Your Step-by-step Guide to

Primary Cell Culture

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Principle and Protocol

Introduction

To master the basic operation process of primary culture and

subculture of mammalian cells, lay a foundation for the
application of bioengineering in medicine.

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Principle of experiment

Cell culture is a simulation of physiological conditions in the body. The cells

are removed from the body and allowed to survive, grow, reproduce, and
pass under artificial conditions. The process of cell life, cell transformation,
cell engineering, etc. is studied. In recent years, it has been widely used in
molecular biology, genetics, immunology, oncology, cell engineering and
other fields to develop into an important biotechnology and has achieved
remarkable success. Tissues or cells taken directly from the body are called
primary cultures. Primary cultured cells have a short in vitro time and their
traits are similar to those in vivo and are suitable for research. In general,
naive tissue and cells, such as embryos and larvae of animals, are more likely
to undergo primary culture.

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In vitro cultured primary cells or cell lines must be passaged in vitro in order to obtain a stable cell line
or to obtain a large number of identical cells, and maintain the continuity of cell types. After the
cultured cells form a confluent monolayer, the nutrient is depleted due to the insufficient density of
the excessively large living space. The cultured cells are dispersed, removed from the container, and
transferred to another container at a ratio of 1:2 or 1:3, which is, passaging culture.

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Cell “one generation” refers to a period of time from cell seeding to isolation and reculture, which is
different from cell generation or multiplication. In one generation, cell culture was increased 3 to 6
times. After cells are passaged, they usually go through three phases: free, exponential, and stop. The
commonly used cell division index indicates the degree of cell proliferation, that is, the number of
dividing phases of the cell population/100 cells. The general cell division index is between 0.2% and
0.5%, and tumor cells can reach 3 to 5%. Cell proliferation is vigorous and prosperous for 2 to 3 days
after inoculation, and is the best period for activity. It is called exponential proliferative phase
(logarithmic growth phase) and is suitable for various tests.

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The main reagent

1. 5% calf serum in MEM medium

2. 0.01mol/L PBS
3. 0.25% Trypsin-0.02% EDTA Mixed Digestion Solution
4. 75% ethanol

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Major equipment

1. Surgical instruments
2. Plates
3. Bottles
4. Sucker
5. Centrifuge tubes (after sterilization)
6. Alcohol lamp
7. Beaker
8. Clean bench
9. Carbon dioxide incubator
10. Inverted microscope

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Experimental Materials

Rabbit, HeLa cells (human cervical cancer cells)

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1. Primary cell culture

1) The rabbit was quickly killed by cervical dislocation. Then, the entire
animal was immersed in a beaker containing 75% ethanol for several
seconds. After removal, it was placed in a large petri dish and carried into a
clean bench. The sterilized skin was excised with iodine and ethanol using
sterile scissors, and the liver or kidney was removed by laparotomy. Place in
a sterile petri dish.

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1. Primary cell culture

2) The excised organs were washed three times with sterile PBS, and the
tissue was carefully cut with an ophthalmic surgical scissors until it became
small pieces of about 1 mm 3, washed with PBS until the tissue became
white. Move into a sterile centrifuge tube and let it stand for a few minutes
to allow the tissue mass to naturally settle to the bottom of the tube and
discard the supernatant.

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1. Primary cell culture

3) Pipette 1 ml of 0.25% trypsin-0.02% EDTA mixed digestion solution into

the centrifuge tube, mix with the tissue block, add the tube plug, and digest
it in a 37°C water bath for 8 to 10 minutes. Shake the tube every few
minutes. Bring the tissue into full contact with the digestive juice, stop and
suck the supernatant, add 5 to 10 ml of MEM medium containing 5% calf
serum to the centrifuge tube, mix by pipetting, and move into two culture
flasks and incubate in a CO2 incubator.

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2. Passage cell culture

1) Take out the monolayer of primary cultured cells or HeLa cells from the
CO2 incubator. Pour out the culture solution from the flask in a clean bench
and add a little digestive juice. (It is better to cover the cells with liquid
surface), and let it stand for 5 to 10 minutes.

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2. Passage cell culture

2) Observe the digested cells under an inverted microscope. If the cells

become gardens and they are no longer connected to each other, then the
digestive juice should be immediately discarded at the super-clean bench,
and 3 to 5 ml of fresh culture fluid should be added and blown to make cell
suspension.

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2. Passage cell culture

3) Aspirate the cell suspension by about 2 ml and add it to another flask.

Add 3 ml or so of the culture solution to each flask, cap the stopper, return it
to the CO2 incubator, and continue the culture.

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