Cell Biology Laboratory PCB 4023

Laboratory 3: Cell Culture and Cell counting

Mayoral, adapted from Mustafi et al 2014

WHY CELL CULTURE?

Cell biologists have successfully established many cell lines from primary tissues to explore
the structure and function of cells, investigate the macromolecules important for cellular
physiology, study pathways and the manifestation of severe disease like cancer, diabetes or
even bacterial invasion. HeLa, MCF7 or HEK293 are some common human cell lines where
as RAW, CHO, Ec1one or 3T3 are other mammalian cell lines popularly used. Cell lines grow
in vitro until they have covered the surface area available (confluency) or the medium
(solution in which cells are grown) is depleted of nutrients. Mass culture of animal cell lines
is also fundamental to the manufacture of viral vaccines and many products of
biotechnology. Vaccines for polio, measles, mumps, rubella, and chickenpox are currently
made in cell cultures. In addition, biological products produced by recombinant DNA (rDNA)
technology in animal cell cultures include enzymes, synthetic hormones, monoclonal
antibodies and anticancer agents.

EQUIPMENT USED IN CELL CULTURE

1.- Biological Safety Cabinet: out of the

different types of hoods it is the best for
working with hazardous organisms since the
aerosols that are generated in the hood are
filtered out before they are released into the
surrounding environment (see diagram on the
right). The air passes through a HEPA (high
efficiency particle) filter that removes
particulates from the air. It is equipped with UV
light to sterilize the surfaces of the hood. Do not
put your hands or face near the hood when the
UV light is on as the short wave light can cause
skin and eye damage. It should be turned on
about 10-20 minutes before being used. Wipe
down all surfaces with ethanol before and after
each use. Keep the hood as free of clutter as possible because this will interfere with the
laminar flow air pattern. Keep as low as possible the protection glass to guarantee proper
airflow, and the safety of the operator and samples. A biological safety cabinet is designed to
provide three basic types of protection: i) Personnel protection from harmful agents inside
the cabinet; ii) Product protection to avoid contamination of the work, experiment, or
process; iii) Environmental protection from contaminants contained within the cabinet.

NOTE: these are not fume hoods and should NOT be used for volatile or explosive chemicals.

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2.-Incubator: in cell biology, an incubator is a device used to provide an ideal environment

for culturing cells, and most commonly used to simulate the physiological conditions of
mammalian cells. The incubator maintains optimal temperature, humidity and other
conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
The risk of microbial contamination of cell cultures in the incubator is high due to the
frequent and improper use of the incubator. Always spray with 70% ethanol your gloves
before you take anything in or out the incubator.

3.- Microscopes: Inverted phase contrast microscopes are used for visualizing the cells.
Microscopes should be kept covered and the lights turned down when not in use. Before
using the microscope or whenever an objective is changed, check that the phase rings are
aligned. After you will mount cells on slides in this laboratory, you will also use the
fluorescence compound microscope to visualize different molecules and organelles.

MEDIA AND GROWTH REQUIREMENTS FOR CELLS

1.- Physiological parameters

Temperature: maintaining ideal culture temperatures is vital for optimal cell growth.
Overheating can be a much more serious problem than under-heating for a cell culture, so it
is good practice to set the temperature just below optimal. An incubator will provide a stable
environment for the cells to grow. Exactly what constitutes an optimal temperature depends
upon your cells; a general rule of thumb is:
• Most mammalian cells thrive at around 37 °C
• Insect cells require lower temperatures of ~ 27 °C
• Avian cell lines normally require 38.5 °C for maximum growth
• “Cold-blooded” animals (e.g., amphibians, cold-water fish) can be cultured
anywhere between 15 - 26 °C
pH Levels: most mammalian cell lines will grow well at pH 7.4, and there is very little
variability among different cell strains. Most of the commercially available culture media
include phenol red as a pH indicator, which allows constant monitoring of pH. During the cell
growth, the medium changes color as pH is changed due to the metabolites released by the
cells. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns
the medium purple. Medium is bright red for pH 7.4, the optimum pH value for cell culture.
CO2 Levels: the cells are grown in an atmosphere of 5-10% CO2 (our incubator is set
to 5%) because the medium used is buffered with sodium bicarbonate/carbonic acid and the
pH must be strictly maintained. Because the pH of the medium is dependent on the balance
of dissolved carbon dioxide (CO2) and bicarbonate (HCO-3), changes in the atmospheric CO2
can alter the pH of the medium. Therefore, we use an external source of CO2 when using
media buffered with a CO-2 bicarbonate based buffer, especially if the cells are cultured in
open dishes or transformed cell lines are cultured at a density. Culture flasks should have
loosened caps to allow for sufficient gas exchange.

