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The Complete Guide To

CELL CULTURE

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WELCOME
What’s Inside

6 - 10Introduction to cell culture
11

Cell culture workflow
13

Considerations before starting cell culture
14 - 15

Cell types & culture characteristics
16 - 17

Cell contamination

18 - 23

Cell culture protocol
24 - 25

Cell culture troubleshooting

4Western
Cell Blotting
CultureIntroduction to

CELL CULTURE
What is cell culture?

Cell culture is the growth of cells from an animal or plant inan artificial, controlled environment. Cells are removed either
from the organism directly and disaggregated before cultivation
or from a cell line or cell strain that has previously been
established.

Cell culture applications

Cell culture:
Is a major consistent and reproducible tool in molecular andcellular biology (Figure 1).
Helps to study normal cell homeostasis, cell biochemistry,metabolism, mutagenesis, diseases, and compound effects.
Is a model system for diseases and drug screening.

Figure 1. Cell culture laboratory applications: western blot, ICC/IF, ELISA, IP, IHC, and FACS analysis

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Basic cell culture
equipment

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The specific equipment of a cell culture laboratory depend on the type of
research conducted, however, all cell culture laboratories have the samecommon equipment being free from pathogenic microorganisms (Figure
2).

1. Wrapped disponsable pipettes
2. Waste container
3. Glass Pipette (if using)
4. Cell culture ﬂasks

4.

5.

6.

7.

1.

5. Wste Liquid
6. Reagants and media

3.
8.

7. Pipettor
8. Work surface

2.

Figure 2. Basic cell culture equipment.

The extended list below is corresponding to the equipment and supplies
for the majority cell culture laboratories, that allows the work to be more
efficient and accurate.
- 70% ethanol antiseptic- Aspiratory pump
- Autoclave- Cell counter- Cell culture flasks- Cell culture-grade Petri dishes- Cell culture-grade tubes of various sizes- Cell culture hood
- Cell culture microwell plates- Cell incubator- Centrifuge- Fridge/freezer- Gloves- Media, sera, cell media additives- Pipettes of various capacities- Serological pipettor- Sterile filters- Waste container- Water bath
*Please note: The specific cell culture equipment depends on the cell typeand aim of the study

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Western
CellBlotting
CultureCell culture safety

Work in a cell culture laboratory is associated with different
risk factors and hazards such as toxins or mutagenic reagents.
The human/animal material may contain viruses and otherdangerous biological agents. Therefore, while manipulating
human/animal material it is important to follow general safety
guidance for laboratory practices.
1.

Wash hands when entering and before leaving the laboratory.

2.

Wear safety clothes (gloves, closed shoes, lab coat).

3.

No eating, drinking, smoking.

4.

No or low aerosol creation.

5.

Decontamination of all surfaces before and after theexperiment.

6.

Work in accordance with the facility guidelines.

7.

Dispose of all waste in an appropriate way.

8.

Restricted access to authorized personnel only.

9.

Avoid using sharp objects.

10. Always clearly label all samples.
11. Report all incidents to the safety officer.

Aseptic techniques
required whileworking with cell
cultureTo be successful in cell culture, it is essential to remain a contamination
free environment (bacteria, fungi etc) Aspetic techniques ensure that no
microorganisms enter the cell culture. Cell culture steriity is ensured by
set of procedures.

Handling

Reagents/Media

Workplace

Slow/careful handling.

Pre-sterilation of all reagents/
equipment.

Cell culture hood works properly

No contamination in reagents(expiration date, appearance
normal).

Frequent de-contamination(hood, fridge etc)

Sterilization of all items before
starting.
Sterile pipettes
No touching of sterile items to
non-sterilized surfaces

Table 1. Aseptic techniques required while working with cell culture.

Work area: sterile and tidy

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Cell culture environment

Cell culture is an amazing tool that allows for easy controlling andmanipulation of all physiochemical and physiological cell factors, suchas, temperature, osmotic pressure, pH, gas, hormones, and nutrients.

Media

pH

Temperature

C02

Contains nutrients, growth
factors, and hormones.

