9 expression in MDA-MB-231 cells8. Tumoroids were exposed to multiple concentrations of each
inhibitor for a period of five days.
5 26
A.
B.
Figure 6. Demonstration of Selective MMP Activity Detection via Inhibition. Total Cy5 signal quantified within object
masks following image capture of tumoroids treated for five days with (A.) GM 6001 or (B.) oridonin. MMP-14, -2, or
NoPro negative control substrates were added to invasion matrix.
When the results for GM 6001 are examined (Figure 6A), it can be seen that the compound inhibits substrate cleavage
and subsequent signal production in a dose dependent manner when both the MMP-2 or MMP-14 substrates are
incorporated, again proving its pan-MMP inhibitory effect. The data from the test using oridonin, by comparison, displays
two disparate outcomes (Figure 6B). Again, Cy5 signal is inhibited in a dose dependent manner in the presence of the
MMP-2 substrate. However, when the MMP-14 substrate is incorporated, no discernible inhibition is seen until the highest
oridonin concentration (31.25 µM) is reached. By examining images captured from the well containing this concentration,
the loss in signal was determined to be from a cytotoxic effect from the compound, rather than due to true inhibition, which
agrees with published literature stating that oridonin exhibits an apoptotic effect on MDA-MB-231 breast cancer cells8.
27
6
Finally, only low levels of signal are generated when the Conclusions
NoPro substrate is incubated in the presence or absence
of each compound, indicating that the substrate is 3D tumor invasion assays are an important addition in the
not cleaved in an appreciable manner and further quest to accurately quantify the invasive characteristics
validating the selectivity of the included substrates. of cancer cells and their response to test molecules.
Being able to perform simple, definitive assessments
A further analysis of the results from each well was also of MMP activity and potential inhibition increases
carried out to ensure that the reduction in captured Cy5 the value of the assay procedure by allowing live cell
signal was the result of an actual decrease in total signal mechanism of action determinations to be made in
from well, and not solely due to a contraction in the the same well. Monitoring the invasion process and
object mask. Image analysis was performed by the Gen5 signal generation following cleavage of the selective
software which allows the signal to be quantified from MMP substrate can be completed in an automated
each pixel in the image through the elimination of the fashion via imaging using the Cytation 5. Finally, Gen5
generated mask. Data Analysis Software allows accurate quantification
of invasion, in addition to MMP activity, in both the
A. original tumoroid and invadopodia. The combination
presents a complete solution to assess target-based
and phenotypic effects of new anti-metastatic drugs.
References
1. Saxe, Charles. ‘Unlocking The Mysteries Of

Metastasis’. ExpertVoices 2013. http://www.cancer.org/
cancer/news/expertvoices/post/2013/01/23/unlocking-
B. the-mysteries-of-metastasis.aspx. Accessed 16 Mar.
2015.
2. Eckert, MA., Lwin, TM., Chang, AT., Kim, J., Danis,
E., Ohno-Machaso, L., Seiki, M. Twist1-induced
invadopodia formation promotes tumor metastasis.
Cancer Cell. 2011, 19, 372-386.
3. Frittoli, E., Palamidessi, A., Disanza, A., Scita, G.
Secretory and endo/exocytic trafficking in invadopodia
formation: The MT1-MMP paradigm. Eur. J. Cell Biol.
Figure 7. Total Cy5 Signal Inhibition. Total fluorescent signal from 2010, 90, 108-114.
stitched 10x images following siz day incubation with varying
concentrations of (A.) GM 6001 or (B.) oridonin. 4. Itoh, Y., Seiki, M. MT1-MMP: a potent modifier of
pericellular microenvironment. J Cell Physiol. 2006, 206,
1-8.
5. Itoh, Y., Nagase, H. Matrix metalloproteinases in
Similar to the results illustrated in Figure 6, the same cancer. Essays Biochem. 2002, 38, 21-36.
decrease in signal is exhibited with increasing GM 6001 6. Mao, Y., Keller, E., Garfield, D., Shen, K., Wang, J.
concentrations in the presence of both the MMP-14 Stromal cells in tumor microenvironment and breast
and MMP-2 substate (Figure 7A). However, the dose cancer. Cancer and Metastasis Reviews. 2013, 32, 303-
dependent decrease with the addition of oridonin is only 315.
seen in the results from the MMP-2 substrate (Figure 7B).
These findings agree with the findings reported above, 7. Kamath, L., Meydani, A., Foss, F., Kuliopulos, A.
and confirm that accurate, reliable data can be generated Signaling from protease-activated receptor-1 inhibits
when cellular analysis algorithms are incorporated. migration and invasion of breast cancer cells. Cancer
Res. 2001, 61, 5933-5940.
8. Wang, S., Zhong, Z, Wan, J, Tan, W., Wu, G., Chen,
M., Wang, Y. Oridonin induces apoptosis, inhibits
migration and invasion on highly-metastatic human
breast cancer cells. Am J Chin Med. 2013, 41, 177-196.
28
7 AN040615_01, Rev. 04/06/15
3D Cell Culture, Adme/Tox, Cell Imaging,
Cell-based Assays, Spheroid Imaging, Stem Cells
Long-Term Hepatotoxicity Studies using Cultured Human iPSC-

Derived Hepatocytes
Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, VT USA
Coby Carlson and Michael Hancock, Cellular Dynamics International, Madison, WI USA
Winy Luty, Enzo Life Sciences, Farmingdale, NY USA
Abstract
Quantitative microscopy was employed to determine the effect of drugs on human iPSC derived
hepatocytes aggregated into spheroids. Three phenotypes of toxicity were measured using
fluorescent probes: reactive oxygen species generation, mitochondrial membrane potential
decline and plasma membrane rupture. Results were compared to hepatocytes cultured in
flat bottom microplates. Spheroid formation enabled much longer kinetic studies, extending
out to 14 days. This capability allowed for measurement of full pharmacology and phenotype.
Introduction
Drug-induced liver injury (DILI), or damage of a potential drug over multiple days to assess
to the liver caused by prescription or non- any cumulative effects. This poses particular
prescription medications, continues to be a challenges when incorporating two-dimensional
growing public health problem and a challenge (2D) cell culture of primary hepatocytes due to the
Key Words: for drug development. Effects can be acute or fact that the cells rapidly dedifferentiate and lose
chronic and are compounded by the growing metabolic activity when cultured in this manner.
market for dietary supplements and herbal Three-dimensional (3D) cell culture models
3D or non-traditional remedies. Most DILI is the exist that allow cells to aggregate and retain
Spheroid result of unexpected responses to a particular the functionality and communication networks
medication, or long-term chronic damage that was found in vivo. The favorable environment created
Hepatocytes
unseen during standard hepatotoxicity testing. by the 3D culture model then allows long-term
Hepatotoxicity dosing experiments to be performed that more
To test new drug entities for potential DILI, in accurately assess the cumulative effects of a drug.
ROS
vivo models remain the gold standard. However,
Superoxide these studies are costly, time-consuming, and Here we demonstrate the suitability of 3D
Apoptosis more importantly, rather poor predictors of human cultured human iPSC-derived hepatocytes for use
toxicity due to the incorporation of mainly murine in hepatotoxicity studies. Hepatocyte spheroids
Mitochondria
hepatocytes. Consequently, in vitro screens using were exposed to multiple concentrations of three
Cytotoxicity primary hepatocytes are less costly, reduce animal drugs with the DILI category I or III: tolcapone,
Stem Cell exposure, and are more amenable to higher- acetaminophen, and mitomycin C. Direct image-
throughput platforms. However, limitations such based assessment of hepatocyte health based
Quantitative Image Analysis
as high inter-individual variability, finite batch on three phenotypes, after short-term and long-
sizes and changes in cell morphology, as well as term exposure to the drugs was performed. These
liver-specific functions during long-term culture phenotypes included generation of reactive
are challenging this model. Human induced oxygen species (ROS) which is indicative
pluripotent stem cell (iPSC)-derived hepatocytes, of oxidative stress; mitochondrial membrane
by comparison, are a promising in vitro alternative potential (MMP) decline, which is an early
because they demonstrate primary tissue- trigger of the apoptotic cascade; and plasma
like phenotype, high levels of consistency and membrane (PM) rupture, which is a sign
unlimited availability. of necrotic cell death. Comparisons were
BioTek Instruments, Inc. also made to iPSC-derived hepatocytes cultured
P.O. Box 998, Highland Park, When performing toxicity studies, hepatocytes in 2D.
Winooski, Vermont 05404-0998 USA are repeatedly dosed with varying concentrations
Phone: 888-451-5171
www.biotek.com
Copyright © 2017
29
Application Note 3D Cell Culture, Adme/Tox, Cell Imaging,

Materials and Methods cells/well. Cells kept for 2D cell culture were added to 384-
well collagen-coated plates at a concentration of 30,000
Materials cells/well. All plates were incubated at 37 ºC/5% CO2,
and media was exchanged every twenty-four hours.
Cells After five days, cells in the 24-well plates were dissociated
and seeded into GravityTRAP plates at a concentration of
iCell Hepatocytes 2.0 (Catalog No. PHC-100-020-001) were
2000 cells/well to allow for spheroid formation (typically
donated by Cellular Dynamics International (Madison,
complete after 48 hours). Media in the 384-well plates
WI). These cells are human iPSC-derived hepatocytes
continued to be changed daily until compounds were
that exhibit typical hepatic functionality and phenotypic
added on Day 7 post-thaw.
stability. Due to their human origin, native cell-like
behavior, and ease of use, iCell Hepatocytes 2.0 represent
an optimal test system for basic hepatic biology in all areas Hepatotoxicity Assay Procedure
of drug development, disease modeling, and toxicology.
Following spheroid formation, 10-point titrations
Assay and Experimental Components of the known hepatotoxins acetaminophen
(5000-0 μM), mitomycin C (10-0 μM), and tolcapone
BioCoat™ Collagen I-coated 24- (Catalog No. 354408) (200-0 μM) were prepared using 1:2 serial dilutions.
and 384-well plates (Catalog No. 354667) were donated by These concentration ranges are reflective of common
Corning Life Sciences (Corning, NY). GravityTRAP™ ULA dosages used for treatment. Spent media was
96-well plates (Catalog No. ISP-09-001) were purchased removed from all test plate wells and replaced with
from PerkinElmer (Waltham, MA). Acetaminophen fresh media and compound titrations. Wells were
(Catalog No. 1706), Mitomycin C (Catalog No. 3258) re-dosed with fresh compound every forty-eight hours.
and Tolcapone (Catalog No. 5864) were purchased from
R&D Systems (Minneapolis, MN). ROS-ID® Total ROS/ Timepoints for assaying the health of the cells were 1
Superoxide Detection Kit (Catalog No. ENZ-51010), and 7 days for 2D plated hepatocytes and 1, 7, and 14
MITO-ID® Membrane Potential Detection Kit (Catalog days for 3D spheroids. Assay measurements included:
No. ENZ-51018) and NUCLEAR-ID® Blue/Red Cell Viability
Reagent (GFP CERTIFIED®) (Catalog No. ENZ-53005) 1. Reactive oxygen species (ROS) generation using
were donated by Enzo Life Sciences (Farmingdale, NY). ROS-ID Total ROS/Superoxide detection kit, which
includes two fluorescent dyes as major components:
Cytation™ 5 Cell Imaging Multi-Mode Reader Oxidative Stress Detection Reagent (Green) for total
ROS detection reagent and Superoxide Detection
Cytation 5 from BioTek Instruments is a modular multi- Reagent (Orange).
mode microplate reader combined with automated
digital microscopy. Filter- and monochromator-based 2. Mitochondrial membrane potential (MMP) decline
microplate reading are available, and the microscopy using MITO-ID, a cationic dye that fluoresces either
module provides up to 60x magnification in fluorescence, green or orange depending upon membrane potential
brightfield, color brightfield and phase contrast. The status. A reduction of orange fluorescence associated
instrument can perform fluorescence imaging in up to with MMP decline indicates early stages of apoptosis.
four channels in a single step. With special emphasis on
live-cell assays, Cytation 5 features temperature control 3. Plasma membrane (PM) rupture using NUCLEAR-ID,
to 65 °C, CO2/O2 gas control and dual injectors for kinetic a mixture of a blue fluorescent cell-permeable nucleic
assays. Z-stacking and projection can also be performed acid dye for live cell imaging and a red fluorescent cell-
to support 3D cell biology. The integrated Gen5™ impermeable nucleic acid dye that is suited for staining
Microplate Reader and Imager Software was used to dead nuclei.
control the imager and for automated dual-masking
The assay workflow began with media removal followed
analysis.
by media replacement containing probes from either the
multiplexed ROS-ID and NUCLEAR-ID, or MITO-ID and
Methods NUCLEAR-ID fluorescent microscopy kits, and incubated
for 5 hours at 37 ºC/5% CO2. Wells were then washed
with PBS to remove unincorporated probes, followed
Cell Culture Preparation
by image-based detection, also in PBS, using the
Cytation 5. A 10x objective was used for 2D cellular
iCell Hepatocytes 2.0 were thawed and cultured according
imaging and a 4x objective for 3D cellular imaging.
to the manufacturer’s protocol. See CDI User's Guide for
more details and media information. Cells intended for
3D spheroid formation were first seeded into 24-well
collagen coated plates at a concentration of 600,000
2 30