Humidity: cells should be left out of the incubator for as little time as possible and the

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incubator doors should not be opened for very long. The humidity must also be maintained
for those cells growing in tissue culture dishes so a pan of water at the bottom of the
incubator is kept filled at all times.

Visible Light: can have an adverse effect on cells; light can induce the production of
toxic compounds in some media. Cell should be cultured in the dark and be exposed to room
light as little as possible.

2.- Medium requirements (extracted from Mater Methods 2013;3:175)

Animal cells can be cultured using either a completely natural medium or an

artificial/synthetic medium along with some natural products. Natural Media: consist solely
of naturally occurring biological fluids. Natural media are very useful and convenient for a
wide range of animal cell culture. The major disadvantage of natural media is its poor
reproducibility due to lack of knowledge of the exact composition of these natural media.
Artificial Media: or synthetic media are prepared by adding nutrients (both organic and
inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates and
cofactors.

Media Type Examples

plasma, serum, lymph, human placental cord

Biological Fluids
serum, amniotic fluid

Natural Extract of liver, spleen, tumors, leucocytes and

media Tissue Extracts bone marrow, extract of bovine embryo and
chick embryo

Clots coagulants or plasma clots

Balanced salt
PBS, DPBS, HBSS, EBSS
solutions
Artificial
media Basal media MEM DMEM

Complex media RPMI-1640, IMDM

3.- Basic Components of Culture Media

Culture media contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients,
and its specific composition vary among cell lines. Each component performs a specific
function, as described below:

Inorganic salt: help to retain the osmotic balance and help in regulating membrane
potential by providing sodium, potassium, and calcium ions.

Amino Acids: are the building blocks of proteins, and thus are obligatory of all known
cell culture media. Essential amino acids must be included in the culture media as cells
cannot synthesize these by themselves. They ae required for the proliferation of cells and
their concentration determines the maximum achievable cell density. L-glutamine, an

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essential amino acid, is particularly important. L-glutamine provides nitrogen for NAD,
NADPH and nucleotides and serves as a secondary energy source for metabolism.

Carbohydrates: in the form of sugars are the major source of energy. Most of the
media contain glucose and galactose; however, some contain maltose and fructose.

Proteins and Peptides: the most commonly used proteins and peptides are albumin,
transferrin, and fibronectin. They are particularly important in serum-free media. Serum is
a rich source of proteins and includes albumin, transferrin, aprotinin, fetuin, and fibronectin.
The binding capacity of albumin makes it a suitable remover of toxic substances from the cell
culture media. Aprotinin is a protective agent in cell culture systems since it has the ability
to inhibit several serine proteases such as trypsin. Fetuin is a glycoprotein found in fetal and
newborn serum at larger concentrations than in adult serum and it is also an inhibitor of
serine proteases. Fibronectin is a key player in cell attachment, and transferrin is an iron
transport protein that acts to supply iron to the cell membrane.

Fatty Acids and Lipids: they are particularly important in serum-free media as they
are generally present in serum.

Vitamins: many are essential for growth and proliferation of cells. Vitamins cannot
be synthesized in sufficient quantities by cells and are therefore important supplements
required in tissue culture. Serum is the major source of vitamins in cell culture, however,
media are also enriched with different vitamins making them suitable for a particular cell
line. The B-group vitamins are most commonly added for growth stimulation.

Trace Elements: often supplemented to serum-free media to replace those normally

found in serum. Trace elements like copper, zinc, selenium and tricarboxylic acid
intermediates are needed in minute amounts for proper cell growth. These micronutrients
are essential for many biological processes, e.g. the maintenance of the functionality of
enzymes.

Media Supplements: the complete growth media recommended for certain cell lines
requires additional components which are not present in the basal media and serum. These
helps sustain proliferation and maintain normal cell metabolism. Although supplements like
hormones, growth factors and signaling substances are required for normal growth in some
cell lines, it may change the osmolality of the complete growth media. Shelf-life of the growth
media changes after the addition of supplements. Complete media containing protein
supplement tend to degrade faster than basic media alone.

Antibiotics: although not required for cell growth, antibiotics are often used to
control the growth of bacterial and fungal contaminants. Routine use of antibiotics for cell
culture is not recommended since antibiotics can mask contamination by mycoplasma and
resistant bacteria. Moreover, antibiotics can also interfere with the metabolism of sensitive
cells.

