University of Indonesia / Jakarta

February 27th 2019

Special Topics in Cell Biology and Cell Culture

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

 Special Topics in Cell Biology and Cell Culture

1. Basics methods of of cell culture

– Prerequirements / structure of a cell culture lab
– Basic techniques of cell culture

2. Cloning of cells
– Separation of cells (MACS, FACS)
– Limited dilution
– Isolation of Adherend Cell Clones

3. Special devices / methods in cell culture

– AVISO CellCelector
– CASY – Cell Analysis System
– MUSE MiniFACS
– Xcelligence – measurement of electric resistance of cells

4. Stem cells

5. CRISPR/Cas9 system for precise genetic engineering of cells

 Literature:

1. Zell- und Gewebekultur: Von den Grundlagen zur Laborbank

Toni Lindl und Gerhard Gstraunthaler
Spektrum Akademischer Verlag (2008)

2. Zell- und Gewebekultur: Allgemeine Grundlagen und spezielle Anwendungen

Gerhard Gstraunthaler und Toni Lindl
Spektrum Akademischer Verlag (2013)

3. Culture of Animal Cells: A Manual of Basic Technique

Robert I. Freshny
Wiley & Sons (2005)

4. Turns R.M and Turns M.P. (2014). CRISPR-Based Technologies: Prokaryotic

Defense Weapons Repurposed. Trends Genet. 30: 111-118

5. Sander J.D. and Joung J.K. (2014). CRISPR-Cas systems for genome editing,
regulation and targeting. Nat. Biotechnol. 32: 347-355

6. Stoltz J.F. et al. (2015). Stem Cells and Regenerative Medicine: Myth or Reality of
the 21th Century. Stem Cells Intern.

Many more review articles and websites

Basic methods of cell culture
 Reasons / Applications for / of cell culture

– Production / isolation of native substances

– Production of recombinant proteins
– Production of antibodies
– Establishment of screening systems
– Functional analysis at the cellular level (e.g. comparative ex-
pression analysis, promoter analysis)
– Infection assays
– Chromosomal localization experiments
– Generation of transgenic animals
– „Tissue engineering“ (tissue cultivation) for transplantation
medicine
– Alternatives for animal experiments (e.g. cytotoxicity tests)
Prerequirements / structure of a cell culture lab

Cleaning area
Hand washbasin, tables, benches
Washer
Drying oven
Autoclave

Preparation area
Working surface, boards, drying ovens
Ultrapure water system (pyrogen free)
Balances
Standard minor devices (pH-meter, agitators)
4°C- and -20°C-refrigerators, optionally in combination, optionally chill room
-80°C-refrigerator
Variable microliter pipettes
Standard glassware (duran flasks, graduates cylinders etc.)
Prerequirements / structure of a cell culture lab
Working area
Optionally ventilation
Clean bench (usually class II according to the German standard / DIN) with UV bulbs and
burner (operable via foot pedal)
Vacuum pump, electric pipetting aids
CO2-incubators
Inverse microscope with phase contrast facility
Neubauer-counting chamber, coverslips
Cooling centrifuge (at least 1000 x g)
Storage boards
Variable microliter pipettes
4°C- and -20°C-refrigerators, optionally -80°C-refrigerator
Liquid nitrogen container for cell storage
Lab water bath (temperature adjustable)
Optionally autoclave
Waste container
Aerosol cans with 70% ethanol (for disinfection)
Sterile culture vessels (T-flasks, Petri dishes, microtiter plates)
Sterile plugged pipettes (glass or disposable)
Sterile Pasteur pipettes
Sterile Falcon tubes, pipette tips etc.
Paper towels
Class II Clean Bench
Foot-Switchable Bunsen Burner
T-Flask
CO2 Incubator
Inverse Microscope
Cryotubes and Liquid Nitrogen Containers
 Sterilization techniques
Sterilization

