The first is the experimental steps:
. Culture medium configuration
. Cell passage
3. Culture medium replacement
4. Cell cryopreservation
The most important one Part of that is doing cell passaging.
What is cell passage?
. Subculture refers to the process of dividing the culture into small parts, re-inoculating it into another culture vessel (bottle), and then cultivating it. For monolayer culture, 80% confluent or newly confluent cells are the ideal passage stage.
. Cell subculture in subculture. After the primary culture is successful, as the culture time prolongs and the cells continue to divide, on the one hand, contact inhibition occurs due to the mutual contact between cells, and the growth rate slows down or even Stop; on the other hand, it may also be detrimental to growth or poisoning due to insufficient nutrients and accumulation of metabolites.
Why is cell passaging necessary?
. Increase the number of cells
Conduct cell culture experiments under different environments and conditions to replace bad and necrotic cells and maintain cell activity.
. Prevent cell poisoning and death
Because cells will continue to reproduce in the culture dish until the nutrients are consumed or metabolites are excessively accumulated. In order to ensure that the cells can continue to reproduce, it needs to be carried out when the cell density reaches 80%-90%. Passage processing. If passage is not performed, the cells will be poisoned and die due to problems such as depletion of culture in the culture dish, excessive accumulation of metabolites, and lowering of pH value.
3. Purified cells
Eliminate the cell types that account for a small proportion of the cultured cells, and purify the final cell type.
What kind of living environment do experimental cells need? The conditions required for
cells in vitro culture are basically the same as those for cells in vivo, requiring four basic conditions.
. Pollution-free environment
A non-toxic and sterile culture environment is the primary condition to ensure the survival of cells. Compared with cells in the body, cells cultured outside the body lose their ability to defend against microorganisms and toxic substances. Once they are contaminated or their own metabolic substances accumulate, it can lead to cell death. Therefore, during culture, keeping the cell living environment pollution-free and timely removal of metabolites are the basic conditions for maintaining cell survival.
. Constant temperature
To maintain the vigorous growth of cultured cells, a constant and appropriate temperature is necessary. The standard temperature for human cell culture is 36.5°C (±0.5°C). If it deviates from this temperature range, the normal metabolism of cells will be affected or even die. Cultured cells are more tolerant to low temperatures than to high temperatures. When the temperature rises no more than 39°C, cell metabolism is proportional to the temperature. Human cells can be damaged to a certain extent at 39-40°C for 1 hour, but they may still recover. ; At 40-41°C for 1 hour, cells will be generally damaged, and only a small half may recover; at 41-42°C for 1 hour, cells will be severely damaged, most of the cells will die, and individual cells may still recover; when the temperature Above 43°C for 1 hour, all cells died.
3. Gas environment
gas is one of the necessary conditions for the survival of human cell culture. The required gases mainly include oxygen and carbon dioxide . Oxygen participates in the tricarboxylic acid cycle , producing energy for cell growth and proliferation and synthesizing various components required for cell growth. During open culture, cells are generally placed in a mixed gas environment of 95% air and 5% carbon dioxide.
Carbon dioxide is both a product of cell metabolism and a component required for cell growth and reproduction. Its main role in cell culture is to maintain the PH value of the culture medium . The suitable pH for most cells is 7.2-7.4. Deviation from this range will have harmful effects on cell culture. However, cells are more acid-resistant than alkaline-resistant, and are more conducive to cell growth in an acidic environment. Some data show that primary amniotic fluid cell culture is optimal when the pH is 6.8.
The most commonly used method to adjust the pH concentration of cell culture fluid is to add NaHCO3, because NaHCO3 can provide CO2, but carbon dioxide easily escapes, so it is most suitable for closed culture, and hydroxyethylpiperazineethanesulfonic acid (HEPES) because of its It is non-toxic to cells and also plays a buffering role. It has the property of preventing rapid changes in pH and is used in open cell culture technology. Its biggest advantage is that it can maintain a relatively constant pH value during open culture or cell observation.
4, cell culture medium
culture medium is not only the basic material that supplies cell nutrition and promotes cell reproduction and proliferation in cultured cells, it is also the living environment for the growth and reproduction of cultured cells. There are many types of culture media, which are divided into two categories: , semi-solid culture media, and liquid culture media according to their material state; they are divided into synthetic culture media and natural culture media according to their sources.