The signal from all multiplexed fluorescent probes was captured in a single imaging step using the following imaging
channels:
ROS-ID/NUCLEAR-ID multiplex assay – DAPI channel: NUCLEAR-ID live cell probe; Texas Red channel: NUCLEAR-ID
dead cell probe; RFP channel: ROS-ID superoxide probe.
MITO-ID/NUCLEAR-ID multiplex assay – DAPI channel: NUCLEAR-ID live cell probe; Texas Red channel: NUCLEAR-
ID dead cell probe; GFP channel: MITO-ID membrane potential probe cytosolic monomers; RFP channel: MITO-ID
mitochondrial aggregates.
Results and Discussion
2D Hepatotoxicity Testing
When assessing the potential of a drug or its metabolites to cause DILI, it is common not only to examine the ability
to induce overt cell death, but also to determine the cause of the observed hepatotoxicity. Two commonly measured
mechanisms include induction of oxidative stress and ROS generation, in addition to loss of mitochondrial membrane
potential (MMP) as an early indicator of apoptotic activity. The capacity of acetaminophen, mitomycin C and tolcapone
to induce oxidative stress and apoptosis, leading to downstream necrosis, following short-term and long-term
treatment of 2D plated iCell Hepatocytes was determined using fluorescence microscopy-based probes (Figure 1).
A. B. C.
D. E. F.
C.
G. H. I.
Figure 1. Images of 2D plated iCell Hepatocytes 2.0 expressing phenotypes of cellular distress after treatment
with acetaminophen. Images captured using a 10x objective. Top row: ROS generation detected as an increase
in orange puncta using the ROS-ID probe, whereas NUCLEAR-ID stained live cells blue or dead cells pink/red.
(A) Low; and (B) intermediate ROS generation after one day of acetaminophen exposure; and (C) high ROS levels
following seven days of acetaminophen treatment (625 µM). Middle row: MMP decline visualized as a loss of
orange aggregates and a simultaneous increase in smaller, rounded-up green stained cells due to compromised
MMP as detected by the MITO-ID reagent. Cells were also stained with NUCLEAR-ID probes to stain live cells
blue and dead cells pink/red. (D) Control cell population exhibiting stable mitochondrial membrane potentials.
(E) Partial and (F) complete loss of orange mitochondrial aggregates indicative of all cells having compromised
MMP following one and seven days of acetaminophen exposure (625 µM). Bottom row: PM rupture indicated
by a loss of green cytosolic staining using the green-fluorescent MITO-ID probe along with an increase in pink/
red stained dead cells versus blue live cells via NUCLEAR-ID. (G) Low amount of PM rupture following one-day
exposure versus (H) high amount of cells with PM rupture following seven days of acetaminophen exposure (625
µM). (I) Loss of cell attachment after seven days of treatment with 5 mM acetaminophen.
3 31

Consistent with previously reported mechanisms of acetaminophen toxicity1, these multiplexed fluorescent as-
says enabled detection of drug induced hepatotoxicity effects following exposure to high doses of acetaminophen.
Increasing acetaminophen concentrations and repetitive dosing resulted in detection of ROS formation (Figures 1A-C),
loss of mitochondrial membrane potential associated with apoptosis (Figures 1D-F), and eventual loss of cell membrane
integrity and necrotic cell death (Figures 1G-I).
2D Hepatocyte Quantitative Image Analysis
Quantification of superoxide expression, induction of apoptosis, and induction of necrotic activity, was performed
for all compound treatments and incubation periods using the cellular analysis features of Gen5™ and the
parameters found in Tables 1-3.

Channel DAPI
Threshold 10000
Background Dark
Split Touching Objects Checked
Include Primary Edge Objects Unchecked
Analyze the Entire Image Checked
Background Flattening Size 50 µm (Rolling Ball Diameter)
Image Smoothing Strength 0 Cycles of 3x3 average filter
Primary Mask Use Threshold Mask
Channel RFP
Measure Within a Secondary Mask Include Secondary Area Only in Analysis
Distance from Primary Mask 0 µm
Ring Width 75 µm
Threshold 5000
Smoothing 0
Method Threshold in Mask
Fill Holes in the Mask Unchecked
Table 1. 2D Superoxide Expression Cellular Analysis Parameters.
4 32


Channel DAPI
Threshold 10000
Background Dark
Channel RFP
Measure Within a Secondary Mask Include Secondary Area Only in Analysis
Distance from Primary Mask 0 µm
Ring Width 100 µm
Threshold 10000
Smoothing 0
Method Threshold in Mask
Table 2. 2D Apoptotic Activity Cellular Analysis Parameters.

Channel DAPI
Threshold 10000
Background Dark
Table 3. 2D Necrotic Activity Cellular Analysis Parameters.
33
5

For superoxide expression and apoptotic activity, primary masks were placed around Hoechst 33342 stained
nuclei (Figure 2A). Secondary masks were then placed around areas of the target probe exceeding set threshold values
(Figure 2B).
For necrotic activity, primary masks again were placed around nuclei stained with the live cell probe from the NU-
CLEAR-ID Blue/Red Cell Viability Reagent (Figure 2C). As both the live and dead cell probes were localized to
the nucleus, the fluorescence from the dead cell probe was also analyzed within the primary mask (Figure 2D).
A. B.
C. D.
Figure 2. 2D hepatotoxicity primary and secondary object mask placement. Untreated hepatocytes following addition of
MITO-ID superoxide probe showing (A) Primary nuclear object mask and (B) secondary object masks around target probe
signal. Hepatocytes following seven day acetaminophen exposure (625 µM) showing (C) primary mask placement using
signal from NUCLEAR-ID live cell probe, and (D) dead cell probe signal within primary masks.
Finally, subpopulation criteria were set to identify cells statistically responding to compound treatments (positive
responder cells). Table 4 describes the specific calculated cellular analysis metric and subpopulation criteria used to
identify the cells exhibiting the three different phenotypic effects from the compound treatment. The fraction of respond-
ing to total cells, expressed as a percentage, indicated the effect each compound treatment had on the hepatocytes
in the well (Figure 3).
Positive Responder Cell Identification Parameters

Phenotypic Response Identification Metric Subpopulation Criteria
Superoxide Expression RFP Secondary Mask Peak RFU (Peak_2) >12000 RFU
Apoptotic Induction RFP Secondary Mask Peak RFU (Peak_2) <25000 RFU
Necrotic Induction Texas Red Primary Mask Peak RFU (Peak) <40000 RFU
Table 4. 2D Positive Responder Cell Criteria.
6 34

A.
B.
2D Hepatocyte EC50 Values (µM)
Superoxide Expression
1 Day 7 Days
Acetaminophen 350 58
Mitomycin C >10 1.6
Tolcapone 9.7 1.0
Apoptosis
1 Day 7 Days
Acetaminophen 320 170
Mitomycin C >10 0.8
Tolcapone 24 34
Necrosis
1 Day 7 Days
Acetaminophen >5000 300
Mitomycin C >10 0.9
Tolcapone >200 14
Figure 3. 2D hepatotoxicity results. (A) Compound dose response curves, and (B) calculated EC50 values.
7 35

The over-the-counter pain reliever acetaminophen and the Parkinson’s disease drug tolcapone demonstrated the
phenotypes of ROS generation and MMP decline after only 24 hours treatment in a similar fashion. The expression
of these phenotypes was increased and evident at lower doses after 7 days treatment. PM rupture, however, was only
significantly evident after 7 days treatment. The chemotherapeutic agent mitomycin C demonstrated relatively muted
phenotypic responses compared to the other drugs, especially after only one day of treatment. However, still induces a
long-term toxic response on the 2D plated hepatocytes.
3D Hepatotoxicity Testing
The toxic effects of acetaminophen, mitomycin C and tolcapone were also examined in 3D cultured iCell Hepatocytes 2.0
spheroids (Figure 4). The same three phenotypes were assessed following 1, 7, and 14 day exposures to each compound.
A. B. C.
D. E. F.
C.
G. H. I.
Figure 4. Images of iCell Hepatocytes 2.0 spheroids expressing phenotypes of cellular distress after treatment with
acetaminophen. Images captured using a 4x objective. Top panel: ROS generation was detected as an increase in orange
signal within the spheroid using the ROS-ID probe. (A) Low; (B) intermediate; and (C) high ROS levels following fourteen
days of acetaminophen treatment. Middle panel: MMP decline was visualized as a loss of orange signal within the spheroid
while maintaining consistent green signal as detected by the MITO-ID reagent. (D) Control cell population exhibiting stable
MMP. (E) Partial; and (F) complete loss of orange mitochondrial aggregates following fourteen days of acetaminophen treat-
ment. Bottom panel: PM rupture was indicated by an increase in pink/red stained dead cells within the spheroid versus blue
live cells via NUCLEAR-ID. (G) Untreated hepatocyte spheroid; (H) intermediate; (I) high amounts of PM rupture following
fourteen-day acetaminophen treatment.
Similar to that seen with imaging of 2D plated hepatocytes, the signal from target probes was also detected from
hepatocytes aggregated into 3D spheroids. By collecting images at multiple z-planes and then projecting a final image
for each imaging channel containing the most in focus portions of the stack, accurate analysis of the impact of compound
treatment was performed.
3D Hepatocyte Spheroid Z-Projected Image Analysis
Levels of ROS generation, MMP decline and PM rupture were calculated in a similar manner as with the 2D plated
hepatocytes. For optimal calculations when using images of the 3D spheroids, Gen5™ cellular analysis parameters were
adjusted appropriately (Tables 5-7).
8 36


Channel DAPI
Threshold 8000
Background Dark
Background Flattening Size Auto (Rolling Ball Diameter)
Channel RFP
Measure Within a Secondary Mask Include Primary and Secondary Area in Analysis
Threshold 8000
Smoothing 0
Method Propagate Mask
Table 5. 3D Superoxide Expression Cellular Analysis Parameters.

