EXPERIMENT5_ Cell Culture Spring2024

BIO 332 Spring 2024
EXPERIMENT 5: SETTING UP A MAMMALIAN CELL CULTURE
1. Introduction:
Cell culture is a process where cells (animal or plant cells) are removed from the organism
and introduced into an artificial environment with favorable conditions for growth. This
allows for researchers to study and learn more about the cells.
The most important thing is the prevention of contamination. Contamination in cell cultures
is one of the main problems for in vitro experimentation. Cell culture contaminants can be
divided into two main categories; chemical contaminants such as impurities in media and
the biological contaminants such as bacteria, mycoplasma and cross contamination from
other cell lines. It is possible to minimize or reduce frequency and seriousness of cell
culture contamination. However, this requires better understanding of contamination
sources and following good aseptic techniques.
Basic equipment for cell culture
A B C
A basic cell culture hood (i.e., laminar-flow hood or biosafety cabinet), allows us to work
under sterile conditions (Fig. 1A). Additionally, incubators (Fig. 1B), water baths (Fig.1C),
centrifuges, refrigerators and freezers (–20°C), cell counters (e.g., Automated cell counter
or hemocytometer), inverted microscopes, liquid nitrogen freezer or cryo storage container,
sterilizer (i.e., autoclave) and aspirators are needed.
BIO 332 Spring 2024
Incubator is a device used to grow and maintain microbiological cultures or cell cultures.
The incubator maintains optimal temperature, humidity and other conditions such as
the carbon dioxide (CO2) and the oxygen content.
Further supplies and materials include cell culture vessels (e.g., flasks, petri dishes, roller
bottles, multi-well plates), pipettes and pipettors, syringes and needles, waste containers,
culture media, sera and reagents.
Subculturing Cells
Subculturing, also referred to as passaging or splitting, is the transferring of some or all

cells from previous culture to fresh growth medium, a procedure that enables the further
propagation of the cell line. Adherent cultures should be passaged when the cells are
approximately 80% confluent (80% of the surface of the flask covered by cell monolayer).
They should still be in the log phase of growth. Majority of cell lines stop growing when
they reach full confluency (contact inhibition), and it takes them longer to recover when
reseeded. Transformed cells can continue proliferating even after they reach confluence,
but they usually deteriorate after about two doublings.
Cells are growing under certain conditions, and they need nutrients for healthy growth.
There are different kinds of growth mediums which are used in the cell culture and the type
of the medium is changed according to cell type.
Phosphate-buffered saline (PBS) is a water-based salt solution containing disodium

hydrogen phosphate and sodium chloride. The osmolarity and ion concentrations of the
solutions match those of the human body (isotonic). PBS has many uses because it is
isotonic and non-toxic to most cells. In the cell culture, it is mostly used for substance
dilution and cell container washing.
Trypsin is a serine protease and used to cleave proteins bonding the cultured cells to the
dish, so that the cells can be suspended in fresh solution and transferred to fresh dishes.
BIO 332 Spring 2024
Because some cell types tend to stick or adhere to the sides and bottom of a dish when
cultivated in vitro.
Cell Counting
For over 100 years, the hemocytometer has been used by cell biologists to count cells. It’s
a modified microscope slide containing two identical wells into which a small volume of cell
suspension is pipetted. Trypan blue is used to dilute the cell suspension. It helps us
distinguish between living and dead cells. The dye passes through the membranes of dead
cells, so they appear blue under a microscope. Living cells appear mostly clear. 100 ul cell
suspension and 10 ul trypan blue are mixed and loaded.
After loading, hemocytometer is placed under the microscope. Each chamber is divided
into a grid pattern consisting of 9 large squares. Each square has the same dimensions
and contains 10-4 ml of suspension. Then, the living cells at the 4 large corner squares are
counted and calculated like this:
Concentration (viable cells/ml): Total number of living cell/4 x Dilution factor x 1.1 x 104
While counting cells, the basic principle is

that any 2 adjacent borders should be
counted, and the remaining 2 borders
should be rejected.
If the solution of the sample is too highly

concentrated, the cells will overlap and
thus the counting will be wrong.
Therefore, such concentrated cell
solutions need to be diluted with an
appropriate solution. This dilution also
needs to be factored into the calculations.
For instance, if the sample has been
diluted by 10x, the final answer obtained
from the calculations must be multiplied
by 10.
BIO 332 Spring 2024
2. Materials
● 70% Ethanol
● Tissue paper
● Micropipette and micropipette tips
● 5 mL, 10 mL serological pipets
● Confluent cells in 10 cm tissue culture dish
● Tissue culture media (RPMI1640) with 10% serum (FBS) and 1% antibiotics
● Trypsin
● 1X PBS
● 10 cm tissue culture dish/plate
● 15 mL centrifuge tube
● Electronic pipettors
● Waste box
● Incubator
● Hemocytometer
● Trypan Blue
3. Procedure
3.1. Cell passaging
● Clean hood with ethanol.

● Sterilize all materials which are loaded into the hood. Spray hands with ethanol.
● Check cells in a 10cm dish under a microscope to confirm that the cells are
80% confluent.
● Warm media, PBS and trypsin to 37°C in a water bath.
● Remove the old media. Be careful to not touch anything outside of the dish with
a pipette.
● Add 5 mL of PBS into the dish. Lightly swash PBS and remove back.
● Add 1 mL trypsin into the dish. Lightly swash trypsin. Make sure that the trypsin
spreads all over the dish.
● Place dish in incubator for 3 min or until detached.
● Remove cells from the incubator and tap the side of the dish.
● Visually check to ensure lumps of cells are dispersed.
● Check cells under a microscope to confirm that cells are detached.
● Add 9 mL of media into the dish to inhibit trypsin. (Note: The liquid suspension
now contains the cells.) Carefully resuspend cells.
● Put the media into a 15 ml falcon.
● Count the cells for passaging.
● Add the required number of cells to the new plate.
BIO 332 Spring 2024
● Add the fresh media to 10 ml.

● Place the dish in the incubator.
● Clean hood with ethanol. Spray ethanol over surfaces and wipe clean with
tissue paper.
3.2. Cell Counting
● Add 10 µl of the Trypan Blue Solution into 100 µl cell suspension, gently mix. (If
the cells are too dense dilute to the cell culture mix appropriately. Ideal counting
is a about total of 80-100 cells)
● Fill the chamber underneath the coverslip with 10 µl of cell suspension with
Trypan Blue in the hemocytometer.
● Count both live and dead cells under a microscope with a 10X objective and
calculate how many live cells you have and the ratio of live cells.
Live cells : light shiny without Trypan Blue uptakes.
Dead cells : stained by the Trypan Blue
Percent of Viable Cells = (Viable Cells #) / (Viable Cells # + Dead Cell #) x100.
Number of Viable Cells = # of Viable Cells x 104 x 1.1 = Viable cells/ml culture.
BIO 332 Spring 2024

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BIO 332 Spring 2024

EXPERIMENT 5: SETTING UP A MAMMALIAN CELL CULTURE

Basic equipment for cell culture

Subculturing, also referred to as passaging or splitting, is the transferring of some or all

Phosphate-buffered saline (PBS) is a water-based salt solution containing disodium

While counting cells, the basic principle is

If the solution of the sample is too highly

3.1. Cell passaging

● Clean hood with ethanol.

● Add the fresh media to 10 ml.

3.2. Cell Counting

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