Average pH for mammatiancells is pH 7.4.

Depends on bodytemperature of host.

Controlled by media.

Sera source of growth,
tipids,hormones.

Organic or C02
bicarbonate buﬀer
systems are popular.

Mammalian cell lines
036-37 C.
0

Insert cell lines 27-30 C.

Can impact pH.
4-10% C02 is mostcommon.

Table 1. Cell culture environment.

Media supplements forcell culture

Media supplements help to optimize cell growth for specific applications
depending on the chosen tissue or cell type. The advantages of using
media supplements such as growth factors or cytokines are that they
may improve cell viability and growth and keep cells healthier for longer(Table 3). For example, fibroblast growth factor (FGF) plays important roles
in diverse biological functions in vivo and in vitro and can maintain cell
culture over the weekend as Proteintech thermostable FGF (HZ-1285) does
not require media changes every day (Figure 3).

Figure 3. The stability of FGFbasic-TS and FGF basic (E. coli-derived) in xeno-free, chemicallydefined cell culture media at 37˚C. The protein concentration was determined by ELISA
each day for 3 days. After one day of incubation at 37˚C, FGF basic was undetectable, while
FGFbasic-TS was present at levels of 60%, 35%, and 20% of its starting concentration atdays 1, 2, and 3.

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Western
CellBlotting
CultureGrowth factors play an essential role to maintain the in vitro culture
growth. Depending on the environment, certain cells can give rise toa variety of lineage-specific cell types. Table 3 contains a summary of
the key growth factors needed for normal cell growth, metabolism, cell
development in culture and their differentiation process.

Supplement

Cat. No.

Table 3. Selected media supplements and their role in cell culture.

Involved in/used for:

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Cell culture

WORKFLOW
Cell culture workflow
in 4 simple steps

ISOLATE• Separate fresh tissue of interest.
• Treat tissue with enzyme(s) (e.g., trypsin, collagenase,protease) and/or mechanically to isolate cells.
• Wash, count, and seed cells.

VERIFY• Examine cells under a brightfield microscope to assess their
growth state, attachment to culture vessels/flasks, and tocheck for any signs of infection.
• Monitor cells for the following days until they reach 		confluence.
• Verify isolated cell types by their morphology and expressed 		
biomarkers.

CULTURE• Passage cells to propagate the cell line.
• Make master and working cell banks.
• Immortalize cells if necessary.

INVESTIGATE• Plan and execute experiments.
• Keep monitoring cell state and possible infections using abrightfield microscope.

		Cell Blotting
CultureWestern

10Considerations before starting

CELL CULTURE

36–37°C for mammalian cells.
Temperature variability >0.5°C in the incubator.
4–8% if the medium uses CO2-bicarbonate
buﬀers to maintain optimal pH.
Check cell culture on a regular basis – visually andunder a brightﬁeld microscope.
A sudden change in medium color (when pH
indicators are used) and/or clarity or a foul smell
may indicate infection.
A brightﬁeld microscope with 20–60x magniﬁcation
will allow you to spot developing bacterial/fungal
infections.
In case of infection decontaminate and discard cells
and clean the working area. The removal of infection
with antimicrobial agents should be used as a lastresort if no cell banks of the cell line exist.
Check cell culture on a regular basis – visually andunder a brightﬁeld microscope.
Sudden detachment/rounding up of adherent cells,
excessive clumping of suspension cells, or thepresence of cell debris is indicative of apoptosis.
Plate cells at 10–30% density during standard
passaging. Plating cells too sparsely may impede
their growth.
Cells should be passaged at 80–90% density, toward
the end of the logarithmic growth phase.
Do not allow your cells to overgrow – this will slow
down their recovery after passaging.
Table 4. Considerations before starting cell culture.

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Cell types & culture

CHARACTERISTICS
Working with theprimary cell culture

Primary culture cells are isolated from the tissue and proliferated
under the appropriate conditions until they occupy all of the available
substrate (i.e., reach confluence). To allow growth of the culture tocontinue past this point, cells need to be subcultured by transferring
them to a new culture dish with fresh growth medium. Primary cell
culture: cell lines directly expanded from tissues. Unless they undergo
an immortalization procedure, primary cells have a limited lifespanand usually reach senescence after 10–20 passages.