Channel DAPI
Threshold 7000
Background Dark
Background Flattening Size Auto (Rolling Ball Diameter)
Channel RFP
Measure Within a Secondary Mask Include Primary and Secondary Area in Analysis
Threshold 40000
Smoothing 0
Method Propagate Mask
Table 6. 3D Apoptotic Activity Cellular Analysis Parameters.
9 37


Channel DAPI
Threshold 5000
Background Dark
Background Flattening Size 1000 (Rolling Ball Diameter)
Table 7. 3D Necrotic Activity Cellular Analysis Parameters.
By adjusting primary analysis criteria, such as minimum and maximum object size, primary masks were placed
around the entire spheroid as a single object (Figure 5A). Secondary masks were then placed around the target
fluorescent probe signal emanating from all cells within the spheroid meeting threshold criteria indicative
of responding cells to compound treatment (Figures 5B). The percentage of area covered by the secondary
masks divided by the entire spheroid area represented the portion of responding cells (Figure 5C and D).
A. B.
C.
10 38

D.
2D and 3D Hepatocyte EC50 Values (µM)
Acetaminophen
1 Day 7 Days 14 Days
2D 3D 2D 3D 2D 3D
Superoxide 350 >5000 58 >5000 n.d. 64
Apoptosis 320 >5000 170 1100 n.d. 29
Necrosis >5000 >5000 300 810 n.d. 73
Mitomycin C
2D 3D 2D 3D 2D 3D
Superoxide >10 >10 1.6 13 n.d. 0.7
Apoptosis >10 >10 0.8 >10 n.d. 1.3
Necrosis >10 >10 0.9 >10 n.d. >10
Necrosis
2D 3D 2D 3D 2D 3D
Superoxide 9.7 >200 1.0 44 n.d. 4.8
Apoptosis 24 >200 34 45 n.d. 9
Necrosis >200 >200 14 26 n.d. 10

Figure 5. 3D hepatotoxicity results. Primary nuclear object masks, and secondary object masks around (A) low; and (B) high target probe signal. (C)
Compound dose response curves. (D) Calculated EC50 values for 2D and 3D hepatocytes.
Analysis of the projected 3D images, dose-response Conclusions

curves, and generated EC50 values (Figures 5A-B, 5C,
5D, respectively) indicates that the hepatotoxins have 3D spheroid cultures of iPSC-derived human hepatocytes,
a minimal short-term effect and right-shifted such as iCell Hepatocytes 2.0, provide a relevant cell
pharmacology in 3D compared to 2D plated model to perform long-term in vitro hepatotoxicity
hepatocytes. The images also demonstrate that testing, while incorporation of fluorescent probes allow
induced necrosis in spheroidal hepatocytes does not quantification of cell toxicity phenotypes in 2D and 3D
cause large-scale cell loss. This feature of 3D cultures can cell models. Additionally, the optimized capabilities
allow long-term hepatotoxicity results to be generated of the Cytation™ 5 and Gen5™ Software provide
with greater accuracy. Finally, the ability to perform automated, dependable imaging and analysis of
extended compound treatments with the 3D hepatocyte incorporated cell models and fluorescent probes.
cell model allow for the elucidation of potential The combination of appropriate cell models, assay
mechanisms of action not possible in 2D. This is seen methodology, and imaging and analysis create an
by the induced ROS generation following a prolonged optimal method to determine the potential
fourteen day mitomycin C treatment (Figure 5C). chronic hepatotoxic effects of test molecules.
References
1. Hinson, J.A.; Roberts, D.W.; James, L.P. Mechanisms

of Acetaminophen-Induced Liver Necrosis. Handb Exp
Pharmacol. 2010, (196), 369-405.
11 39
AN081417_11, Rev. 08/16/17
Cell-Based Assays
The Effect of Cell Culture Method on Long-Term Primary

Hepatocyte Cell Health
Brad Larson, Applications Department, BioTek Instruments, Inc., Winooski, VT

Grant Cameron, TAP Biosystems, Royston, Hertfordshire, UK
Stewart Hunt, InSphero, Inc., Cambridge, MA
Diana Long, Promega Corporation, Madison, WI
Timothy Moeller, BioreclamationIVT, Baltimore, MD
Introduction
Hepatotoxicity studies are an important part The ability to culture, characterize and challenge
of the drug discovery process following lead cells over longer periods is of key importance
molecule generation. Whereas the initial during dosing and safety research. Here, we use
screening process identifies those molecules with two common screening tests to demonstrate
target efficacy, hepatotoxicity studies uncover differences between primary hepatocytes cultured
whether a potential drug causes drug-induced using traditional 2D culture methods and those
liver injury (DILI) in spite of any therapeutic effect. cultured using two novel 3D culture methods.
Cell function was measured via cytochrome
While in vivo studies are still the gold standard; P450 isoform 3A4 (CYP3A4) enzyme activity
in vitro screening using primary hepatocytes, the analysis using the P450-Glo™ CYP3A4 Assay with
cells predominantly responsible for the liver’s mass Luciferin-IPA, while cell viability was assessed by
and metabolic functions, has gained importance means of cellular ATP measurement using the
for reducing animal exposure, is amendable to CellTiter-Glo® Assay or CellTiter-Glo 3D Assay. All
high-throughput platforms and better equipped assays are manufactured by Promega Corporation.
to determine toxicity mechanisms of action.
Typically, this testing involves repeatedly dosing
the hepatocytes with the potential drug over Materials and Methods
Key Words: multiple days to assess cell viability, metabolism
and toxicity. However, the challenge remains Materials
in that hepatocytes cultured in traditional two-
3D dimensional (2D) formats undergo rapid de- Cells
Spheroid differentiation and loss of key functions1,2,3. Cryopreserved plateable human hepatocytes
Additionally, 2D cultured cells are less able to (Lot IZT) were provided by BioreclamationIVT
RAFT form the cell-cell and cell-matrix communication (Baltimore, MD). 3D liver microtissues from
networks seen in vivo4. Therefore, hepatotoxicity the aforementioned human hepatocyte lot,
Hepatocytes
studies are usually limited in duration, and results and created using proprietary hanging drop
Hepatotoxicity may not provide a complete understanding of the technology, were purchased from InSphero, Inc.
drug’s cumulative and long-term in vivo effects. (Cambridge, MA) and supplied in ready-to-use 96-
Cytochrome P450 well GravityTRAP™ plates.
Newer three-dimensional cell culture models
CYP3A4
exist, allowing cells to grow as aggregates Reagents, Kits, Consumables
Cell Viability thereby better resembling the functionalities
and communication networks found in vivo4. Two P450-Glo CYP3A4 Assay with Luciferin-IPA
such models will be discussed here. One model (Catalog No. V9002), CellTiter-Glo Assay (Catalog
uses a standard-sized 96-well microplate with a No. G7571), and CellTiter-Glo 3D Assay were
small cavity at the bottom of each tapered well. donated by Promega Corporation (Madison, WI).
When media and cells are added to the wells, BioCoat™ Collagen I 384-well black, clear bottom
the cells self-aggregate into microtissue spheres plates (Catalog No. 354667) and BioCoat Collagen
BioTek Instruments, Inc. in the hanging media drop formed by the hole. I 96-well black, clear bottom plates (Catalog No.
P.O. Box 998, Highland Park, The other uses a collagen hydrogel scaffold with 356649) were donated by Corning Life Sciences
Winooski, Vermont 05404-0998 USA (Corning, NY). The collagen hydrogel RAFT™
Phone: 888-451-5171 tissue-like properties that again encourages cells
to aggregate into three-dimensional structures 3D Cell Culture System was donated by TAP
Email: customercare@biotek.com (3D). Both methods offer a higher degree of Biosystems (Hertfordshire, UK).
www.biotek.com biomimicry, and encourage the re-establishment
Copyright © 2014
of cell-cell and cell-ECM communication.
40
Application Note Cell-Based Assays
Instrumentation Following incubation, 50 μL of supernatant was transferred

to a separate white 96-well assay plate, and an equal
MultiFlo™ FX Microplate Dispenser
volume of P450-Glo Luciferin Detection Reagent (LDR)
MultiFlo FX Microplate Dispenser was used to was added to the same wells. The assay plate was then
dispense the cell/collagen mix for RAFT hydrogel shaken for 60 seconds and incubated for 20 minutes at 37
creation, perform medium exchanges and test ºC/5% CO2, followed by luminescent signal measurement.
compound removal, and dispense assay components. 3D Liver Microtissues
Cytation™ 3 Cell Imaging Multi-Mode Microplate The aforementioned procedure was repeated using 30
Reader µL of pre-warmed medium containing Luc-IPA substrate
on selected wells. 50 µL of LDR was then added to those
Cytation 3 Cell Imaging Multi-Mode Microplate wells, and the wells were mixed five times by aspirating/
Reader was used to perform all luminescence dispensing. The contents of those selected wells, including
microplate reads using a 0.25 second integration time. microtissue, were then transferred to a white, 96-well assay
plate and incubated and read as previously described.
2D Hepatocytes
Methods
The aforementioned procedure was repeated using 25
Cell Culture and Propagation µL or 50 µL of pre-warmed medium for the 384-well and
96-well culture plates, respectively. Following incubation,
RAFT 3D Cultured Hepatocytes 12.5 µL of supernatant from the 384-well cell plate was
transferred to a separate white 384-well assay plate along
Cryopreserved hepatocytes were thawed and added to
with an equal amount of LDR, while 50 µL of supernatant
the prepared collagen solution provided in the RAFT 3D
from the 96-well cell plate was transferred to a separate
Cell Culture System. The mixture was then dispensed to
white 96-well assay plate along with an equal amount
a 96-well microplate in a volume of 240 µL per well at a
of LDR. The plates were shaken for 60 seconds and
final concentration of 100,000 cells/well. The cell plate
incubated for 20 minutes at room temperature followed
was then incubated at 37 ºC/5% CO2 for 15 minutes. After
by luminescent signal detection.
incubation, a 96-well RAFT plate containing individual
sterile absorbers was inserted into the cell plate wells and
Cell Viability
the combined system was incubated at 37 ºC/5% CO2
for 15 minutes to allow media absorption and collagen
RAFT 3D Cultured Hepatocytes
concentration. After incubation, the absorbing plate was
removed and 100 µL of new medium was added to the Using the original cell plate, 50 μL of CellTiter-Glo reagent
120 µm thick cell/collagen hydrogel. The cell plate was was added, the plate was shaken at room temperature
incubated at 37 ºC/5% CO2 for three days with daily for 5 minutes, then incubated at room temperature for
medium exchanges. 25 minutes. After incubation, the luminescent signal was
quantified.
3D Liver Microtissues
3D Liver Microtissues
Medium was exchanged in 96-well GravityTRAP plates
containing 3D liver microtissues from the supplier, and Using the original cell plate, 35 µL of CellTiter-Glo 3D
the plate was incubated overnight at 37 ºC/5% CO2. reagent was added to selected wells. The wells were mixed
five times by aspirating/dispensing, and the contents
2D Hepatocytes of those selected wells, including microtissue, were
transferred to a white, 96-well assay plate. The plate was
Cryopreserved hepatocytes were thawed, diluted with
shaken for 5 minutes at room temperature, followed by a
media provided by BioreclamationIVT, and plated in
25 minute room temperature incubation and luminescent
384-well black, clear bottom plates at concentrations of
signal detection.
2000, 5000, and 10,000 cells/well or in 96-well black, clear
bottom plates at a concentration of 50,000 cells/well and 2D Hepatocytes
incubated overnight 37 ºC/5% CO2.
After cell activity was measured, 12.5 μL of CellTiter-Glo
CYP3A4 Activity reagent was added to the original 384-well cell plate,
and 50 μL of CellTiter-Glo reagent was added to the
RAFT 3D Cultured Hepatocytes original 96-well cell plate. The plates were shaken at room
temperature for one minute, followed by an additional
Medium was removed from the wells and replaced with 25-minute room temperature incubation and subsequent
50 μL of 37 ºC pre-warmed medium containing 3 µM Luc- luminescent signal detection.
IPA substrate. The cell plate was then incubated at 37
ºC/5% CO2 for four hours.
2 41
Results and Discussion These findings demonstrate that enzyme activity