– Autoclave 15 min, 121°C, 2 bar

Flasks and filter devices, thermostable solutions

– Hot air

After drying at least 30 min, 180°C

Pipettes, glass wool, metal cases

– Sterile filtration

Thermolabile solutions, media

Pore size 0.1 µM
Small filters for 1-50 ml
Larger devices for 50-500 ml, membrane pump
In the case of foaming liquids pressure filtration recommended
Autoclave
Autoclave
Autoclave
 Sterilization techniques
Sterilization

– Autoclave 15 min, 121°C, 2 bar

Flasks and filter devices, thermostable solutions

– Hot air

After drying at least 30 min, 180°C

Pipettes, glass wool, metal cases

– Sterile filtration

Thermolabile solutions, media

Pore size 0.1 µM
Small filters for 1-50 ml
Larger devices for 50-500 ml, membrane pump
In the case of foaming liquids pressure filtration recommended
Hot Air Sterilizer
 Sterilization techniques
Sterilization

– Autoclave 15 min, 121°C, 2 bar

Flasks and filter devices, thermostable solutions

– Hot air

After drying at least 30 min, 180°C

Pipettes, glass wool, metal cases

– Sterile filtration

Thermolabile solutions, media

Pore size 0.1 µM
Small filters for 1-50 ml
Larger devices for 50-500 ml, membrane pump
In the case of foaming liquids pressure filtration recommended
Sterile filters
 Some important terms

– Adherent cells: cells which adhere to the bottom of the culture

vessels generating a monolayer

– Monolayer: layer of adherent cells with a height of one cell

– Suspension culture: culture of non-adherent cells in suspension

– Proliferation: growth (reproduction) of cells

– Confluence: state of a densely grown monolayer (no space between

cells left)

– „Shake off“: „technique“ of knocking against the buttom of the culture

vessel to loosen the cells when passaging them

– Generation time: time range between two mitotic divisions of cells

Media frequently used in cell culture
DMEM - F12 medium

Usually complemented with

10% FCS and antibiotics
RPMI - 1640 medium

Usually complemented with

10% FCS and antibiotics
FCS (fetal calf serum) amongst others
contains growth factors important for
proliferation of the cells
Acid-base-equilibrium

NaHCO3  Na+ + HCO3-

HCO3-  CO2 + OH-

If CO2 concentration increases (in CO2 - incubator)

CO2 + H2O  H2CO3  H+ + HCO3-

If H+ - concentration increases:

HCO3- + H+  [H2CO3]  CO2 + H2O

If OH- - concentration increases):

OH- + H2CO3  HCO3- + H2O

pH values in media approximately pH 7.1 - 7.4

 Primary cells

– Taken directly from living tissue (e.g. biopsy material)

– Dissociation from tissue by proteolytic enzymes (e.g. trypsin, pronase,

collagenase) and EDTA (loosening of cadherin-mediated cell-cell
connections)

– Elaborate and time-consuming, results heterogenous

– Cells are in parts still able to differentiate, limited life span

 Cell lines

– Establishment by spontanous transformation / mutation or directed

mutation, e.g. with viral genes

– More than 4000 cell lines from more than 150 animal species
(including human) available (collections: ATCC, DSMZ, ECACC)

– Easier to handle

– High mitotic activities, unchanged properties for a high number of

generations

– Defects in signal transduction pathways involved in regulation of cell

cycle and differentiation => thus, not suitable for investigation of
corresponding problems
Frequently used cell lines (selection)
 Thawing of cells and taking them into culture

– Carefully open N2-container, using gloves take the cryotubes out of the
buck and instantly transfer to 37°C water bath.

– Keep the cells just as long in the water bath as the last ice clot needs to
be melted.

– Rapidly go to the clean bench, briefly disinfect tube with 70% ethanol,
using a 2 ml-pipette transfer the cells into a sterile centrifuge tube filled
with at least 20 ml of fresh medium. Centrifuge for 10 min at 300 x g
and aspirate medium.

– Fill up with medium (10-30 ml).

