Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Long-term, live cell imaging of host-pathogen interactions using the CellASIC™ ONIX System

Long-term, live cell imaging of host-pathogen interactions using the CellASIC™ ONIX System

Ratings: (0)|Views: 7|Likes:
The CellASIC™ ONIX Microfluidic System is well suited for host-pathogen studies by providing a stable, long term culture environment for host cells (including primary cells) with controlled pathogen exposure. The continuous perfusion format and the ability to switch media solutions enable wash-out of pathogens from the chamber and subsequent monitoring of host cell response over many days. The enclosed small volume of the culture chamber also provides practical advantages for working with infectious agents during live cell imaging. The design of the microfluidic plates enables the dynamics of infection to be tracked using live cell imaging on standard inverted microscopes.
The CellASIC™ ONIX Microfluidic System is well suited for host-pathogen studies by providing a stable, long term culture environment for host cells (including primary cells) with controlled pathogen exposure. The continuous perfusion format and the ability to switch media solutions enable wash-out of pathogens from the chamber and subsequent monitoring of host cell response over many days. The enclosed small volume of the culture chamber also provides practical advantages for working with infectious agents during live cell imaging. The design of the microfluidic plates enables the dynamics of infection to be tracked using live cell imaging on standard inverted microscopes.

More info:

Categories:Types, Research
Published by: EMD Millipore Bioscience on Jan 19, 2013
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

05/14/2014

pdf

text

original

 
Data Sheet
Introduction
Host-pathogen interactions represent a signicant areao biomedical research, encompassing the study o howviruses, bacteria, and other organisms cause inectionsand diseases or alter normal physiology o host cellsand tissues
1
. As the eld o cellular systems biologyadvances, there is increasing interest in using
in vitro 
cellculture models to study host-pathogen interactions
2
.The proper study o host-pathogen interactions requirescareul control o the cell environment to simulatephysiologic conditions. An
in vitro 
model that canreplicate inection parameters, including fow rate,exposure time, solution type, and timed introductiono therapeutic agents while sustaining long-term livecell microscopy enables experiments that are dicultor impossible with standard methods. In many cases,it is crucial to precisely manipulate the exposureo pathogen to the cultured host cells: too low anexposure may prevent appropriate interactions, whiletoo high an exposure could cause conounding eects(such as bacterial overgrowth and nutrient starvation)independent o the desired host-pathogen interaction.In addition, inormation on the eect o exposuretimes and fow conditions could prove valuable orunderstanding the mechanisms o disease or manyinectious agents. A urther challenge in host-pathogenresearch is the ability to sustain inections
in vitro 
, sincecultured host cells oten de-dierentiate in culture andno longer support pathogen responses ound
in vivo 
. Forcertain pathogens, the time scale o inection even underoptimal conditions could take days or weeks, placingadditional burdens on the cell culture method. Recentadvances in microfuidics-based cell culture technologiesoer potential solutions to these problems
3
.The CellASIC™ ONIX Microfuidic System is well suitedor host-pathogen studies by providing a stable, long-term culture environment or host cells (includingprimary cells) with controlled pathogen exposure. Thecontinuous perusion ormat and the ability to switchmedia solutions enable wash-out o pathogens romthe chamber and subsequent monitoring o host cellresponse over many days. The enclosed small volume o the culture chamber also provides practical advantagesor working with inectious agents during live cellimaging. The design o the microfuidic plates enablesthe dynamics o inection to be tracked using live cellimaging on standard inverted microscopes.Here, we demonstrate a host-pathogen experimentusing human intestinal cells inected with engineered
E. coli 
strains (Figure 1). Both an invasive and non-invasive bacterial strain were monitored or long-terminection with time-lapsed imaging up to 24 hours inthe CellASIC™ M04S microfuidic plate.
Application Note
Long-term, live cell imaging o host-pathogen interactions usingthe CellASIC
ONIX System
AB
Figure 1.
Human colonadenocarcinoma cells(HT-29) cultured inthe M04S microuidicchamber and inectedwith an invasive strain o 
E. coli.
(A) Phase contrastimage showing apoptoticcells, and (B) uorescenceimage showing invadedbacteria (red) and cellviability (green). Imageswere acquired with a100X objective lens.
EMD Millipore is a division o Merck KGaA, Darmstadt, Germany
 