remains constant in both hepatocyte 3D culture
CYP3A4 Activity models over the entire culture period, thus confirming
that 3D cell cultures provide a superior cellular model
CYP3A4 activity in 3D and 2D cultured hepatocytes was for maintaining long term CYP enzyme activity
assessed for approximately two weeks, with daily media relative to conventional microplate cell seeding.
exchanges, and calculated as normalized percentages
from RLU values using the following formula: It is also interesting to note the effect of cell seeding
density in 2D cell culture evident in 96-well plates,
RLU(Day X)/RLU(Day 1)*100 (Figure 1B) 50,000 cells/well is sufficient cell density to
ensure cell:cell contact, which appears to better maintain
When comparing the liver microtissues to the 2D long term CYP activity relative to that demonstrated
cultured cells in 2000, 5000 and 10,000 cells/well at lower cell densities in 384-well plates (Figure 1A).
concentrations (Figure 1A), 3D CYP activity remains
unaffected throughout the incubation period, while Cell Viability
the 2D culture activity decreases significantly across
all three cell seeding densities, with the lowest activity Cell viability, as measured by ATP activity, in 3D and 2D
being seen with the 2000 cells/well concentration. cultured hepatocytes was also assessed for the same
Enzyme activity in the RAFT 3D cultured cells also time period, with daily media exchanges, and calculated
remains steady over the culture period, while the enzyme using the aforementioned formula from RLU values.
activity in 2D cultured cells decreases starting by the
third day and continuing to the tenth day (Figure 1B). Comparing the normalized ATP activity percentages
in the liver microtissues versus the 2D cultured cells in
A. varying concentrations (Figure 2A), it can be observed
that 3D cultured cell ATP levels remain constant. ATP
levels for the 2D cultured 2000 and 5000 cells/well
fall to 50% or lower by the end of the culture period,
while the ATP levels of the highest 2D cell culture
concentration remain similar to those seen in the 3D
hanging drop cultured cells. When comparing the
RAFT 3D cultured cells to 2D cells (Figure 2B), the 3D
cultured cell viability is again constant, while a loss of
viability is seen starting at day 7 in the 2D cultured cells.
A.
B.
B.
Figure 1. CYP450 Enzyme Activity Assessment in Long-Term 2D

and 3D Hepatocyte Cell Cultures. (A) 3D liver microtissues and
2D cultured cells in 2000, 5000, 10,000 cells/well concentrations.
(B) Collagen hydrogel cultured 3D cells and 2D cultured cells at a
concentration of 50,000 cells/well.
Figure 2. Cell Viability in Long-Term 2D and 3D Hepatocyte Cell

Cultures as calculated from ATP levels. (A) 3D liver microtissues and
2D cultured cells in 2000, 5000, 10,000 cells/well concentrations.
3 (B) Collagen hydrogel cultured 3D cells and 2D cultured cells at a
42
concentration of 50,000 cells/well.
The viability data also confirms that 3D cultured cells

retain plasma membrane integrity better relative.
Also, similar to the CYP3A4 activity data, cell

density has a demonstrable effect on cell viability.
Conclusions
Both the liver microtissue and collagen hydrogel 3D cell

culture models retained viability and normal function, as
witnessed by CYP3A4 activity and cell viability data, over
extended culturing conditions. The variability between
2D and 3D cultured hepatocytes highlights the necessity
to incorporate relevant 3D cell models when performing
long term toxicity studies that incorporate multiple
week dosing periods in order to fully understand any
cumulative hepatotoxic effects of a drug.
References
1. Kim, Y.; Rajagopalan, P. 3D Hepatic Cultures

Simultaneously Maintain Primary Hepatocyte and
Liver Sinusoidal Endothelial Cell Phenotypes. PLOS
ONE. 2010 Nov 12, http://www.plosone.org/article/
info%3Adoi%2F10.1371%2Fjournal.pone.0015456
(accessed February 4, 2014).
2. Chen, Y.; Wong, P.P.; Sjeklocha, L.; Steer, C.J.; Sahin,

M.B. Mature hepatocytes exhibit unexpected plasticity
by direct dedifferentiation into liver progenitor cells in
culture. Hepatology. 2012 Feb, 55(2), 563-574.
3. Elaut, G.; Henkens, T.; Papeleu, P.; Snykers, S.; Vinken,

M.; Vanhaecke, T.; Rogiers, V. Molecular mechanisms
underlying the dedifferentiation process of isolated
hepatocytes and their cultures. Curr Drug Metab. 2006
Aug, 7(6), 629-660.
4. Przyborski, S. 3D Cell Culture developments in

technology to improve in vitro analyses. Drug Discov
World. 2011 Spring, 67-72.
43
4 AN021914_01, Rev. 02/19/14
3D Cell Culture, Automation & Liquid Handling, Biomarkers, Cell Biology,
Cell Imaging, Cell-based Assays, Spheroid Imaging, Stem Cells
An Automated Walk-Away System to Perform Differentiation of

3D Mesenchymal Stem Cell Spheroids
Brad Larson, BioTek Instruments, Inc., Winooski, VT USA
Glauco Souza, Nano3D Biosciences, Inc., Houston, TX USA
Jan Seldin, Greiner Bio-One, Monroe, NC USA
Introduction
Human mesenchymal stem cells (hMSCs) are oxide and poly-L-lysine, which magnetizes the
multipotent and found in multiple areas of the cells without eliciting deleterious biological effect.
body including bone marrow, skeletal muscle, The cells are then placed into a microplate well
dermis, and blood. The cells are known for their and levitated by placing a magnet above the well,
ease of isolation and ability to differentiate where they aggregate and form extra-cellular
and mature into multiple lineages including matrix (ECM) within a few hours. After levitation,
adipocytes, chondrocytes, and osteocytes. the magnet is removed, and the 3D aggregates
hMSCs also play a critical role in adult tissue are dissociated into a dispersed cell suspension
repair, therefore are of great interest in tissue of single cells and small cell aggregates by gentle
engineering applications. For example, as adult pipetting action. Cells are then transferred to
cartilage cannot repair itself, chondrocyte- a 384-well assay plate and a spheroid magnet is
derived hMSCs may be used for cartilage repair positioned below the plate for an appropriate
applications, and in fact, transplantation of incubation period, allowing the cells within
spheroidal chondrocytes is already being studied each well to be patterned into a spheroid
as a treatment for hip joint cartilage defects1. Initial configuration. The magnetized spheroid can
hMSC experimentation involved two-dimensional be held intact during regular media exchanges.
(2D) cell culture in a monolayer. However,
Key Words:
culturing the cells in this manner results in a loss
of replicative ability, and differentiation capability
Stem Cell over time2,3. A number of techniques to culture
Mesenchymal Stem Cell hMSCs in a three-dimensional (3D) format were
then incorporated, such as pellet and micromass
Chondrocyte culture4,5. These methods better exemplified
Automation the differentiation process, but disadvantages
Differentiation included requiring large numbers of cells, difficult
manual processing steps, and a high overall cost
3D per method. Recently developed 3D cell culture
Spheroid technologies, which have the ability to create
spheroids from smaller cell numbers in high density
Magnetic Bioprinting
microplates, can overcome the limitations of
Nanoshuttle earlier methods while still providing the necessary
Antibody environment for proper stem cell differentiation.
Biomarkers
Complete differentiation from multipotent hMSCs
Quantitative Image Analysis to final target lineages, such as chondrocytes,
typically takes 14-28 days. With media exchanges
required every 2-3 days, a manual process is not
only tedious, but when working with nonattached Figure 1. BiO Assay Kit protocol. The 384-Well BiO Assay Kit
uses the NanoShuttle-PL nanoparticle assembly to magnetize
cells, increases the risk of accidental spheroid cells. After incubation, cells are detached, resuspended in a
removal. Automating the processing steps and cell-repellent plate, and magnetically levitated to aggregate
and induce ECM. After breaking up the aggregates, single
incorporating a 3D magnetic bioprinting method cells are transferred to a 384-well cell-repellent plate placed
atop a 384-well magnet, where they are printed at the well
frees researchers to perform other tasks and bottom.
BioTek Instruments, Inc. increases repeatability with little to no risk of
P.O. Box 998, Highland Park, spheroid loss. In this method (Figure 1), cells are
Winooski, Vermont 05404-0998 USA first incubated with a biocompatible magnetic
Phone: 888-451-5171
nanoparticle assembly consisting of gold, iron
www.biotek.com
Copyright © 2016
44
Application Note 3D Cell Culture, Automation & Liquid Handling, Biomarkers, Cell Biology,
Here we demonstrate the validation of a solution to Repellent Surface 6-Well (GBO Catalog No. 657860),
perform automated chondrocyte differentiation from were generously donated by Nano3D Biosciences, Inc.,
3D hMSC spheroids, where all instrumentation was (Houston, TX) and Greiner Bio-One, Inc., (Monroe, NC).
contained within a laminar flow hood. A combination
washer/dispenser with magnetic plate adapter was used Cytation™ 5 Cell Imaging Multi-Mode Reader
for media exchanges, while an automated incubator
maintained proper microplate environmental conditions Cytation 5 is a modular multi-mode microplate reader
between exchanges. Label-free cellular imaging under combined with automated digital microscopy. Filter-
environment control was performed following media and monochromator-based microplate reading are
exchanges to confirm maintenance of spheroids available, and the microscopy module provides up to
during processing. Immunofluorescence following 60x magnification in fluorescence, brightfield, color
differentiation confirmed the effectiveness of the brightfield and phase contrast. The instrument can
system for use with critical stem cell differentiation. The perform fluorescence imaging in up to four channels
combination of 3D magnetic bioprinting, automated in a single step. With special emphasis on live-cell
liquid handling and incubation, and image-based analysis assays, Cytation 5 features temperature control,
provides easy-to-use, robust methods to optimize CO2/O2 gas control and dual injectors for kinetic assays,
hMSC spheroid creation and differentiation processes. and is controlled by integrated Gen5™ Microplate
Reader and Imager Software. The software was also
used for dual-masking and automated analyses.
Materials and Methods BioSpa™ 8 Automated Incubator
Materials The BioSpa 8 Automated Incubator links BioTek readers

or imagers together with washers and dispensers for full
Cells workflow automation of up to 8 microplates. Temperature,
CO2/O2 and humidity levels are controlled and monitored
Poietics™ Normal Human Bone Marrow Derived through the BioSpa software to maintain an ideal
Mesenchymal Stem Cells (Catalog No. PT- 2501), environment for cell cultures during all experimental
Mesenchymal Stem Cell Growth Medium (MSCGM) stages.
BulletKit™ (Catalog No. PT-3001), and hMSC
Chondrogenic BulletKit (Catalog No. PT- 3003) EL406™ Combination Washer Dispenser
were donated by Lonza (Basel, Switzerland).
The EL406 offers fast, accurate media removal and plate
Antibodies and Inhibitor Compounds washing capabilities through its Dual-Action™ Manifold.
It also offers reagent dispensing capabilities through
Goat anti-ITGB1/CD29 antibody (Catalog No. ED08199) the use of its peristaltic or syringe pumps, with volumes
was purchased from Everest (Oxfordshire, UK). Rabbit ranging from 500 nL – 3000 μL/well. A specialized Magnet
anti-CD44 monoclonal antibody [EPR1013Y] (Catalog Adapter (Catalog No. 7182104) and 384-well Flat Magnet
No. ab51037), mouse anti-CD166 monoclonal antibody (Catalog No. 7103017) were used to secure placement
[8E12C7] (Catalog No. ab175428), rabbit anti-Collagen of the 3D spheroids during media transfer steps.
II polyclonal antibody (Catalog No. ab34712), donkey
anti-goat IgG H&L (Alexa Fluor® 488) polyclonal
Methods
antibody (Catalog No. ab150129), and donkey anti-
rabbit IgG H&L (Alexa Fluor 647) polyclonal antibody
(Catalog No. ab150075) were purchased from abcam Cell Preparation and Spheroid Formation
(Cambridge, UK). Goat anti-mouse IgG H&L (Alexa
Fluor 594) polyclonal antibody (Catalog No. A-11032) hMSCs were thawed from cryopreservation, resuspended
and Alexa Fluor 488 Phalloidin (Catalog No. A-12379) in complete MSCGM medium, and dispensed into
were purchased from ThermoFisher Scientific three separate T-75 flasks at a concentration of 5000
(Waltham, MA). Hoechst 33342 (Catalog No. 14533) cells/cm2, per the vendor’s recommended protocol. Cells
was purchased from Sigma-Aldrich (Saint Louis, MO). propagated in the flasks for seven days while the
cells reached a confluency of 80%. A 600 µL volume
Assay and Experimental Components of NanoShuttle-PL was then added to each flask and
incubated overnight. Cells were removed from the
The 384-Well BiO Assay™ Kit (GBO Catalog No. 781846, flasks and added to the 6-well cell repellent plate at a
consisting of 2 vials NanoShuttle™-PL, 6-Well Levitating concentration of 1.2x106 cells/mL in a volume of 2 mL/
Magnet Drive, 384-Well Spheroid and Holding Magnet well. A 6-well magnet drive was placed atop the plate to
Drives (2), 96-Well Deep Well Mixing Plate, 6-Well and levitate the cells, where they aggregated and induced
384-Well Clear Cell Repellent Surface Microplates), ECM formation during an eight-hour incubation at
prototype 384-Well Ring Drive and additional Cell 37 ºC/5% CO2. After incubation, cells and ECM were
2 45
broken up and resuspended. A total of 5000 cells were added to wells in a 384-well cell repellent microplate intended
for 3D spheroid formation, while a total of 10,000 cells were added to wells in a 384-well cell culture microplate intended
for 2D cell culture in a volume of 50 µL mesenchymal stem cell complete growth media. The process was replicated for a
total of four microplates intended for each cell culture method. A magnet was placed under each 3D spheroid plate. All
microplates were incubated at 37 °C/5% CO2 for approximately 48 hours to allow the 2D cells to attach to the microplate
well bottom, and to allow the 3D cells to aggregate into spheroids within each well.
hMSC Confirmational Imaging
Prior to initiating the differentiation process, immunofluorescent staining was performed on a subset of
undifferentiated 3D cultured spheroids to confirm proper hMSC functionality via expression of common
biomarker proteins using the procedure outlined in Table 1. Undifferentiated hMSC staining was also performed
on 2D cultured cells using generally accepted staining methods. Expression of hMSC CD29, CD44, and CD166
surface antigen markers was assessed using the specific primary and secondary antibodies detailed in Table 2.
Number Step Explanation Iteration/Incubation time
Incubate 5 minutes following