(1) Synthetic medium: The synthetic medium is strictly formulated according to the type and quantity of substances required by the cells. It contains carbohydrates , amino acids , lipids, inorganic salts, vitamins, trace amounts of minerals and Cell growth factors etc. The cells can survive but cannot grow and proliferate well when used alone.
(2) Natural culture medium: The most common natural culture medium is serum , among which calf serum is the most common. Since serum contains a variety of cell growth factors, adhesion-promoting factors and various active substances, combined with synthetic culture media, it can enable cells to proliferate and grow smoothly.
How to do cell passaging specifically?
Before the experiment, the reagents (culture medium, trypsin and PBS) were put into the water bath and heated 30 minutes in advance.
experiments should be performed as sterile as possible. Clean your hands with 75% ethanol and then open the biosafety cabinet . All experimental operations must not leave the biosafety cabinet to facilitate the disconnection of the cells to form a single cell suspension.
Note: Single cell suspension refers to the suspension formed after digesting and pipetting adherent cultured cells.
. Passage when the cell density reaches 80-90%
. Prepare: complete culture medium, trypsin, PBS (separately placed to prevent contamination). Use alcohol lamp to burn at any time. Prepare centrifuge tubes and petri dishes of different sizes
3. Aspirate away the old culture medium, add 2-3ml PBS to rinse 1-3 times, discard PBS
4. Add 1ml 0.25% trypsin along the wall (to interrupt the connections between cells), and adjust the gun Head to 3ml
5. Observe under the microscope. When the intercellular space begins to enlarge, immediately add 3ml of culture medium to terminate the digestion. Single cell suspension, collected in centrifuge tubes.
7. Centrifuge at 900rpm for 5 minutes and resuspend. Discard the supernatant and use a pipette tip to suck up the remaining liquid.
8. Add 1ml of culture medium and pipet 40 times until the liquid level does not come out of the pipette tip.
9. Add 3.65ml of culture medium to the medium dish; glass piece, shake it open, and add 1.5ml of culture medium
0. Add 350 μl of cells in a centrifuge tube to a medium dish, shake it back and forth and left and right; add 100-170 μl
1. label to a small dish, and place it in an incubator for storage.
Note: When the cell gaps become more and more When the gaps are randomly dispersed and uniform in size, add culture medium to stop digestion and form a single-cell suspension, which is collected in a centrifuge tube. Because of the difference in density, cells will eventually settle and adhere to the bottom of the centrifuge tube. Use the pipette to remove the liquid, add culture medium and pipet to form a new single cell suspension, and then form a new generation of cells.
Note: Due to aging and mutation during reproduction, cells are generally only used for 40 generations, and do not shake vigorously within 12 hours after completing the operation to cause new cells to detach from the bottom of the culture dish. A small dish is required for subsequent experiments. At this time, place 7-8 glass coverslips on the bottom of the dish and spread them to avoid overlapping. Add 1.5mL and 175uL of culture medium and cells respectively.
In addition to passage, cells also need to change the culture medium from time to time to clean up suspended dead cells and metabolites.
(2) Recovery
. Liquid nitrogen Take
. Tighten the bottle cap
3. 37℃ water bath, shake vigorously until completely dissolved, avoid touching the bottle cap with water and hands. touch
4. 75% alcohol Fully disinfect the bottle cap
5. Prepare a 10ml centrifuge tube, transfer all cells, about 1ml
6. Add 4ml culture medium
7. Centrifuge
8. Add 4ml
9. For centrifugation, observe the amount of centrifugation. If it is less, don’t use it.
0 . Pour off the culture medium and use a pipette tip to absorb the excess liquid
1. Add 1ml of culture medium from the medium dish into the centrifuge tube, pipe it repeatedly, and add the culture medium
2. Shake the culture dish and culture it in the incubator
(3) Cryopreservation
. pass down generations After centrifugation
. Add 1ml cryopreservation solution and pipet evenly
3. Put it into a freezing bottle
4. Freeze it at -70℃ for 1-2 days
5. Then transfer it to liquid nitrogen tank
in biological cytology research , sometimes it is necessary to culture and analyze cancer cells in vitro. To maintain cells well, it is necessary to use good serum, such as Ausbian® special imported fetal bovine serum , whose endotoxin is less than 3Eu/ml, making the cells healthier and customers' experiments smoother.
The article comes from: Heartinker
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