– Leave the cells alone for 24 h.

Adhesion and proliferation of adherent cells
Mouse fibroblast cell line
Mouse fibroblast cell line
Mouse fibroblast cell line
Human bladder
carcinoma cell line
 Changing medium of monolayer culture
(usually in intervals of 2-3 days)

– Microscopically check sterility of the cultures.

– Prewarm medium (water bath, 37°C).

– Disinfect the outer faces of all vessels.

– Open culture vessel, lay down cap with its hole downwards.

– Using a Pasteur pipette (connnected to vacuum pump) carefully

aspirate medium, do not touch the monolayer!

– Pour fresh medium into the culture vessel, take care not to flush away
cells! Use side opposite to the monolayer! Medium may not touch the
neck of the vessel!

– Close the vessel.

 Trypan blue staining (required for counting of cells)

– Trypan blue only enters damaged or dead cells - not able to enter living
cells.

– Treat cell (if adherent) with trypsin, resuspend in medium or PBS pH 7.4.

– Adjust concentration of cells to approximately 105-106 cells per ml.

– Dilute 0.1 ml cell suspension with 3.6 ml PBS and add 2.7 ml prewarmed
0.5% trypan blue solution.

– Mix gently using a pipette, incubate 2-5 minutes at 37°C, again mix and
immediately count the cells by means of a Neubauer counting chamber
(even slightly stained cells are considered to be dead).

– % living cells = 100 x unstained cells / (unstained cells + stained cells).

PBS (phosphate-buffered saline)
1 X PBS

8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4

in 800 ml of distilled H2O.

Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Sterilize by autoclave.

10 X PBS

– Dissolve the following in 800ml distilled H2O:

80g of NaCl
2.0g of KCl
14.4g of Na2HPO4
2.4g of KH2PO4
– Adjust pH to 7.4.
– Adjust volume to 1L with additional distilled H2O.
– Sterilize by autoclaving.
Neubauer counting chamber
 Passaging of cells of a monolayer culture
(usually when monolayer has reached 80-90% of confluence,
dilution 1:3 - 1:10)

– Aspirate medium by means of a Pasteur pipette (connected to vacuum

pump).
– Rinse 2 x with PBS (removal of medium / FCS).
– Add trypsin / EDTA solution to the cells, incubate for approximately 7
min in CO2 incubator => loosening of cells (optionally "shake off").
– Addition of medium (FCS inhibits trypsin).
– Sediment cells by centrifugation in 50 ml Falcon tubes (7 min, 700
rpm, RT).
– Aspirate medium.
– Resuspend cell pellet in 10 ml of medium, optionally cell counting.
– Transfer desired number of cells in new culture vessel.
 Freezing of cells

– Chill freezing medium, culture medium and cryotubes.

– Treat cell (if adherent) with trypsin.

– Adjust number of cells to approximately 1x107 cells per ml. Fully

resuspend the cells - avoid clumps.

– Mix cells with equal volume of double concentrated cold freezing

medium. Fill each cryotube with 1.6 ml of the suspension.

– Put cryotubes in 4°C refrigerator for 15 min, then in -20°C

refrigerator for 2 h, then overnight in gas phase of the N2 container.

– At the next day transfer cryotubes into liquid nitrogen.

– Freezing medium (100 ml, double concentrated) is prepared by mixing

of 50 ml culture medium (with FCS), 30 ml FCS, 20 ml DMSO.
 Sending of cells

– Cryotubes on dry ice

– T25 or T75 flasks with living cells:

- Fill up T-flasks (cell density 60-80% confluence) up to the

border with culture medium and tightly close the cap (without
aeration). Additionally seal with parafilm.

- Pad the flask(s).

- Upon reception remove 7-20 ml using a pipette, check cells,

passage.
 Transfection of cells

– Cells may be genetically engineered by transfecting them with DNA (usually

recombinant vector).

– Applications include the recombinant production of a protein and the

establishment of systems for screening for drug candidates.