1 Wash Aspirate media and add 1mL PBS
each addition/Repeat 2x
2 Fixation Aspirate PBS and add 1 mL 4% paraformaldehyde 60 Minutes
Aspirate 4% paraformaldehyde and add 1 mL 0.2% Triton-
3 Permeabilization 60 Minutes
x100
4 Wash Aspirate media and add 1 mL PBS
Blocking buffer
5 Aspirate PBS and add 1 mL 1% BSA / 5% goat serum in PBS 60 Minutes
addition
Primary antibody
6 Dilute primary antibody according to specifications
preparation
Primary antibody
7 Aspirate PBS and add 1 mL diluted primary antibody Overnight @ 4 oC
addition
8 Wash Aspirate primary antibody and add 1 mL PBS Repeat 3x
Secondary antibody
9 Dilute secondary antibody according to specifications
preparation
Secondary antibody
10 Aspirate PBS and add 1 mL diluted secondary antibody 5 Hours @ RT
addition C.
11 Wash Aspirate secondary antibody and add 1 mL PBS Repeat 3x
Store @ 4 oC until time of
12 Imaging Preparation Aspirate final wash and add 1 mL PBS
imaging
Table 1. Spheroid Fixing and Staining Procedure.
Antigen Primary Antibody Dilution Secondary Antibody Dilution

Marker
Donkey anti-goat IgG H&L
Goat anti-ITGB1/
CD29 1:100 (Alexa Fluor® 488) polyclonal 1:200
CD29 antibody
antibody
Rabbit anti-CD44 Donkey anti-rabbit IgG H&L
CD44 monoclonal antibody 1:100 (Alexa Fluor® 647) polyclonal 1:200
[EPR1013Y] antibody
Mouse anti-CD166 Goat anti-mouse IgG H&L
CD166 monoclonal antibody 1:100 (Alexa Fluor® 594) polyclonal 1:200
[8E12C7] antibody
Table 2. Antigen Primary and Secondary Antibodies.
3 46
After the 3D spheroids or 2D cultured cells were immunostained, they were imaged using a 20x or a 10x objective, respec-
tively, using the fluorescence channels listed in Table 3.
Channel Fluorescent Probe

DAPI Hoechst 33342
GFP Alexa Fluor 488
Texas Red Alexa Fluor 594
CY5 Alexa Fluor 647
Table 3. Fluorescent probe and Cytation 5 imaging channel setup.
Automated Stem Cell Differentiation
After incubation to allow 2D cell attachment and 3D spheroid creation, the plates were placed into the BioSpa 8 at
37 °C/5% CO2 for up to twenty days during the differentiation period. The BioSpa 8 method was programmed such
that plates were automatically moved to the EL406 on day 0 and every three days subsequent to replace the re-
spective media. The EL406 was fitted with a specialized magnet adapter and 384-well flat magnet to secure the 3D
spheroids during liquid handling. A one-minute resting period allowed the spheroids to magnetically secure at the
well bottom, after which EL406’s aspirate pins removed 75% of the spent media from the wells of each microplate,
and new growth media was added via the peripump to negative control wells for a total volume of 50 µL per well,
while chondrocyte differentiation media was dispensed in the same manner to positive control wells. Following me-
dia exchange, the BioSpa 8 arm then automatically moved each plate from the EL406 to Cytation 5, where bright-
field imaging was performed to confirm successful media exchange without loss of cells. The parameters listed in
Table 4 were used to accurately focus on the hMSC spheroids and stitch together the montage tiles into a final image.
Brightfield Imaging Parameters

Objective 4x
Imaging Channel Brightfield
Image Focusing Autofocus
Image Montage 3x2
Delay after Plate Movement 30 msec
Montage Autofocus Option Only focus on center of montage
Tile Overlap Columns: 197 µm / Rows: 300 µm
Table 4. Cytation 5 imaging parameters.
Expression of the collagen II protein, a prominent component of healthy cartilage6 and a validation of chondrocyte
differentiation7, was determined using the antibodies in Table 5.
Differentiation Primary Antibody Dilution Secondary Antibody Dilution

Marker
Donkey anti-rabbit IgG
Rabbit anti-Collagen
Collagen II 1:100 H&L (Alexa Fluor® 647) 1:200
II polyclonal antibody
polyclonal antibody
Table 5. Collagen II Primary and Secondary Antibodies.
Additionally, at Day 5, 10, 20, one microplate each containing 2D cells and 3D spheroids was removed from BioSpa 8, and
fluorescent immunostaining per the aforementioned procedure was performed to detect collagen formation. A place-
holder microplate was substituted for each removed assay plate to maintain the robotic protocol.
4 47
Results and Discussion
hMSC Confirmational Imaging
Proper hMSC function was validated by confirming the presence of commonly expressed surface antigen markers. As seen
in Figure 2, fluorescent signals corresponding to CD29, CD44, and CD166 surface antigen markers were detected in 2D
cultured cells and 3D cultured spheroids. Signal from bound primary and fluorescently labeled secondary antibodies ap-
pear as punctuate spots within each image, indicating distinct areas of antigen expression within 2D or 3D cultured cells.
A. B.
Figure 2. hMSC biomarker expression as imaged by Cytation 5. Arrows indicate immunofluorescence

identification of protein expression to confirm proper cell function in (A) 2D cultured cells using 10x
objective and; (B) 3D cultured spheroids using 20x objective. DAPI: Hoechst 33342 stained nuclei, GFP:
CD29 expression, Texas Red: CD166 expression, CY5: CD44 expression.
Automated Stem Cell Differentiation
During designated media exchange periods, brightfield imaging was performed to confirm that cells and spher-
oids remained intact during the aspiration and dispense procedure. As seen in Figure 3A-C, 3D spheroids are con-
firmed to remain intact in the wells during media exchanges over the entire twenty-day incubation. The same can
be said for 2D cultured cells up to 10 days of incubation (Figure 3D and E). However, after 10 days of culture in the
plates, visible cell loss is witnessed following media exchange (Figure 3F). This observation confirms previous re-
search findings that 2D cultured cells lose integrity, detach, and become non-viable following ten days of incubation8.
5 Days 10 Days 20 Days

A. B. C.
3D
D. E. F.
2D
Figure 3. Post-media exchange imaging validation. Day 5, Day 10 and Day 20 3x2 montage brightfield images
captured using a 4x objective of (A-C) 3D spheroids; and (D-F) 2D cultured cells demonstrating visible cell loss
starting at Day 10.
48
5
Chondrocyte differentiation in 2D cultured cells was then examined by comparing cells cultured in differentia-
tion media to those remaining undifferentiated in growth media. Per Figure 4, initial chondrocyte differentiation
(Figure 4D) is seen within five days of incubation, and rapidly peaks at ten days (Figure 4E), compared to no differ-
entiation in cells cultured in growth media (Figure 4A-C). After ten days, loss of viability occurs in all 2D cultured
cells; and in the differentiated cells, the collagen II fluorescent probe is leached into the surrounding media (Fig-
ure 4F). This confirms the limitations associated with incorporating 2D differentiated hMSCs in long-term studies.

A. B. C.
Undifferentiated
D. E. F.
Differentiated
Figure 4. 2D cultured hMSC chondrocyte differentiation over time. Day 5, Day 10 and Day 20 images of (A-C)
undifferentiated cells; and (D-F) differentiated cells, captured using 10x objective. DAPI: Hoechst 33342 stained
nuclei, GFP: AlexaFluor 488 phalloidin stained actin filaments, CY5: Collagen II expression.
In the same manner, chondrocyte differentiation in 3D cultured spheroids was examined by comparing spher-
oids cultured in differentiation media to those remaining undifferentiated in growth media. Per Figures
5A-C, no discernible collagen expression is seen in undifferentiated spheroids, while a steady increase in col-
lagen expression over time is seen in differentiated spheroids (Figures 5D-F). This confirms the suitability of 3D cul-
tured and differentiated hMSC spheroids for long-term studies. Additionally, the differentiated spheroid images
were overlaid at individual z-planes (Figure 6) to improve image clarity and enable quantification of differentiation.

A. B. C.
Undifferentiated
D. E. F.
Differentiated
Figure 5. 3D cultured hMSC spheroid chondrocyte differentiation over time. Day 5, Day 10 and Day 20 images
of (A-C) undifferentiated spheroids; and (D-F) differentiated spheroids, captured using 20x objective. DAPI:
Hoechst 33342 stained nuclei, GFP: AlexaFluor 488 phalloidin stained actin filaments, CY5: Collagen II expression.
6 49
A. B. C.
D.
Figure 6. Z-stacking and projection of 3D spheroid images. (A-C) Images captured at individual z-planes. (D)
Final z-projected image of chondrocyte differentiated hMSC spheroid. Arrows indicate nuclei, collagen and
protein expression. DAPI: Hoechst 33342 stained nuclei, GFP: AlexaFluor 488 phalloidin stained actin filaments,
CY5: Collagen II expression.
Quantification of Chondrocyte Differentiation via

Cellular Analysis
Using the z-stacked image, Gen5 software automatically pre-processed the samples to remove excess background signal
and prepare the image for quantitative analysis. Primary cellular analysis criteria were applied to place an object mask
around the entire spheroid. Secondary analysis criteria were then used to automatically mask areas within the spheroid
where the CY5 signal from collagen II antibody labeling was greater than background threshold levels as indicated by the
arrows in Figure 7.
Figure 7. Automated dual-mask analysis, with primary mask placed

around the entire spheroid, and secondary mask placed around
7 discontinuous areas of increased CY5 signal. 50
The percent of CY5 area coverage, indicating greater References

differentiation and collage II expression can then be cal-
culated as a ratio of the secondary mask to the primary 1. Körsmeier, K.; Claßen, T.; Kamminga, M.; Rekowski,
mask, expressed as a percentage. The final percentage J.; Jäger, M.; Landgraeber, S. Arthroscopic three-dimen-
values in Figure 8 indicate a significant increase in col- sional autologous chondrocyte transplantation using
lagen II production in chondrocyte differentiated hMSC spheroids for the treatment of full-thickness cartilage
spheroids compared to undifferentiated hMSC spher- defects of the hip joint. Knee Surg Sports Traumatol
oids, thus validating that 3D cultured hMSC spheroids Arthrosc. 2016, 24(6), 2032–2037.
can be successfully differentiated into chondrocytes.
2. Park, E.; Patel, A.N. Changes in the expression pat-
tern of mesenchymal and pluripotent markers in human
adipose-derived stem cells. Cell Biol Int. 2010, 34,
979–984.
3. Baer, P.C.; Griesche, N.; Luttmann, W.; Schubert, R.;