Serum: serum is a complex mix of albumins, growth factors and growth inhibitors. It
is one of the most important components of cell culture media and serves as a source for

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amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K),
carbohydrates, lipids, hormones, growth factors, minerals, and trace elements. Serum from
fetal and calf bovine sources are commonly used to support the growth of cells in culture.
Normal growth media often contain 2-10% of serum. Due to the presence of both growth
factors and inhibitors, the role of serum in cell culture is very complex.

CONTAMINATION AND PROPER ASEPTIC TEHCNIQUES

Contamination of cell cultures is the most common problem encountered in cell culture
laboratories, always with very serious consequences. The main cell culture biological
contaminants are bacteria (i.e. mycoplasma), molds, yeasts, viruses, as well as cross
contamination by other cell lines. Basic good lab practice, including proper aseptic
techniques (see document attached to this laboratory) when working with cells and
reagents, is the most important factor in reducing the risk of contamination.

COUNTING CELLS IN A CELL CULTURE DISH

Cell culture, microbiology and other

applications require determining the cell
concentration. A device used for determining
the number of cells per unit volume of a
suspension is called a counting chamber. The
most widely used type of chamber is called a
haemocytometer, since it was originally
designed for performing blood cell counts
(see picture on the right).

Preparing the counting chamber:

To prepare the counting chamber the mirror-like polished surface is carefully cleaned with
lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially
made and are thicker than those for conventional microscopy, since they must be heavy
enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the
counting surface prior to putting on the cell suspension. The suspension is introduced into
the V -shaped wells with a micropipette. The area under the coverslip fills by capillary action.
Enough liquid should be introduced so that the mirrored surface is just covered. The charged
counting chamber is then placed on the microscope stage and the counting grid is brought
into focus at low power.

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Counting grid

It is essential to be
extremely careful
with higher power
objectives, since the
counting chamber is
much thicker than a
conventional slide.
The chamber or an
objective lens may be
damaged if the user is
not careful. One
entire grid on
standard
haemocytometers
with Neubauer
rulings can be seen at
40x (4x objective).

The main divisions separate the grid into 9 large squares (see figure on the right). Each
square has a surface area of one square mm, and the depth of the chamber is 0.1 mm (red
square). Thus, the entire counting grid lies under a volume of 0.9 mm3.

Counting cells

Suspensions should be dilute enough so that the cells or other particles do not overlap each
other on the grid, and should be uniformly distributed. Now systematically count the cells in
selected squares so that the total count is in the order of 100 cells (minimum number of cells
needed for a statistically significant count). For large cells this may mean counting the four
large corner squares and the middle one (total 5 squares). For a dense suspension of small
cells you may wish to count the cells in the four 0.2 mm2 (blue area) plus the middle square
in the central square. Always decide on a specific counting pattern to avoid bias. For cells
that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it
overlaps the bottom or left ruling.

Determining the cell count

Here is a way to determine a particle or cell count using a hemocytometer. Suppose that you
conduct a count as described above, and count 187 particles in the five small squares
described (5 of the blue squares). Each square has an area of 0.04 mm-squared and depth of
0.1 mm. The total volume in each square is (0.04) x (0.1) = 0.004 mm3. You have five squares
with combined volume of 5 x (0.004) = 0.02 mm3. Thus you counted 187 particles in a volume
of 0.02 mm-cubed, giving you 187/ (0.02) = 9350 particles per mm3. There are 1000 mm3 in
1 cm3, which is the same as 1 mL. In other words, 1 mm3 = 1µl = 0.001 mL. Therefore, your
cell count is 9,350,000 per mL or 9,350,000 cells/mL

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Counting over a larger surface area

Cells are often large enough to require counting over a larger surface area. For example, you
might count the total number of cells in the four large corner squares plus the middle
combined (so five of the red squares in the figure above). Each square has surface area of 1
mm3 and a depth of 0.1 mm, giving it a volume of 0.1 mm3. Suppose that you counted 125
cells (total) in the five squares. You then have 125 cells per 0.5 mm3, which is 250 cells/mm3
or 250,000 cells/mL).

Counting on a diluted sample

Sometimes you will need to dilute a cell suspension to get the cell density low enough for
counting. In that case you will need to multiply your final count by the dilution factor. For
example, suppose that for counting you had to dilute a suspension of macrophages 10-fold.
Suppose you obtained a final count of 250,000 cells/mL as described above. Then the count
in the original (undiluted) suspension is 10 x 250,000 which is 2,500,000 cells/mL.