Working with the cellline culture

After the first subculture, the primary cells start to become a cell lineor subclone. Cell lines derived from primary cultures have a limited
lifespan as a consequence of being outside of their tissue niche. Whilecell culturing, those cells with the highest growth capacity predominate,resulting in a degree of genotypic and phenotypic uniformity in the
population over time.
Continuous cell culture: Cells are immortalized and can be grown formany passages with no significant loss of viability. Many are well-known
cell lines with very defined characteristics (summary Figure 4).

Cell line
Transformation

2nd subcultre

1st subcultre

1016

Seeding

Total cell yield (theoretical)

Continuous cell line
Primary
CultureSenescence& DeathSubcultural interval

Serial passage

106
0

4

6

8

10

12

Figure 4. Primary cells culture vs. cell line culture.

14

20 Weeks

12Working with adherent
cells

Western
Cell Blotting
CultureAdherent cells: types of cell lines that grow in the monolayer attached tothe surface. While passaged (see standard passaging), a detaching agent(e.g., trypsin) needs to be used to detach them from the surface. They
re-attach to the surface within a few hours upon plating. Based on theirmorphology, adherent cells can be divided into two main categories:•

Fibroblast-like: elongated shape, usually migrate on the dish•Epithelial-like: polygonal shape, stationary, grow in patches

Suspension cells (lymphoblast-like): type of cell lines that grow insuspension and do not form monolayers on the surface. Cells formclumps, especially at high density (summary Figure 5).

Working with cells insuspension

Figure 5. Mammalian cell lines differ in shape. Left: adherent fibroblast-like cells with
an elongated shape; Middle: adherent epithelial-like with a polygonal shape; Right:
suspension lymphoblast-like cells with a rounded shape.

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Cell

CONTAMINATION
Contamination of cell cultures is a serious risk to your samples.
Consequently, safety measures, including aseptic techniques, should be
put in place to actively prevent it and your samples should be regularly
checked for any sign of infections. Cell contamination may severely affecttheir metabolism, growth, and viability, as well as decrease transfection
efficiency and threaten the reproducibility of cell-based experiments.
Contamination in cell culture may be due to chemical and biologicalagents. Chemical contamination, such as the presence of endotoxins,
detergents, or impurities of used media, sera, or other media supplements
can be controlled by using trusted suppliers that guarantee rigorous
batch testing. Biological contamination can arise from improper aseptic
techniques and most often comes from the direct environment in the cell
culture area. The most common biological contaminants are bacteria, fungi(yeast and mold), viruses, and mycoplasma (summary Figure 6).

Bacterial contamination:

Many bacterial strains can grow in commonly used media in mammaliancell culture. Due to rapid logarithmic growth, the signs of contamination areeasily detectable within a few days of the initial contamination.
• Loss of medium clarity.
• Faster acidification of medium (medium turns orange/yellow if phenol
red is present).
• A thin bacterial film appears on the surface of cell culture vessels/flasks.
Bacteria can be identified by a brightfield microscope check (20–60x
magnification) as slowly moving, dark, round- or rod-shaped objects thatare significantly smaller than mammalian cells.

Mycoplasma
contamination:

Mycoplasma are very small (0.2um) bacteria characterized by their lack of acell wall. Unlike standard bacterial contamination, mycoplasma often growat a slow rate in infected cultures and can be present with no visible signs ofinfection such as loss of clarity or change of pH.
•

Mycoplasma do not contain cell walls, so they cannot be seen with astandard brightfield microscope.

•

Due to their slow growth rate, mycoplasma infection may develop for along time without any signs.

•

Mycoplasma infection often affects cell growth rates, metabolism, andcan even lead to chromosome aberrations of samples.