2
Plate design
The M04S microfuidic plate is built on the CellASIC™ ONIXMicrofuidic Platorm. The plate has a standard 96-wellootprint to t typical microscope stage holders. The customwell layout was designed to maximize live cell imagingcapabilities. The M04S plate has our independent units(A-D), with each unit containing eight wells (one gravity inlet,our switching inlets, one cell inlet, two waste outlets). Theour cell culture chambers are centralized under a single largeimaging window (Figure 2). The chamber-to-chamberdistance is 5.2 mm, reducing objective travel time and ocusdrit. The bottom surace o the plate is a #1.5 thickness (170µm) optical glass slide to maximize quality o high resolution,high numerical aperture imaging. The plate houses allexperiment solutions allowing control with an externalpneumatic maniold (Figure 3). The maniold lets the userdirect fow rates and select exposure solutions withoutperturbing the microscope stage. The programmablesotware interace automates fow switching times. A gasline allows control o the environment within themicrochambers through a network o gas-permeable airdiusion channels. Temperature is regulated through anon-board heater/chiller on the maniold.
Bacterial inection model
To demonstrate time-lapsed imaging o host-pathogeninteractions in the microfuidic culture system, a humancolon adenocarcinoma cell line (HT-29, ATCC®) was exposedto two
E. coli 
strains (courtesy o Tim Lu, MIT). The strainstested included one capable o HT-29 inection and one thatcould not. A schematic o how the perusion system wasused to monitor invasion is depicted in Figure 4.
Figure 3.
Side view schematic o the microuidic plate with micro-incubation maniold on a microscope stage. The bottomsurace o the microuidic plate is a thin glass sheet, allowinghigh quality cell imaging. The plate is sealed to a pneumaticmaniold, allowing user control o the ow profle duringimaging. Additional air channels allow control o the gasenvironment.
Figure 2.
The M04S plate contains our independent ow units (A-D),each with our upstream solution inlets, a gravity ow inlet,a cell inlet, and two waste wells. The culture chamber is 2.8mm in diameter (120 µm height) and is surrounded with amicroabricated perusion barrier (4 µm pores). Inlet 1 is agravity ow well, allowing long term cell culture in a standardincubator without a pressure system. Continuous ow o solutions rom the inlets creates a dynamic exposure profleduring live cell imaging.
Figure 4.
Schematic o host-pathogenexperiment. (A) Human cellsare cultured in the M04Smicrouidic plate to establisha healthy, dierentiated,and stable host populationunder continuous mediumperusion. (B) Pathogens(bacteria) are introducedby ow to expose the hostcells. (C) Medium perusionis re-established, allowinglong-term monitoring o host-pathogen interactionvia live cell microscopy.
ABC
ADCB17654328
5.2 mm
Imaging WindowFlow Inlets (2,3)Cell Inlet (6)Cell Culture Chamber(2.8 mm diameter)Waste(7-8)PerfusionBarriersUnit MarkerAir ChannelsGravity Inlet (1)Flow Inlets (4,5)Gravity Inlet (1)
Gas-permeablemembraneCellsRecirculatinggas mixtureManifoldAir pressure-driven flowConvective Peltierheat exchanger#1.5 thickness(170 µm) glassGlass windowMicrofluidicplate
 
3
The experiment was prepared by culturing HT-29 cells in theM04S microfuidic plate in a standard cell culture incubatoror three days with gravity perusion o McCoy’s 5A medium+ 10% etal bovine serum (FBS). This allowed the cells toorm close cell-cell contacts and enter growth phase. On theday o the experiment,
E. coli 
cells were cultured in LuriaBroth (LB) on a shaker to achieve log phase growth. TheM04S plate was then loaded with culture medium (McCoy’s5A + 10% FBS) in well 2 and bacterial suspension in well 3.Within each plate, the invasive strain, non-invasive strain,and no bacteria control were run in parallel chambers. Forlive cell imaging, the M04S plate was sealed to the CellASIC™ONIX Microincubator Controller maniold (or perusion,temperature, and gas control) and imaged using an OlympusIX71 microscope. Conditions were set at 37 °C and 5% CO
2
 or the duration o the experiment. The cells were initiallyperused with culture medium or 1 hour at a fow rate o 5 µL/hr to stabilize imaging, and then exposed to the bacteriasolution or 30 minutes, ollowed by wash-out by mediumfow at 5 µL/hr or the remainder o the experiment. Figure 5shows images comparing the invasive strain (a and b) againstthe non-invasive strain (c and d). During bacterial exposure,there was a high abundance o both strains visible in thesolution (t = 0). The subsequent wash-out removed themajority o the bacteria, allowing the HT-29 cells to continue
Figure 5.
Time-lapsed imaging o bacterial invasion into human HT-29 cells. A) Phase contrast and (B) uorescence images o an invasivestrain o 
E. coli 
. (C) Phase contrast and (D) uorescence images o a non-invasive
E. coli 
strain. Both strains are designed toexpress mCherry ollowing invasion into HT-29 cells
.
A
-1 hr0 hr1 hr6 hr12 hr
CDB
healthy culture. In the non-invasive condition, the HT-29 cellswere able to continue growth and exhibit a healthymorphology. In the invasive strain case, the HT-29 cellsshowed clear signs o stress by 6 hours, with widespread celldeath occurring by 12 hours. The fuorescence channelsshowed mCherry expression by the bacterial strains triggeredby invasion, veriying the cell death as a result o invasioninstead o nutrient competition.Figure 6 shows higher magnication images o the HT-29cells exposed to the invasive and non-invasive strains. Here,the cells were stained with the green fuorescent Calcein AMviability marker, and the bacteria were red fuorescent. In theinvasive case, there was clear co-localization o the bacteriawith the human cells, whereas in the non-invasive case, thebacteria were eectively excluded rom the HT-29 colony.When tracked over time, the invasive bacteria triggeredapoptosis and eventually cell rupture, promoting bacterialgrowth and continued invasion. By 15 hours ater initialexposure, almost all o the HT-29 cells in the invasivechamber had died, while the majority o the HT-29 cells inthe non-invasive chamber remained viable.The continuous perusion culture ormat was highlybenecial in promoting long-term host-pathogen response.

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->