Any of these protocols can also be combined with other protocols like trypan blue to estimate
the % of viable cell in culture. You just need to take in consideration the dilution factors you
include with every new step.

EXPERIMENTAL PROTOCOL:

After the cell lines have covered the surface area available, they should be sub-cultured in
order to prevent the cultures from dying. To subculture the cells they need to be brought
into suspension. In the majority of cases proteases, e.g. trypsin, are used to release the cells
from the flask.

This exercise will introduce students to basic practices in the cell biology laboratory such as
manipulation of cultures, handling culturing equipment, pipetting of cells, aseptic techniques
working with cells in the lab. In addition, students will perform an experiment in which they
determine the cell count and % of viable cells in the presence and absence of H2O2.

EQUIPMENT: provided by the lab coordinators

- Culture plates and cover slips
- 37°C incubator
- Glass slides
- Forceps and needles
- Inverted microscope
- Haemocytometers + thick coverslips (not disposable!)

REAGENTS:
- L1 mouse fibroblast
- PBS (buffer solution)
- 0.25%Trypsin, 0.1% EDTA in HBSS w/o Ca2+ , Mg2+ and NaHCO3
- Antibiotics stock solution
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- Stock 1 M Hydrogen peroxide

- Stock 0.4% (w/v) Trypan Blue dye

PROCEDURE:
1. Start with a cell culture dish of 3T3-L1 fibroblasts. Check their morphology under the
microscope. What microscope do you use for checking cells in a culture dish?

2. Take your culture dish to the biosafety hood.

3. For half of the student groups who have the control, add 25 µL of water. Those groups
assigned to H2O2 will add 25 µL of that solution to their dishes.
4. Transfer the cells into the 37°C incubator, wait 15 minutes. Make sure you label the dish
with your group number and treatment.
5. Remove growth media from one side of the culture dish using the vacuum trap.
6. Gently, add 1 mL of PBS to your culture plate, swirl it around and then take it out using the
vacuum trap.
7. Add 0.5 mL of trypsin solution to the plate. Gently swirl the plate and incubate into the
37°C incubator for 3-4 minutes. When trypsinization process is complete, check the cells
under the microscope; the cells will be in suspension and appear rounded.
8. Block/Re-suspend the cells with 1 mL of complete media (note that the final volume in
the plate now is 1.5 mL). Cells can be re-suspended by gently pipetting the cell
suspension to break up the clumps. Check under the inverted microscope to ensure
that all the cells are dislodged.
9. Before the cells have a chance to settle and using the 1 mL pipette, take out 200 µL of cell
suspension and place it in a clean new Eppendorf tube. Add 200 µL 0.4%-Trypan Blue
dye and mix gently (note for your calculations later that this is a two-fold dilution).
10. Allow the Eppendorf to incubate for 5 minutes at room temperature (on your bench).
11. Kindly ask your instructor for a haemocytometer. Please handle the haemocytometer
and its cover slip with both hands, they break very easily and this equipment is
very expensive.
12. Place 10 µl on the haemocytometer (between the counting slide and the glass cover slip).
13. Count the total number of cells under the microscope as described in the introduction
and as explained by your instructor. Make sure you also record the number of blue cells

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and the number of white/transparent cells. Carefully, clean and return to your
instructor BOTH the cover slip and the haemocytometer.
14. Calculate the cell concentration in the suspension you obtained in step 7.
15. Calculate the % of viable cells in your suspension, and write this result up on the board
as directed by your instructor/TA to facilitate discussion of the class dataset overall.
16. Based on the cell concentration you calculated in step 13, transfer 1x105 cells to a 35 mm
cell culture dish, and add cell culture medium to make the total volume 2 mL.
17. Gently swirl the plate to spread the cells evenly, and incubate the cell culture dishes in
the CO2 incubator at 37 °C.
18. Normally, the cells and the media should be checked at day 3 and day 5.

QUESTIONS: Testing and applying your understanding.

1.) Calculate how many cells/mL you have in your cell culture dish. What is the total cell
number you would have in the 5 mL?

2.) Calculate the % of non-viable cells in your suspension and compare it with the results
obtained by the other treatment groups in class. Is there any difference between those cells
treated with H2O2 and those that were not?

3.) Name 3 cell lines, the tissue they were isolated from and when they were isolated.

4.) How is new media stopping the trypsinization process?