•

Routine testing using immunofluorescence with DNA stain, ELISA, or PCR
is recommended to monitor possible infections

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Fungal (yeast/mold)contamination:

Western
CellBlotting
CultureSimilar to bacterial contamination, yeasts can grow rapidly in mammalianculture media, leading to a loss of medium clarity, albeit at a slower rate.
•

Relatively rapid progression from initial infection to a serious threat.

•

pH is unchanged or slightly increases at later stages of the contamination,
which can affect your experiments.

•

Brightfield microscope check (20–60x magnification): rod-shaped orbranched filamentous objects that become visible to the naked eye atlater stages of contamination.

Many fungi can produce spores capable of surviving for a long time in adormant state in hostile environments and, therefore, in the event of fungal
contamination it is particularly important to decontaminate all working
surfaces and incubators to prevent repeated contaminations.

Virus contamination:

Viral contaminations are not very common but can potentially pose a serious
health threat to cell culture personnel.
•

Viruses are hard to detect because they do not affect cell culture growthand cannot be seen with a brightfield microscope.

•

Dedicated assays (e.g., ELISA, PCR) can be used to test for infections.

Viruses
Bacteria

Electronmicroscopy

Yeast

Figure 6. Cell culture contamination. Cells can be contaminated with bacteria (small, dark,
rod-shaped that can form strings), yeast (elongated, branched structures, light with darker
edges), or viruses (visible only with electron microscopy).

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Cell culture

PROTOCOL
Standard passaging(subculture):

Passaging refers to the diluting of cells that have reached high confluence
to supplement cells with fresh medium to enable continuous culture
propagation.
For mammalian cells, passaging should be performed when cells aretoward the end of the logarithmic growth phase, so before they reach thestationary phase (Figure 3). For adherent cells that usually means reaching
80–90% confluence (there is still space for growth on a culture dish); forsuspension culture, it is when cells start to clump and the culture becomescloudy, especially if a dish is shaken slightly.
Passaging of cells at the stationary phase is not recommended because theytend to take longer to begin the logarithmic growth phase upon seeding.
Additionally, the build-up of lactic acid in dense cultures may impact cell
metabolism.
Adherent cells:
1. Remove medium.
2. Wash cells with calcium- and magnesium-free balanced salt 		
solution (phosphate buffered saline, Hanks’ balanced salt solution,
etc...
3. Add detaching agent (e.g., trypsin). Incubate at 37°C until cells are
fully detached from the dish (2–20 min depending on the cell line).
4. Resuspend cells in fresh medium, pipette thoroughly to obtain single
cell suspension. If your medium does not contain serum, you needto inactivate detaching agents, e.g., by addition of trypsin inhibitors.
Measure total number of cells (see: counting cells).
5. Plate cells onto a new dish at the desired cell density.
Suspension cells:
1. Collect cells with medium and briefly centrifuge (150–300 xg, 3–5 min)to pellet cells.
2. Remove medium and gently resuspend cells with a balanced salt
solution. Briefly centrifuge (150–300 xg, 3–5 min) to pellet cells.
3. Remove salt solution and resuspend cells in fresh medium. Measure
total number of cells (see: counting cells).
4. Plate cells onto a new dish at desired cell density.

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Western
CellBlotting
CultureIt should be standard procedure to keep a record of the number of cellpassages for all cell lines. For primary cell lines with a finite lifetime, it
helps to monitor their viability and plan experiments before they reachsenescence and cease dividing. For immortalized cells, it helps to monitor
their age and design experiments up to a certain passage number.