Luttmann, A.; Geiger H. Human adipose-derived mes-
enchymal stem cells in vitro: evaluation of an optimal
expansion medium preserving stemness. Cytotherapy.
2010, 12, 96–106.
4. Chang, C.H.; Lin, H.Y.; Fang, H.W.; Loo, S.T.; Hung,

S.C.; Ho, Y.C.; Chen, C.C.; Lin, F.H.; Liu, H.C. Chondro-
genesis from immortalized human mesenchymal stem
cells: comparison between collagen gel and pellet
Figure 8. Change in CY5 signal compared to background threshold
over time.
culture methods. Artif Organs. 2008, 32(7), 561-566.
5. Ahrens, P.B.; Solursh, M.; Reiter, R.S. Stage-related

capacity for limb chondrogenesis in cell culture. Dev
Biol. 1977, 60(1), 69–82.
Conclusion
6. Yoon, H.J.; Kim, S.B.; Somaiya, D.; Noh, M.J.; Choi, K.;
The 384-Well BiO Assay Kit and NanoShuttle-PL particles Lim, C.; Lee, H.; Lee, Y.; Yi, Y.; Lee, K.H. Type II collagen
manufactured by nano3D Biosciences, combined with and glycosaminoglycan expression induction in primary
Greiner Bio-One Cell-Repellent Surface 6-Well and 384- human chondrocyte by TGF-β1. BMC Musculoskelet
Well Microplates, provide a simple and robust method Disord. 2015, 16(141), 1-12.
to create biomimetic hMSC spheroids. Through incorpo-
ration of the BioSpa™ 8 and magnetic adapter on the 7. Bhang, S.H.; Jeon, J.Y.; La, W.G.; Seong, J.Y.; Hwang,
EL406™, the differentiation process can be automated J.W.; Ryu, S.E.; Kim, B.S. Enhanced chondrogenic
to simplify and increase the repeatability of included marker expression of human mesenchymal stem cells
procedures. Automation and differentiation confirma- by interaction with both TGF-β3 and hyaluronic acid.
tion can then be performed using brightfield and fluo- Biotechnol Appl Biochem. 2011, 58(4), 271-276.
rescent imaging with the Cytation™ 5. The combination
provides a proven method to carryout differentiation of 8. Maguire, T.; Novik, E. Methods in Bioengineering:
3D cultured stem cells. Alternative Technologies to Animal Testing; Artech
House methods in bioengineering series; Artech House:
Boston, MA, 2010.
8 51
AN121616_22, Rev. 121616
3D Cell Culture
An Image-Based Method to Detect and Quantify T Cell Mediated

Cytotoxicity of 2D and 3D Target Cell Models
Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA
Wini Luty, Courtney Noah, BioreclamationIVT, Westbury, NY USA
Olivier Donzé, AdipoGen Life Sciences, Epalinges, Switzerland
Glauco R. Souza, University of Texas Health Science Center at Houston and Nano3D Biosciences, Inc.,
Houston, TX USA
Abstract
T cell mediated cytotoxicity plays an important role in a suite of new methods being
developed with the goal of boosting a patient’s immune system to combat cancer. In
order to evaluate and optimize adoptive T cell immunotherapies, sensitive in vitro methods
must be included in the testing process. In the procedure described here, phenotypic and
quantitative assessments of 2D and 3D target cell necrotic induction were made using
automated live cell imaging. It was found that direct activation of T cells produced a
significantly greater cytotoxic effect than general activation suggesting that T cells can be
"taught" to target and destroy specific target cells.
Introduction
CD3+CD8+ cytotoxic T lymphocytes (CTL) are cells4, newer methods were developed using
the effector cells responsible for T cell mediated microplate-based optical methods generating
Key Words: cytotoxicity that can act by cell-to-cell contact luminescence or fluorescence. These techniques
either by releasing granzymes and perforin or were optimized to detect the signal from target
Adoptive Immunotherapy
through Fas ligand mediated toxicity1. As part of cells plated in a uniform two-dimensional (2D)
T Cell Mediated Cytotoxicity the adaptive immune system, these cells mount monolayer in microplate wells. With increasing
Cancer Immunotherapy targeted attacks to rid the body of a variety of adaptation of cells aggregated into a three-
T Cell Activation compromised cells, such as cancer cells, without dimensional (3D) configuration to create a more
harming healthy cells. Counteracting this natural in vivo-like model, cells are no longer evenly
Directed Activation
defense is the widely known fact that tumors spread throughout the bottom of a
Immuno-Oncology develop multiple methods to avoid immune well. Through the incorporation of
Cytotoxic T Lymphocyte detection and create a level of tolerance against microscopic imaging and cellular analysis,
T Cell the immune cells designed to seek out and destroy sensitive detection of induced cytotoxicity from
cells containing foreign antigens2. For many years, 2D and 3D plated target cells, as well as
the development of treatments avoided use of visualization of the interplay between CTL and
a patient’s immune system to kill cancer cells, target cells, can be achieved.
as immunotherapy-based treatments met with
multiple clinical failures. Developing methods Here, we demonstrate an automated method
offer renewed hope for cancer patients. Adoptive to monitor and measure CTL cell mediated
immunotherapy techniques activates a patient’s cytotoxicity kinetically using digital widefield
T cells ex vivo against tumor antigens before microscopy. Co-cultured target MDA-MB-231
infusing the activated T cells back into the patient breast cancer and fibroblast cells were plated
to target and destroy tumor cells selectively3. in 2D and 3D format and dosed with a live cell
apoptosis/necrosis reagent. T cells, activated
The most popular in vitro method to monitor using general or directed methods and stained
CTL effect on target cells is the cell mediated with a far red tracking dye, were then added in
cytotoxicity (CMC) assay where T cells and ratios of 20, 10, 5, or 0:1 to the target cells.
target cells are added to a microplate well The plates were then added to an automated
BioTek Instruments, Inc.
P.O. Box 998, Highland Park, as a co-culture. Traditionally toxicity was incubator and shuttled to the digital
Winooski, Vermont 05404-0998 USA measured using chromium (51Cr) release from widefield microscope, using a robotic arm,
Phone: 888-451-5171 preloaded target cells. Due to problems with every four hours where brightfield and
Outside the USA: 802-655-4740 fluorescent images were captured for a total
radioactivity disposal, and low sensitivity due to
www.biotek.com spontaneous release of the isotope from target of seven days. Visual observation of the
Copyright © 2017
52
kinetic images enabled monitoring of CTL:target cell interactions for 2D and 3D cultured cells, while cellular image
analysis allowed for calculation of CTL induced cytotoxicity during the entire incubation period.
Materials and Methods
Materials
Cells and Media
MDA-MB-231 epithelial breast adenocarcinoma cells (Catalog No. HTB-26) were obtained from ATCC (Manassas,
VA). Human Neonatal Dermal Fibroblast cells stably expressing RFP (Catalog No. cAP-0008RFP) were purchased from
Angio-Proteomie (Boston, MA). Human purified CD3+ T cells, isolated via negative selection from peripheral blood
mononuclear cells (Catalog No. HM-PBMC-TCELLCD3-M) were donated by BioreclamationIVT (Westbury, NY). Advanced
DMEM (Catalog No. 12491-015), RPMI 1640 medium (Catalog No. 11875-093), Fetal bovine serum, (Catalog No. 10437-
036), and penicillin-streptomycin-glutamine (100X) (Catalog No. 10378-016) were purchased from ThermoFisher Scientific
(Waltham, MA).
Assay and Experimental Components
IL-2 Superkine (Fc) (Catalog No. AG-40B-0111-C010), anti-CD3 (human), mAb (UCHT1) (Catalog No. ANC-144-
020) and anti-CD28 (human), mAb (ANC28.1/5D10) (Catalog No. ANC-177-020) were donated by AdipoGen
Life Sciences (San Diego, CA). SCREENSTAR® 190 µm cycloolefin filmbottom 384-well microplates (GBO
Catalog No. 789836), CELLSTAR® µClear 384-well cell-repellent surface microplates (GBO Catalog No. 781976) and
the 384-Well BiO Assay Kit (GBO Catalog No. 781846, consisting of 2 vials NanoShuttle-PL, 6-Well Levitating Magnet
Drive, 384-Well Spheroid and Holding Magnet Drives (2), 96-Well Deep Well Mixing Plate, 6-Well and 384-Well Clear
Cell Repellent Surface Microplates), prototype 384-Well Ring Drive, and additional Cell Repellent Surface 6-Well (GBO
Catalog No. 657860) were donated by Nano3D Biosciences, Inc., and Greiner Bio-One, Inc., (Monroe, NC). The Kinetic
Apoptosis Kit (Microscopy) (Catalog No. ab129817) was donated by Abcam (Cambridge, MA). CellTracker™ Deep Red
Dye (Catalog No. C34565) was purchased from ThermoFisher Scientific (Waltham, MA).
Cytation 5 is a modular multi-mode microplate reader combined with an automated digital microscope.
Filter- and monochromator-based microplate reading are available, and the microscopy module provides up to
60x magnification in fluorescence, brightfield, color brightfield and phase contrast. The instrument can perform
fluorescence imaging in up to four channels in a single step. With special emphasis on live cell assays, Cytation 5
features shaking, temperature control to 65 ºC, CO2/O2 gas control and dual injectors for kinetic assays, and is
controlled by integrated Gen5™ Microplate Reader and Imager Software, which also automates image capture,
processing and analysis. The instrument was used to kinetically monitor CTL:target cell interactions as well
as cytotoxicity induction within the 2D and 3D plated target cells.
BioSpa™ 8 Automated Incubator
The BioSpa 8 Automated Incubator links BioTek readers or imagers together with washers
and dispensers for full workflow automation of up to eight microplates. Temperature, CO2/
O2 and humidity levels are controlled and monitored through the BioSpa software to maintain
an ideal environment for cell cultures during all experimental stages. Test plates were incubated in the
BioSpa to maintain proper atmospheric conditions for a period of seven days and automatically transferred to
the Cytation 5 every four hours for brightfield and fluorescent imaging.
Methods
Overview
This work uses three workflows which are depicted pictorially in Figure 1.
2 53
Figure 1. T Cell activation and cell mediated cytotoxicity assay workflow.
The first workflow on the left side of Figure 1 involves

