Cell culture refers to a method that simulates the internal environment (sterile, suitable temperature, pH, and certain nutritional conditions, etc.) in vitro to make it survive, grow, reproduce and maintain its main structure and function. Aseptic operation is important to cell culture. Aseptic operation is the key to the success of cell culture. All non-disposable items placed in the ultra-clean table need to be autoclaved in advance; items that cannot be autoclaved also need to be sterilized with 75% ethanol. Including alcohol lamps, straws, pipettes, test tube racks, petri dishes, waste tanks, etc.
1. The pH of the culture medium changes too fast
1.1 Reason
1). The CO2 tension is wrong
2). The cap of the culture bottle is too tight
3). NaHCO3 buffer system has insufficient buffering power
4). The salt concentration in the culture medium is incorrect
5). Bacteria, yeast or fungus contamination
1.2 Solution
1). Increase or decrease the CO2 concentration in the incubator according to the concentration of NaHCO3 in the culture solution. The concentration of NaHCO3 from 2.0 g/L to 3.7 g/L corresponds to a CO2 concentration of 5% to 10%. Or switch to non-CO2 medium
2). Loosen the cap 1/4 turn
3). Add HEPES buffer to the final concentration of 10 to 25 mM
4). Switch to a culture solution prepared based on Earle’s salt in a CO2 culture environment, and switch to a culture solution prepared based on Hank’s salt in an atmospheric culture environment
5). Discard the culture or sterilize with antibiotics
2. The culture solution has precipitated, and the pH value does not change
2.1 Reason
1). After washing with detergent, the residual phosphate will precipitate the medium components
2). Freeze preservation of culture medium
2.2 Solution
1). Rinse glassware repeatedly with deionized water, and then sterilize
2). Heat the culture solution to 37°C, shake to dissolve it if the precipitate still exists, discard the culture solution
3. The culture solution has precipitated and the pH changes at the same time
3.1 Reason
Bacterial or fungal contamination
3.2 Solution
Discard the culture or sterilize it with antibiotics
4. Cultured cells do not adhere to the wall
4.1 Reason
1). Excessive trypsin digestion;
2). Mycoplasma pollution;
3). There is no adhesion factor in the culture medium.
4.2 Solution
1). Shorten the trypsin digestion time or reduce the trypsin concentration.
2). Isolate the culture and detect mycoplasma. Clean the stand and incubator. If contamination with mycoplasma is found, discard the culture.
5. Clusters of suspension cells
5.1 Reason
1). The culture medium contains calcium and magnesium ions;
2). Mycoplasma pollution;
3). Excessive protease digestion causes cell lysis to release DNA.
5.2 Solution
1). Wash the cells with calcium-magnesium-free balanced salt solution, and gently pipette the cells to obtain a single cell suspension.
2). Isolate the culture and detect mycoplasma. If contamination with mycoplasma is found, discard the culture.
3). Treat the cells with DNase I.
6. Cultured cell death and abnormal growth
6.1 Causes of cell death
1). No CO2 in the incubator;
2). The temperature in the incubator fluctuates too much;
3). Cells are damaged during cryopreservation or resuscitation;
4). The osmotic pressure of the culture solution is incorrect;
5). Accumulation of toxic metabolites in the culture medium.
6.2 Solution
1). Detect CO2 in the incubator;
2). Check the temperature in the incubator;
3). Take new preserved cell species;
4). Detect the osmotic pressure of the culture medium. Note: Most mammalian cells can tolerate an osmotic pressure of 260-350 mOsm/kg;
5). Adding additional reagents such as HEPES or drugs, which may affect the osmotic pressure of the culture medium;
6). Replace with fresh culture medium.
6.3 Reasons for slowed growth of cultured cells
1). After replacing the serum or culture medium, the cells need a period of time to adapt;
2). Due to the special types of cells, some growth factors are lacking in the culture medium;
3). There is a small amount of fungus or bacteria contamination in the culture medium cell contamination
4). The cell density is too low during passaging;
5). The cell state is aging; cell aging
6. Mycoplasma pollution. Mycoplasma contamination
6.4 Solution
1). If the laboratory does not have the most suitable medium for cells, you can freeze some of the original culture conditions first, and then gradually increase the proportion of the new medium, 25%, 50%, 75% to 100%, after cell subculture 3-5 times, let the cells adapt slowly.
2). To judge whether the culture medium is contaminated, you can observe the cell status without adding antibiotics.
3). Increase the seeding density of cells.
4). When the cells are found to be aging, replace with new cells in time.
5). If it is suspected to be mycoplasma contamination, be sure to isolate the suspected contaminated petri dish and clean and disinfect the incubator in time.
7. How to determine whether a cell is contaminated by mycoplasma?
Can choose direct culture method, fluorescence staining or PCR method to detect, the more commonly used is PCR. When the cells are contaminated by mycoplasma, in principle, the mycoplasma can be removed, but the whole process is time-consuming and labor-intensive, and it also tests the personal operation ability. Therefore, if it is not in a situation where the cell stock is very small, it is recommended to discard it immediately and re-cultivation when it is found to be contaminated.
8. What is the best way to preserve serum?
It is recommended that the serum should be stored at -5°C to -20°C. However, if stored at 4°C, do not exceed one month. If it is not possible to use up one bottle at a time, it is recommended to aseptically distribute the serum into an appropriate sterilized container, and then put it back into the freezer.