– Frequently used methods for transfection are lipofection (use of cationic

lipids) and electroporation (application of high voltage in a special cuvette).
Some examples for cationic lipids used for transfection
Electroporation Device
Recombinant production of peptides / proteins in mammalian cells

– Well charaterized: CHO cells (Chinese hamster ovary)

– Correct glycosylation / modifications
– Transient or stable expression (here, multiple copies may be
integrated into genome)

Transfection

Expression of the green fluorescent protein

(GFP) from jellyfish in CHO cells
 Special Topics in Cell Biology and Cell Culture

1. Basics methods of of cell culture

– Prerequirements / structure of a cell culture lab
– Basic techniques of cell culture

2. Cloning of cells
– Separation of cells (MACS, FACS)
– Limited dilution
– Isolation of Adherend Cell Clones

3. Special devices / methods in cell culture

– AVISO CellCelector
– CASY – Cell Analysis System
– MUSE MiniFACS
– Xcelligence – measurement of electric resistance of cells

4. Stem cells

5. CRISPR/Cas9 system for precise genetic engineering of cells

Cloning of Cells
Definitions

A Clone is a group of cells derived from a single

ancestral cell.

Cell cloning: The process of producing a group of

cells that are genetically identical (clones) to a
single ancestral cell.

medicinet.com
Cloning of primary cells usually requires
separation prior to cloning
Separation of Cells
 Primary Cells

– Direct from organism

– Complete dissociation
1. Mechanical dissociation
2. Enzymatic dissociation
– With exception of monocytes blood cells only grow in suspension
culture

Enzymatic dissociation (method of choice):

Trypsin, collagenase, dispase, pronase, elastase, hyaluronidase,
DNAse and combinations possible
Trypsin and pronase most effective but highest degree of damage
Dispase and collagenase result in less yield but also less damage
DNA is released from necrotic cells – leads to cell aggregation
Different types of cells exhibit different patterns of
cell surface proteins („CD“ – Cluster of Differen-
tiation)

By means of antibodies directed against certain CD

proteins different cell types may be determined and
separated
IgG Antibody

-S-S-: disulfide bond

CDRs: hypervariable regions
blue: heavy chain
green: light chain
red: hinge region
 MACS – Magnetism-Activated-Cell-Sorting

– Antibodies specific for certain CD proteins (depends on desired cell

type) are linked to paramagnetic beads

– After incubation with antibody-labeled beads, heterologous cell

suspension is applied onto MACS column

– Desired cells bound to the beads are retained (positive selection) –

undesired cells are in the flow through

– Subsequently, column is removed from the magnet and desired

cells are eluted

– Also negative selection possible – here, antibodies are directed

against undesired cells
MACS – Magnetism-Activated-Cell-Sorting
 Fluorescence-Activated Cell Sorter (FACS)

– Generation of (electrically charged) drops containing single cells

– Drops move through capillary with laser and detectors at certain positions

– Depending on the specific cell passing the detector, FACS is capable of

detecting the intensities of the forward light scatter (proportional to the size of the
cell) and the side light scatter (proportional to the granularity of the cell)

– Moreover, it is capable of detecting fluorescence of specific wavelengths (e.g.

when fluorescent-labeled antibodies bind to specific cell surface molecules of the
cell)

– Allows a relative precise characterization of cell type

– Upon detection, charged drops with desired cells may at the end of the capillary
be directed into a certain well (tube) by generation of an electromagnetic field =>
separation (and cloning) of cells
FACS Device
Principle of FACS Analysis
Principle of FACS Analysis
FACS Scatter Dot Plot

Green:
Lymphocytes
(small, low granularity)

Blue:
Monocytes
(large, low granularity)