T cell activation where CD3+ T cells are exposed to
the MDA-MB-231 target cells which have been
bioprinted into spheroids using magnetic fields
(see 3D Target Cell Preparation for further detail.)
The activated T cells are then stained with CellTracker
Deep Red dye and used in either a bioprinted 3D
spheroid-based cytototoxicity assay or another using
plated cells. The CellTracker dye allows visualization of
the T cells attacking the target cells, while propidium
iodide dye allows for quantification of target cell
death associated with plasma membrane rupture.
3D Target Cell Preparation
T-75 flasks of MDA-MB-231 or fibroblast cell cultures

were cultured to 80% confluence, then as illustrated
in Figure 2, treated with 600 μL NanoShuttle-PL
overnight at 37 ºC/5% CO2. After incubation, cells
were trypsinized for 3-5 minutes at 37 ºC/5% CO2.
Cells were removed from the flasks and added to the
6-well cell repellent plate at a concentration of 1.2x106
cells/well. A 6-well magnet drive was placed atop the Figure 2. Bioprinting procedure used to create 3D spheroids for T cell
activation.
well plate to levitate the cells, where aggregation and
extracellular matrix (ECM) formation took place during
an eight-hour incubation at 37 ºC/5% CO2. After
incubation, the cells and ECM were broken up,
resuspended, and combined together at equal
concentrations in complete advanced DMEM
medium.
3 54
For T cell activation, 3D spheroids were bioprinted in a 24-well cell repellant microplate using a 384-well spheroid magnet
drive (see Directed and General T cell Activation).
A modified procedure associated with Figure 2 was used to prepare 3D spheroids for the cytotoxicity assay. The
procedure was the same until the spheroid bioprinting was conducted. Instead of bioprinting in a 24-well plate as
for T cell activation, the assay used 384 well plates such that a single spheroid was bioprinted in each well. To each
well of the 384-well cell repellent microplate, a total of 2000 cells (1000 MDA-MB-231 and 1000 fibroblasts) were
added. The microplate was incubated at 37 °C/5% CO2 for 48 hours to allow the cells to aggregate into co-cultured
tumoroids within each well.
2D Target Cell Preparation
T-75 flasks of MDA-MB-231 or fibroblast cell cultures were cultured to 80% confluence. Cells were then
trypsinized for 3-5 minutes at 37 ºC/5% CO2 and removed from the flasks. Following centrifugation, the cells were
resuspended and combined together at equal concentrations in complete advanced DMEM medium. A total
of 2000 cells (1000 MDA-MB-231 and 1000 fibroblasts) were added to wells of a 384-well TC treated microplate
intended for 2D cell culture (Figure 2). The microplate was incubated at 37 °C/5% CO2 overnight to allow the
cells to attach to the wells.
Directed and General T Cell Activation
A total of 10,000 target cells and media were added to 24-well cell repellent plate wells for each experimental
condition as follows (Figure 2). Directed activation: (A) 100% MDA-MB-231; (B) 75% MDA-MB-231 and
25% fibroblasts; (C) 50% MDA-MB-231 and 50% fibroblasts; general activation: (D) no cells. Total volume was
1 mL for wells in each test condition. The 24-well plate was then placed atop a 384-well spheroid magnet drive
and incubated at 37 ºC/5% CO2 for four days where the cells aggregated into multiple 3D spheroids within
each well (Figure 3). Note that the magnet drive is designed for 384-well densities, such that the expanded size
of a 24-well plate well provides nine (9) separate spheroids/well.
Figure 3. 24-well plate well showing co-culture of T cells and bioprinted

magnetized 3D target spheroids prior to commencement of directed activa-
tion. T cells added in a 10:1 ratio to target cells previously aggregated into 3D
spheroids (~ 1000 µm ID).
Following spheroid aggregation, T cells were prepared at a concentration of 100,000 cells/mL in RPMI medium
containing 100 ng/mL IL-2 Superkine (Fc) (superkine) along with 250 ng/mL each of anti-CD3 and anti-CD28
antibodies. Spent media was then aspirated while the plate remained on the magnet drive to secure the
spheroids, and replaced with fresh media containing the T cells, antibodies, and superkine as previously
described. The plate was then placed back into the BioSpa™ to incubate for six days. The
BioSpa was pre-programmed to capture a 12 x 10 image montage from each test well every six
hours. Manual exchange of media, IL-2 Superkine, and antibodies was performed after 72 hours.
The directed activation procedure over the six days serves to not only activate the T-cells, but also teaches
them to recognize target cell antigens allowing for targeted cytotoxicity. General activation follows the
same procedure, but uses no target cells, thus there should be no targeted cytotoxicity5.
4 55
T Cell Staining and Addition
Upon completion of the activation process, the 24-well plate containing the T cells and magnetized
target cells was placed back on the 384-well magnet drive. The T cells were then removed from each well
and transferred to a separate 15 mL conical tube for staining with the CellTracker Deep Red Dye allowing
for differentiation from the target cells during the cytotoxicity experiment. Dye, at a concentration of 1 µM,
was added to the tubes and incubated at 37 oC/5% CO2 for 45 minutes. The tubes were then centrifuged for
15 minutes at 200 RCF. Media containing the excess dye was then removed and replaced with fresh RPMI
medium. Stained T cells from each activation condition were then diluted in RPMI medium containing 10 µL/mL of
the propidium iodide necrosis probe from the Kinetic Apoptosis Kit. The cells were then added to the 384-well
2D or 3D cell culture plates, already containing a total of 2000 target cells, in concentrations equaling 40,000 cells/
well, 20,000 cells/well, or 10,000 cells/well (Figure 1). These concentrations created ratios of 20:1, 10:1, or 5:1 T cells
to target cells in each well. Untreated negative control wells were also included to examine basal target
cell cytotoxicity levels over time. Table 1 illustrates the final plate layout.
Table 1. 2D and 3D Cell Mediated Cytotoxicity Assay Plate Layout.
Cell Mediated Cytotoxicity Assay Automated Procedure
Figure 4. BioSpa Live Cell Imaging System, including BioSpa 8 and Cytation 5.
2D and 3D assay plates, containing T cells and target cells, were added to the BioSpa™ 8, as part of
the BioSpa Live Cell Imaging System (Figure 4), with atmospheric conditions previously set to 37 oC/5% CO2.
Water was also added to the pan to create a humidified environment, which was monitored. The BioSpa
software was set such that the plates were automatically transferred to the Cytation™ 5 for brightfield and
fluorescent imaging of the test wells every four hours for a total of seven days. Table 2 explains the imaging carried
out with each channel. For 2D plated cells, a single 4x magnification image was taken with each channel to
capture a representative population of cells per well. Laser autofocus was incorporated to ensure proper
focusing on the target cell layer as well as the most efficient focusing procedure. For 3D plated cells, since the
cells within the 3D target cell spheroids existed on multiple z-planes, a z-stack consisting of five slices was
captured with each channel. Laser autofocus was again incorporated. Two images each were taken below and
above the decided upon focal plane.
5 56
Imaging Channel Target Necrotic Cell Identification Criteria

Brightfield All Cells 2D Analysis 3D Analysis
PI Necrotic Cells Tsf[Propidium Tsf(ZProj[Propidium
Channel
CY5 T Cells Iodide] Iodide])
Table 2. Cell Imaged per Imaging Channel. Threshold Auto (-6) 5000
Background Dark Dark
Split Touching
2D and 3D Image Processing Checked Checked
Objects
Fill Holes in Masks Checked Checked
Following capture, 2D and 3D images were
Min. Object Size 10 µm 10 µm
processed prior to analysis. 2D images underwent
preprocessing to remove background signal Max. Object Size 4000 µm 100 µm
from each channel using the settings in Table 3. Include Primary
Unchecked Unchecked
Edge Objects
2D Image Pre-processing Parameters Analyze Entire
Checked Checked
Apply Image Rolling Ball Image
Channel Background
Pre-processing Diameter
Brightfield No
Rolling Ball
1000 µm 75 µm
PI Yes Dark Auto Diameter
CY5 Yes Dark Auto Image Smoothing
20 0
Strength
Table 3. 2D Image Pre-processing Parameters.
Evaluate 5% of Lowest 5% of Lowest
Background On Pixels Pixels
For 3D images, first a z-projection of the images cap- Analysis Metric
tured in the z-stack was carried out to create a final image
Object Sum
containing only the most in-focus information (Table 4). Int[Tsf[ZProj
Metric of Interest Cell Count
[Propidium
3D Image Stiching Parameters Iodide]]]
Method Focus Shaking Table 6. Necrotic Cell Identification Criteria.
Size of Max. Filter 11 Pixels
Top Slice 0 µm from focal plane
Bottom Slice -53.8 µm from focal plane An additional image analysis step was performed on
the 3D images to determine the extent to which target
Table 4. 3D Z-projection Criteria.
cell spheroids disintegrated following T cell treatment
(Table 7).
Preprocessing of the projected image was then per-
3D Image Analysis Parameters
formed to again remove background signal from each
channel (Table 5). Data In Tsf(ZProj[Brightfield])
Threshold (Lower Value) Unchecked
3D Image Pre-processing Parameters Threshold (Upper Value) Checked (2500)
Apply Image Rolling Ball
Channel Background Analysis Metric
Pre-processing Diameter
Brightfield No Metric of Interest Confluence
PI Yes Dark Auto Table 7. Spheroid Disintegration Criteria.
CY5 Yes Dark Auto

Table 5. 3D Image Pre-processing Parameters.
Cellular Analysis of 2D and 3D Processed Images
Cellular analysis was carried out on the processed

images to determine the total signal emanating
from necrotic target cells using the criteria in Table 6.
6 57
Results and Discussion A.
Image-Based Detection of Co-Cultured Cell

Interaction
T cells, activated using direct and general activation

procedures, were added to the target cells in con-
centrations equaling 20:1, 10:1, 5:1 and 0:1 to start B.
the cell mediated cytotoxicity assay. To monitor the
interaction of the co-cultured cells, plates were im-
B.
aged immediately following T cell addition and ev-
ery four hours subsequent throughout the entire
seven-day incubation period.
As assay incubation times increase, it is apparent

that activated T cells (red fluorescence) seek out
and cluster around the antigen presenting target
cells through antigen-receptor binding in both
2D and 3D formats (Figure 5A). This T cell aggregation
is in marked contrast to the more even distribution Figure 6. Brightfield/PI Imaging of Cellular
of red fluorescence at time 0. Interaction. 4x brightfield and PI images show-
ing necrotic (A) 2D or; (B) 3D target cells in
response to T cell binding. Time = 24 hours.
A.
Kinetic Imaging of T Cell Mediated Target Cell

Cytotoxicity Induction
In order to determine the kinetics of cytotoxicity

induction within the target cells, imaging must
be carried out at regular intervals throughout
the entire incubation period. As the full cytotoxic
B. effect may not be reached until days after T cell
addition, it is also essential that cells be allowed to
interact for multiple days. The environmental
controls of the Cytation™ 5 and BioSpa™ 8, as well
as automatic transfer of test plates from incubator
to imager, allow kinetic analysis to be completed
without compromising cell health. In the experi-
ments performed here, brightfield and fluorescent
images were captured every four hours for a total of
Figure 5. Brightfield/CY5 Imaging of Cellular seven days. Figures 7 and 8 demonstrate the iterative
Interaction. 4x brightfield and CY5 images cytotoxic effect that T cells, directly activated in
showing T cell clustering and binding to (A) 2D
or; (B) 3D target cells. Time = 24 hours. the presence of 100% MDA-MB-231 cells and
added at a 20:1 ratio, have on 2D and 3D cultured
target cells, respectively.
When images from the PI channel are overlaid

with those from the brightfield channel one can
observe that yellow fluorescent signal from the
propidium iodide necrotic cell probe originates
from the same target cells with bound T cells
(Figure 6). This confirms the downstream cytotoxic
effect of T cell binding to the target cells.
58
7
A. A.
B. B.
C. C.
D. D.
Figure 7. CY5/PI Imaging of 2D Cytotoxic Figure 8. Brightfield/CY5/PI Imaging of 3D