Cell counting

Despite the recent development of automated counters, the Neubauer
chamber is still the most common method used for cell counting. A
Neubauer chamber is used to perform a proper cell count, although thedimensions and volumes of each chamber may differ.
The necessary elements to perform a cell count (Figure 7):a) Cellular dilution to measureb) Neubauer chamberc) Optical microscoped) Cover glasse) Pipette / micropipette with disposable tips

a

c

b

d

Figure 7. The equipment needed to perform a cell count with a Neubauer chambereptglab.com

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While performing the cellcount please remember:-

Avoid counting cells that are obvious cell debris vs. rounded cells-

When using trypan blue, count the cells that have excluded the dye-

Cell suspensions should be dilute enough so that the cells do not
overlap each other on the grid-Mix the cell suspension thoroughly before taking a sample to count-Count the cells in selected squares-

Decide on a specific counting pattern to avoid bias

The formula for calculation of the cell concentration:

Number of cells

Concentration (cel / ml) = ------------------Volume (in ml)-

The number of cells is the sum of all cells counted in all squares-

The volume is the total volume of all the squares counted-

In case a dilution was applied, the final concentration needs to beconverted to the original concentration before the dilution

Figure 8. The equipment needed to perform a cell count with a Neubauer

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Western
CellBlotting
CultureAn example of cell countingwith a Neubauer chamber:

We counted the cells in four squares to obtain a reliable estimate of thenumber of cells.
We obtained the following cell numbers: 54, 48, 52, and 60, which gave
us a total of 214 cells.
The area of one square in the Neubauer chamber used equals 0.01 cm2,
while the depth of the chamber is 0.01 cm.
Multiplying 0.01 cm2 by 0.01 cm gives 0.0001 ml total volume per one
square.
Using the above equation, we divide the total number of cells (214) byvolume (0.0001*4 squares = 0.0004) to obtain a concentration of
535 000 cells per ml.

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Cell banks(cryopreservation)

Mammalian cell lines are suitable for long-term storage at low
temperatures (below -130°C – usually in liquid nitrogen). Cell lines areroutinely frozen to make and keep reference/parental cell lines, newly
produced transgenic cell lines, keep stocks of primary and immortalized
cells, and for shipping purposes. The viability of cell banks is dependent
on the cryopreservation procedure employed when making them and on
the proper storage conditions. It is good laboratory practice to create anumber of master stocks at the lowest possible passage number for longterm storage together with a number of working stocks for general use inexperiments.

New cell line (Handled under quarantine conditions)

Token freeze of 3-5 ampules and initial mycoplasma test

FAIL

PASS

Quarantine Facility
Main cell culture

Abandon banking procedure

Resuscitate one ampule and expand in culture

Cryo-preservation of master bank(10-100 ampules)

Mandatory quality control tests. Cell count and viability. Microbial QC including mycoplasma. Authentication

Resuscitate one ampule and expand in culture

Repeat banking

Cryopreservation of working bank (20-200 ampules)

Quality control tests. Cell count and viability. Microbial QC including mycoplasma. Authentication

Repeat banking released for use

Figure 9.. Cell banking overview. chamber

Released for use

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Preparing cell banks

Western
CellBlotting
Culture1.

Grow cells toward the end of the logarithmic growth phase (~90%
confluence). Ideally, use cells with the lowest possible passage number.

2.

Passage cells as normal – detach adherent cells, collect and washsuspension cells (see: standard passaging).

3.
4.

Measure total cell number (see: counting cells).
Resuspend cells in freezing medium. In general, the freezing medium isroutine culture medium supplemented with a cryoprotectant, an agent
that prevents the formulation of ice crystals from the water present
within cells that would destroy cell integrity. DMSO and glycerol are thetwo most commonly used cryoprotective agents. The exact
concentration is cell-dependent and should be experimentally verified,
but they are usually used at 5–10% (vol/vol) final concentration.
Note: Prepare freezing medium before adding to cells because
concentrated DMSO is toxic to cells.

Thawing cells from banks

5.

Aliquot cells into cryovials (usually 106–107 cells/ml) and gently cool
down to -80°C. The most efficient cooling rate is 1°C per 1 minute,which can be achieved by putting vials into a commercial cryofreezing
container or by wrapping them in a thick layer of cotton wool andplacing in a -80°C freezer.

6.

After 1–3 days place cells in a liquid nitrogen dewar and store in liquid
nitrogen or the gas phase of liquid nitrogen for long-term storage.

1.

Transport frozen cell vial in low temperature (portable liquid nitrogencontainer or dry ice) to cell culture area.

2.

Prepare a culture dish with pre-warmed medium.