Pink:
Neutrophil Granulocytes
(large, high granularity)
 Gating

Cell populations identified in scatter analyis may be further analyzed by „gating“

according to their specific pattern of cell surface proteins (CDs). Here, prior to FACS
analysis cells have been incubated with CD-specific fluorescent-labeled antibodies.
R1 defines lymphocytes. Antibodies are specific for T-lymphocytes (CD3-FITC) and
B-lymphocytes (CD19-PE). The larger portion is represented by T-lymphocytes.
 FACS Analysis (Including Gating) of a Patient Suffering
from a Certain Kind of Leukemia

It is striking, that lymphocytes of the patient compared to a healthy

individual are almost completely represented by B-lymphocytes.
MUSE MiniFACS
Cloning of Cells
96-Well Microtiter Plate
Limited Dilution Cloning (http://www.sciencegateway.org)

The aim is to obtain clones derived from a single cell.

Protocol I

1. Prepare a suspension of cells in 10 ml medium that contains 1000

viable cells.

2. Prepare another suspension of cells in 10 ml that contains 100

viable cells.

3. Dispense 0.1 ml of the higher concentration (10 cell/0.1 ml) into 48

wells of the 96-well plate. Dispense 0.1 ml of the lower
concentration (1 cells/0.1 ml) into the remaining 48 wells.

4. Incubate the plate for 4-7 days without changing the medium.
Observe for growth of isolated clones.
Limited Dilution Cloning (http://www.sciencegateway.org)

The aim is to obtain clones derived from a single cell.

Protocol II

1. Prepare 8 tubes. Dilute cells in 2.5 ml of medium at the

concentrations of 20, 10, 5, 2.5, 1.25, 0.6, 0.3, and 0.15 cells/ml.

2. Use two 96-well plates. Dispense 0.1 ml of each dilution into 3

columns (24 wells).

3. Incubate the plate for 4-7 days without changing the medium.
Observe for growth of isolated clones.
24-Well Plate
 Isolation of Adherend Cell Clones

– Disseminate 60-120 cells into a 10 cm Petri dish with 10 ml culture medium.

– By use of a hole puncher generate 0.5 cm filter paper disks, moisten it with
destilled water and autoclave it in a glass Petri dish.
– After 10-14 days, when cell colonies are macroscopically well visible, mark
them with a pen at the bottom.
– Aspirate medium and wash with 2 x PBS.
– Pipette trypsin solution into Petri dish with filter paper disks.
– Using sterile forceps put filter paper disk onto marked colony and incubate 5-
10 min. at 37° C.
– Add 1 ml culture medium in each well of a 24-well plate.
– Using sterile forceps put one filter paper disk from the culture dish in each
well (cell layer down).
– After two days the growing up of the cells under the filter paper may be visible.
– When cells in the 24-well plate are subconfluent, they may be transferred into a
larger culture vessel.
Other Devices Used in Cell Culture
AVISO CellCelector

Microscopy- and computer-aided isolation of

cells / cell clones
 CASY – Cell Analysis System

– Electrical resistance of cells is measured by means of a measuring

capillary and is digitalized

– Suitable for counting of cells

– Electrical resistance of dead cells is lower compared to living cells

(discrimination)

– => Also suitable for in vitro toxicity tests

CASY
MUSE MiniFACS
 MUSE
SmartFlare
– Gene expression analysis at the single cell level according to
the „SmartFlare“ principle

– Fluorescent-labeled oligonucleotides compete with target-

RNA for probes linked to gold beads

– Upon displacement of the labeled oligonucleotides by specific

mRNA loss of quenching by gold => fluorescence =>
proportional to concentration of target-mRNA => quantification
SmartFlare - Prinzip
 Xcelligence

– Based on measurements of the electric resistance of cells on

micro-electrode-equipped culture plates

– Depending on number, physiological states, adherence,

morphology, migration etc. of the cells electric resistance changes

– „Cell index“ (value without unit) => high electrical resistance = high
cell index

– Cell index is correlated with certain state of the cell (changes when
„something happens“)

– May be used for screening approaches

Example Migration Assay
Thank you very much for your attention !