Target Cell Induction. 4x overlaid CY5 and PI Cytotoxic Target Cell Induction. 4x overlaid
images showing stained T cells and signal from brightfield, CY5 and PI images showing stained
propidium iodide necrotic cell probe following T cells and signal from propidium iodide necrot-
(A) 0; (B) 48; (C) 96; and (D) 168 hour co-culture ic cell probe following (A) 0; (B) 48; (C) 96; and
incubation periods. (D) 168 hour co-culture incubation periods.
Quantification of Target Cell Cytotoxicity
Following image capture, the level of T cell induced target cell cytotoxicity was then quantified.
A. B.
Figure 9. Cellular Analysis of Target Cell Cytotoxicity. 4x images showing fluorescence from propidium iodide necrotic cell
probe following 96-hour incubation. Object masks (in blue) placed around (A) 2D and; (B) 3D cultured target cells meeting cel-
lular analysis criteria.
8 59
Using the optimized image analysis criteria described in Table 6, object masks were placed around cells
meeting minimum threshold signal criteria from the PI necrotic cell probe (Figure 9). As T cells have
a smaller size compared to the target cells in either 2D or 3D format, the minimum object size cutoff
value was set such that single necrotic T cells were not included in the analysis. This can be seen in Figures 9A and B.
A phenomenon also observed in the kinetic images of the 3D CMC assay is that the tumoroid began to disintegrate
in response to increasing cytotoxicity, releasing groups of cells into the surrounding media. While smaller
than the intact tumoroid body, these aggregates remain larger than individual T cells and also emit signal
from the PI necrotic cell probe, therefore are included in the final analysis (Figure 9B).
From the analysis performed, the number of necrotic cells per image was calculated for 2D cultured
target cells. When cultured in 3D, cells within the tumoroid and smaller aggregates exist on multiple z-planes.
Therefore, to quantify induced cytotoxicity with the greatest level of accuracy, the total PI signal within all object
masks per image was quantified. The values (cell count or total PI signal) calculated at each timepoint were
then automatically divided by the value calculated at time 0 in Gen5 software. In this way small variances
between replicates were normalized. Following analysis, the results were plotted to evaluate whether
differences were seen in induced target cell cytotoxicity between test conditions. The graphs
in Figure 10 show the calculated data for T cells added to test wells in a 20:1 ratio, activated in the
presence of 100%, 75%, 50% or 0% MDA-MB-231 cells, compared to unactivated T cells.
A. B.
Figure 10. Activation Protocol Cytotoxicity Induction Analysis. Comparison of cytotoxic target cell induction by T cells
activated in the presence of anti-CD3 and anti-CD28 antibodies, superkine, and 100% MDA-MB-231 cells, 75% MDA-
MB-231/25% fibroblast cells, 50% MDA-MB-231/50% fibroblast cells, or no cells. Data for unactivated T cells also included
and plotted using left y-axis. Necrotic cell count or total PI signal over time from untreated negative control target cells
plotted on right y-axis. Results shown for T cells incubated with (A) 2D cultured target cells; or (B) 3D cultured target cells
for seven days.
From Figure 10 it is evident that T cell induced cytotoxicity increases in terms of the degree of directed cell
activation in both 2D and 3D cell models. T cells activated in the presence of 100% MDA-MB-231 cells elicit the
highest level of cytotoxicity, while those activated only in the presence of antibodies and superkine elicit the
lowest increase in necrotic cell numbers per image over basal necrotic cell numbers. The models differ in their kinet-
ic responses, however. In the 2D model (Fig 10A), T cell-mediated cytotoxicity peaks at about 24 hours after addition
of the activated T cells, as witnessed by the ratio of necrotic cells from wells containing T cells to necrotic
cell numbers from negative control wells. Any further necrosis beyond about 3 days is due to the limitations of
the 2D model as noted by the increased necrosis over time evident in the negative control. Conversely, in the
3D model (Figure 10B), necrotic ratios of total signal from the PI probe continue to increase or plateau over the
course of the kinetic run due to the fact that cell health is much better retained in the untreated 3D cell model.
Analysis of necrotic cell induction was then performed on wells containing T cells directly activated in the
presence of 100% MDA-MB-231 cells and then added to 2D and 3D plated target cells for the CMC assay in ratios
of 20:1, 10:1, 5:1. A negative control was also included where target cells were untreated.
9 60
A. B.
Figure 11. Effect of T Cell Concentration. Comparison of cytotoxic target cell induction by T cells added to wells at
concentrations of 40,000 cells/well (20:1 ratio), 20,000 cells/well (10:1 ratio), 10,000 cells/well (5:1 ratio), and 0 cells/well
(negative control). Results shown for T cells incubated with (A) 2D; or (B) 3D cultured target cells for seven days.
It is evident from Figure 11 that kinetic responses of T cell-mediated cytotoxicity for different ratios of T cell
to target cell for both 2D and 3D models are obtained over time. These findings are consistent with the previ-
ous results from the activation protocol comparison (Fig 10), as well as results reported with in vivo testing6.
Finally, the effects of directed activation can also be measured using the brightfield channel when target-
cells are cultured in 3D. This is dueto the fact that in response to the cytotoxic T cell effect, tumoroids break
apart over time, or explode, releasing cells and ECM within the well. Using the confluence measurement ca-
pabilities of Gen5™ and the optimized metrics in Table 7, the extent of tumoroid disintegration can then be
quantified. Only pixels within each image with a brightfield signal below the upper threshold criteria are included
in the percent confluence calculation. When viewed in Gen5, outlier pixels are seen as white (Figure 12).
A. 3.6% Confluence B. 28.9% Confluence
C. 88.3% Confluence D. 99.9% Confluence
Figure 12. Image Confluence Determination using Brightfield Signal. 4x brightfield images following im-
age analysis and % confluence determination. Pixels not included in confluence calculation appear white.
Images shown after cell interaction and binding with a 10:1 T cell to target cell ratio for (A) 72; (B) 116; (C)
136; and (D) 168 hour incubation periods.
Percent confluence values can then be plotted over time to visualize the kinetics of tumoroid disintegration
in response to increasing T cell to target cell ratios.
10 61
References
1. Janeway, C.A. Jr.; Travers, P.; Walport, M.; Shlom-

chik, M.J. T Cell-Mediated Immunity. Immunobiology:
The Immune System in Health and Disease, 5th edition
[Online]; Garland Science: New York, 2001; https://www.
ncbi.nlm.nih.gov/books/NBK27101/ (accessed Oct 24,
2017).
2. Mahoney, K.M.; Rennert, P.D.; Freeman, G.J.; Combi-

nation cancer immunotherapy and new immunomodula-
tory targets. Nat Rev Drug Discov. 2015, 14, 561-584.
3. Perica, K.; Varela, J.C.; Oelke, M.; Schneck, J. Adop-

tive T cell immunotherapy for cancer. Rambam Mai-
monides Med J. 2015, 6(1), 1-9.
Figure 13. Kinetic Percent Image Confluence Quantification. Plot
of kinetic brightfield image percent confluence due to 3D tumoroid
disintegration. 4. Zaritskaya, L.; Shurin, M.R.; Sayers, T.J.; Malyguine,
A.M. New flow cytometric assays for monitoring cell-
mediated cytotoxicity. Expert Rev Vaccines. 2010, 9(6),
601-616.
The curves in Figure 13 illustrate how plotting
confluence over time explains the kinetics of the 5. Bethune, M.T.; Joglekar, A.V. Personalized T cell-
final effect. As would be expected, higher mediated cancer immunotherapy: progress and chal-
concentrations of activated T cells destroy the tu- lenges. Curr Opin Biotechnol. 217, 48, 142-152.
moroid faster than lower concentrations. Untreated
tumoroids also show little change in confluence 6. Zangle, T.A.; Burnes, D.; Mathis, C.; Witte, O.N.;
due to the fact that little to no cellular toxicity is Teitell, M.A. Quantifying biomass changes of single
seen (Figure 10B) allowing the tumoroid to remain CD8+ cells during antigen specific cytotoxicity. PLoS
intact during the seven day incubation period. One. [Online] 2013, http://journals.plos.org/plosone/
article?id=10.1371/journal.pone.0068916 (accessed Oct
24, 2017).
Conclusions
It was found that direct activation of T cells, where

they were exposed to target cells over extended
periods in vitro, produced a significant increase in
cytotoxicity compared to general activation
using no target cells. Furthermore, a diminishing
effect was evident if the target cells were co-cul-
tured with fibroblasts in the activation process: the
greater the ratio of fibroblasts, the less cytotoxicity
evident. This suggests that T cells can be inctructed
in the activation process to seek out and destroy
target cells.
The 3D cell model was far superior to the 2D cell

model as cell health was maintained throughout
the long kinetic runs. Cytotoxicity could be quantified
using propidium iodide that measure plasma mem-
brane rupture or with brightfield (label-free) that mea-
sured confluence increase by spheroid disaggregation.
The BioSpa™ System, comprised of an automated

CO2 incubator shuffling microplates to the cell imaging
reader, allows for walk-away automation of the 7-day
kinetic cytotoxicity assay.
11 62
AN121117_20, Rev. 12/11/17
Featured Products
Cytation 5 Cell Imaging Multimode Reader,
by BioTek Instruments, Inc.
“It is one of the best and most

useful instruments I’ve ever had.” “A great, versatile instrument.”
Rating: 5.0 Rating: 5.0

Laura McMullan, CDC
Puria Motamed, University of Tehran
Multi-Flo™ FX Multi-mode Dispenser

by BioTek Instruments, Inc.
“Great results – a
must-have for
screening and ELISAs.”
Rating: 5.0
Ryan Bennett, OyGen, Inc.
63

79 BioTek Ebook FINAL - Compressed

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

79 BioTek Ebook FINAL - Compressed

Uploaded by

Copyright:

Available Formats

Long-Term 3D Spheroid

Culture & Analysis

BROUGHT TO YOU BY INDEPENDENT SCIENCE PUBLISHER IN PARTNERSHIP WITH

free) and advanced microscopy analyses Monitoring Toxicity in Multiple-Dosing Studies

Automated Media Exchange for Spheroid Cultures Using a Novel

Spheroidal 3D cellular structures are a mainstay Experimental Components

Application Note 3D Cell Culture, Automation

Figure 2. (A.) AMX Media Exchange Module aspirate tip

Figure 1. AMX Media Exchange Module with

Application Note 3D Cell Culture, Automation

Figure 3. AMX Media Exchange

Qualitative Validation of AMX Media Exchange Module

Quantitative Validation of AMX Media Exchange Module

Spheroid Volume = (4/3) * π * (Object Size/2)3

Application Note 3D Cell Culture, Automation

Quantitative Validation of AMX Media Exchange Module

4 AN072718_09, Rev. 07/27/18 8

Brightfield and Fluorescence Imaging using 3D PrimeSurface®

Introduction Materials and Methods

Culturing cells in three-dimensions (3D) has Materials

Application Note 3D Cell Culture

within Gen5. Following auto focusing, multiple images

Application Note 3D Cell Culture

Spheroid Necrosis Induction Analysis Criteria A. E.

Primary Mask Criteria

Advanced Detection Options

Application Note 3D Cell Culture

Figure 4. Plot and linear regression of Gen5 96- and 384-well

Application Note 3D Cell Culture

Figure 5A. Imaging of camptothecin induced spheroidal

Figure 5B. Primary cellular analysis to determine necrotic

Application Note 3D Cell Culture

A. The images in Figure 7 of object mask

Figure 7. Necrotic cell analysis of treated

When the necrotic cell area was divided by the total

Figure 8. Plot of percent spheroid area covered by affected

3D Spheroid-Based Tumor Invasion Assay

Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA

Application Note 3D Cell Culture, Live Cell Imaging

Materials and Methods MultiFlo™ FX Multi-Mode Dispenser

Materials The MultiFlo FX is a modular, upgradable reagent

Application Note 3D Cell Culture, Live Cell Imaging

Imaging Parameters Tumoroid Invasion Analysis

Complete 3D Invading Primary mask cellular analysis criteria (Table 4) were

Imaging Stitching Parameters Rolling Ball Diameter 2000

Imaging Z-Projection Parameters Analysis Metric

Channel 1 Stitched[Brightfield] Object Area_2[ZProj[Stitched[

Application Note 3D Cell Culture, Live Cell Imaging

Results and Discussion Imaging of U-87/Fibroblast Individual Co-Cultured Cell

Figure 3. Brightfield and fluorescent overlaid

By viewing the fluorescent signal emitted by each co-

Figure 2. U-87/fibroblast 4x brightfield imaging showing (A-D) four

Application Note 3D Cell Culture, Live Cell Imaging

Figure 7. LN-229/fibroblast tumoroid invasive potential over time, 4x

Figure 5. U-87/fibroblast tumoroid invasive potential over time, 4x

Gen5 Image+ Based Cellular Analysis of Tumoroid

Figure 8. Invading tumoroid object masking. Zoomed 4x brightfield

Figure 6. Imaging of LN-229/fibroblast co-cultured cell model. (A)

Application Note 3D Cell Culture, Live Cell Imaging

Figure 9. Kinetic total tumoroid area ratios. Area ratios

From the results seen in Figure 9, it is apparent that

Application Note 3D Cell Culture, Live Cell Imaging

The area covered by invadopodia for U-87/fibroblast