3.

Thaw cells rapidly (e.g., in a 37°C water bath).
Note: Thawing cells rapidly ensures high cell viability.

Cell transfection

4.

Optional step to remove cryopreservant and non-viable cells: resuspend
cells in medium and briefly centrifuge (150–300 xg for 3–5 min.).
Resuspend cell pellet in fresh medium.

5.

Transfer freshly thawed cells into a dish.

Transfection of cells refers to the delivery of nucleic acid (DNA or RNA)
into cultured cells. The most commonly used reagents are cationiclipids that can associate with nucleic acids to form positively charged
complexes that allow interaction of DNA/RNA with the negatively
charged cell membrane. That leads to the efficient entry of nucleic acids
into cells via endocytosis.
Alternatively, nucleic acids can be delivered into cells by electroporation,
co-precipitation of DNA with calcium phosphate, or polybrene/DMSO
shock.
The delivered DNA does not usually integrate with the host genome.
Therefore, DNA expression is generally transient. However, it is possible
to engineer stable cell lines with transfected DNA stably integrated withthe genome, but this requires a method of selecting cells with stable
integration with a dedicated DNA vector design and antibiotic selection.

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Cell culture

TROUBLESHOOTINGMy cells are not
growing.

Check that the medium is recommended for the particular cell type.
Recommendations can be obtained from the biggest cell banks(ATCC and ECACC) and from previous scientific publications
reporting use of the cell line.
Most cell lines require additional supplements for their growth.
The most commonly used are serum (e.g., fetal bovine serum,
concentration depends on the cell type, typically 5–20% (vol/vol)),glutamine, and non-essential amino acids (NEAA).

My adherent cells are
not attaching to cell
culture dishes.

Check the type of dishes that you use.
Many manufacturers provide dishes for suspension cultures withvery hydrophobic surfaces that are not suitable for adherent cell
culture. In this case, adherent cells do not attach to the surface anddie.
Consider whether your cell line requires a special coating to
improve cell adherence.
Most commonly used coating agents include poly-L-lysine (diluted
in water), collagens (diluted in low molar acid solutions such as
acetic acid or hydrochloric acid), and fibronectin (diluted in calciumand magnesium-free PBS).
Specific guidelines are provided by manufacturers, but mostcommonly used protocols involve the preparation of a diluted
coating agent, which is applied to dishes, followed by 1 h
incubation at room temperature in the tissue culture hood andremoval of excess agent by washing surfaces a few times with PBS.

My cells are regularly
infected withmycoplasma or otherpathogens.

Examine your aseptic procedures for possible methods of
optimization.
Always work with cells under dedicated cell culture hoods and wear
appropriate PPE, including gloves and a lab coat.
Regularly clean and decontaminate the working area. Use steriletips and pipettes, sterilize any glassware and tools by autoclaving.
If needed, maintain two cell cultures, one with antibiotics and one
antibiotic-free and examine both cultures regularly for any signs ofinfection.

22

Western
CellBlotting
CultureIdeally, the regular use of antibiotics in routine cell culture should
be limited because they can mask low-level infections, making
them hard to detect, and provide selective pressure for developing
antibiotic-resistant pathogens in your working area.
Consider microbiological testing of your working and master stocks
to validate they are not contaminated.

I cannot recover any/
many cells from my cellbanks.

Cells may not have been frozen or thawed properly; please follow ourguidelines above.
Ensure that frozen vials are stored at the appropriate temperature(below -130°C) at all times.
Consider freezing more cells per one vial and seed freshly thawed
cells at a higher density to allow logarithmic growth from the very
beginning.

I have problems
achieving efficienttransfection of mysamples.

The efficiency of cell transfection depends on many aspects such as
general cell metabolic state and passage number. Also, some medium
supplements, such as antibiotics or serum, can decrease transfection
efficiency. In principle, transfection protocols should be optimized
for every cell line, considering variables such as the selection of atransfection reagent, the ratio of DNA/RNA to transfection reagent, andcell density during transfection.

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Western Blotting

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Western Blotting

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