9. How to thaw serum without impairing its quality?
It is recommended that after taking the serum out of the freezer, put it in a refrigerator at 2～8℃ to melt it, and then melt it completely at room temperature. But it must be noted that during the melting process, it must be shaken regularly and evenly.
10. What should I do if there is flocculent sediment after the serum is thawed?
There are many reasons for the appearance of sediment in the serum, but the most common cause is the denaturation of lipoproteins in the serum, and fibrin (one of the proteins that form clotting) will also exist in the serum after the serum is thawed. Is also one of the main causes of deposits. But these flocculent sediments do not affect the quality of the serum itself. If you want to remove these flocculent sediments, you can aliquot the serum into a sterile centrifuge tube, centrifuge it slightly at 400 g, and then add the supernatant to the culture medium and filter it together. We do not recommend that you use filtration to remove these flocculent sediments, because it may clog your filter membrane.
11. How to control the trypsin digestion time?
The degree of trypsin digestion is a key step in cell culture: excessively digested cell debris increases, black scum increases, cells will fall off in pieces, which seriously affects cell viability, and some cells float and lose with the discarded trypsin; digestion Insufficient, it is difficult for cells to blow down from the bottle wall, and repeated pipetting will also damage cell viability.
The sensitivity of different cells to the digestion fluid is different, and the time of trypsin digestion will also be different. Trypsin digestion time is related to the concentration of pancreatin, whether it contains EDTA, the storage time of pancreatin, the storage temperature of pancreatin, whether it is repeatedly freeze-thaw, the volume of pancreatin added in digestion, the digestion temperature and the density of cells. Digestion For newly purchased cells, it is recommended that customers use a low concentration of pancreatin to carefully explore the digestion time. You can observe whether the cells are rounded every 1 minute and record the best digestion time.
12. Why add EDTA to the trypsin?
EDTA is used to chelate free magnesium ions and calcium ions in order to maintain the activity of inhibiting trypsin. It is recommended to wash the cells with EDTA before trypsinizing the cells to eliminate all divalent ions from the culture medium.
13. Is it possible to use a different type of serum from the original?
Serum is an extremely important source of nutrients for cell culture, so the type and quality of serum will have a great impact on cell growth. Serum from different species differs in the amount or content of some substances or molecules, and incorrect use of serum often results in the inability of cells to survive.
14. How to keep the water tray of the CO2 incubator clean?
Change the water in the water tray regularly (once a week). The water in the water tray must be sterile distilled water or sterile deionized water. 1% copper sulfate can be added to the water tray to prevent mold contamination.
After the CO2 incubator is contaminated by mold, all cells can be temporarily transferred, and the incubator (including the partition and the wall) can be scrubbed with peracetic acid. And put the peracetic acid in the incubator for one hour to diffuse the steam. After the smell of peracetic acid has dissipated, move it into the cells.
15. Some black spots often appear during cultivation. Is it pollution? How to prevent and deal with it?
15.1 Judgment method
Firstly, observe whether the culture medium becomes turbid by naked eyes. If it becomes turbid, it can basically be determined as contamination; if the culture medium does not become turbid by naked eyes observed: observe under the microscope whether the size and shape of the black spots are regular, whether they are moving, whether it is Brownian motion or fast moving through the straight line. If the size of the black dots is irregular, perform Brownian motion, the black dots may be cell debris (it may be caused by poor cell condition or over-digestion), protein precipitation caused by repeated freezing and thawing of serum, or cellular metabolite. If the black spots are the same size and move quickly, it is likely to be bacterial contamination.
15.2 Preventive measures
1). Grasp the best time for cell passage, and don’t pass the cell when it grows old;
2). Master the digestion time to prevent cell debris from being over-digested;
3). Reduce the freezing and thawing times of serum and other reagents; adjust the pH of the culture medium to the best;
4). Strictly control the water quality and the cleaning of utensils.
15.3 Treatment method
If it is determined that the black spots are pollution, please dispose of the cells in time and discard them. In other cases, please refer to the following:
If it is a suspension cell: Collect the cell supernatant by slow centrifugation (500-600 rpm/min, 5-6 min) and replace with a new culture flask;
If the cells are adherent cells: Wash the cells 2-3 times with PBS. When washing, gently tap the culture flask to remove the fragments and particles that are not firmly attached. Then discard the PBS and add low-concentration pancreas during digestion. Enzymes, such as 0.05% trypsin, are digested for about 1 minute to allow particles and debris in the intercellular space to fall off, remove low-concentration trypsin, and then digest the cells normally. Centrifuge the collected cell suspension at a slow speed (500-600rpm/min, 5 -6 min) and replace the culture flask with a new one, and try to increase the serum concentration appropriately for culture.
16. How to distinguish live cells from dead cells? How to count live cells with trypan blue?
Observation under the microscope: the living cells are bright, full and shiny, and the dead cells are darker. Cell viability can also be calculated by staining with trypan blue.
Dilute the cell suspension to 200-2000 cells/ml with serum-free medium, and add 0.1 ml of 0.4% trypan blue solution to 0.1 ml of cell suspension. Mix gently and count the cells with a hemocytometer. Live cells reject trypan blue, so the cells that were stained blue are dead cells. Note that the count should be completed within 10 minutes of adding trypan blue. If there is a cell counter, it can be performed directly with a counter.