Some common problems in animal cell / tissue culture
Due to too many influencing factors, some problems in cell / tissue culture are often difficult to analyze and solve, so they are dubbed "black art" or "witchcraft". This article lists some common problems and solutions in animal cell / tissue culture.
1. Storage of culture reagents:
Serum: -5oC--20oC, avoid repeated freezing and thawing.
Media: 2oC – 8oC, protected from light.
Complete medium: 2oC – 8oC, protected from light and used up within 2 weeks.
Try to shorten the time that serum and medium are exposed to light.
2. How to use glutamine in liquid medium:
Many cells require the addition of glutamine to the growth medium. Glutamine is very unstable in solution and should be stored frozen at -20 ° C and added to the medium before use. When the liquid medium with glutamine is stored at 4 ° C for more than two weeks in the refrigerator, the original amount of glutamine should be added again. Another option is to use L-glutamine derivatives-L-alanyl-L-glutamine dipeptide (such as one of the ingredients contained in the Glutaplex formulation of GenDepot's product represented by Qiwei Yicheng) instead of L-glutamine . Compared with L-glutamine, Glutaplex is more stable, slower degradation, improve cell survival and growth, and greatly reduce the accumulation of ammonia that is toxic to cells.
3. Whether phenol red should be added to the medium:
Phenol red is used as a pH indicator in the medium. It is red when neutral, yellow when acid, and purple when it is alkaline. Studies have shown that phenol red can mimic the effects of steroid hormones, especially estrogen. To avoid sterol reactions, when culturing cells, especially mammalian cells, a medium without phenol red should be used. Because phenol red interferes with the detection, some researchers do not use phenol red-added medium when doing flow cytometry.
4. Issues to be noted when using serum:
1) Before the serum is used for the first time, should the serum be heated at 56oC for 30 minutes to inactivate the complement system?
Activated complement participates in lysis of cells, stimulates smooth muscle contraction, cells and platelets release histamine, and activates lymphocytes and macrophages. When conducting immunological research, culturing ES cells, insect cells and smooth muscle cells, it is recommended to use heat-inactivated serum. Experiments have shown that heat-inactivated serum that is properly processed is unnecessary for most cells. After this treatment, the serum has little or no effect on cell growth, even if the high temperature treatment affects the quality of the serum, and the cell growth rate is reduced. The formation of heat-treated serum precipitates will increase significantly. These precipitates look like "small black spots" under an inverted microscope, which often makes researchers mistakenly think that the serum is contaminated. Putting the serum in a 37 ° C environment will increase this precipitate, which makes the researchers mistakenly believe that it is the division and expansion of microorganisms. If not necessary, this step of heat treatment may not be necessary. Not only save time, but also ensure the quality of serum.
2) The type of serum commonly used:
Newborn Bovine Serum: The blood of new born cattle is made within 5 days. Calf serum: blood is collected within 16 weeks of birth. Fetal Bovine Serum: (Imported Serum) is made by laparotomy. Domestic manufacturers often fail to do so, and often call blood collection within 2 days of birth as fetal bovine serum. According to the production process of serum and the content of hemoglobin and endotoxin, it is further divided into: (1) Special grade fetal bovine serum: 40 nanometers filtered, endotoxin content ≤10EU, hemoglobin content ≤10mg / dl. (2) Superior fetal bovine serum: After three 100-nanometer filtrations, the endotoxin content ≤25EU / ml and the hemoglobin content ≤25mg / ml. (3) Standard fetal bovine serum: 3 times 100-nanometer filtration, low endotoxin and low hemoglobin content.
3) Why does the fetal bovine serum stored in the refrigerator precipitate?
Some fetal bovine serum products are not pre-aged. When stored at 2-8 ° C, various proteins and lipoproteins in the serum, such as cold agglutinin, fibrinogen, and vitronectin, may aggregate to form a precipitate or visible turbidity. This should not affect the quality of the serum. It is recommended to store fetal bovine serum at -20 ° C to avoid repeated freezing and thawing.
4) After the serum is thawed, it is found that there are flocculent precipitates:
There are many reasons for the appearance of precipitates in serum, but the most common cause is due to the denaturation of lipoproteins in serum. Fibrin (one of the proteins that form blood coagulation) will also be present in the serum after it is thawed, which is also one of the reasons for the precipitate. But these flocculent precipitates do not affect the quality of the serum itself. To remove these flocculent precipitates, the serum can be divided into sterile centrifuge tubes and centrifuged at 400g, and the supernatant can be added to the medium and filtered together. It is best not to use filtration to remove these flocculent sediments, as it may clog the filter membrane.
5) How to avoid the generation of sediment?
We recommend that you pay attention to the following operations when using serum: (1) When thawing serum, please follow the recommended stepwise thawing method (-2 ° C to 4 ° C overnight, 37oC water bath thawing). If the temperature changed when the serum is thawed is too large (such as -20 ℃ to 37 ℃), it is very easy to produce precipitates. (2) When thawing the serum, shake it evenly at any time to make the temperature and composition uniform and reduce the occurrence of precipitation. (3) Do not leave the serum at 37 ℃ for too long. If left at 37 ° C for too long, the serum will become cloudy, and many less stable components in the serum will be damaged as a result, affecting the quality of the serum. (4) If it is melted in a water bath higher than 40oC and shaken occasionally to mix well, it is very easy to cause an increase in sediment. Similarly, heat inactivation of serum also causes increased precipitation. If not necessary, heat inactivation can be omitted. (5) If it is necessary to perform heat inactivation of serum, please observe the principle of 56 ° C for 30 minutes, and shake it evenly at any time. If the temperature is too high, the time is too long or the shaking is uneven, it will cause an increase in sediment.
5. The effect of pH of culture solution on cell growth:
Since the most suitable pH for cell culture is 7.2-7.4, deviation from this range will have a harmful effect on the cells. The pH requirements of various cells are not exactly the same. Primary cultured cells generally have poor tolerance to pH changes, and infinite cell lines have strong tolerance. But in general, cells are more resistant to acid than alkaline, and it is more conducive to cell growth in an acid environment. Therefore, when preparing the culture liquid, the pH of the liquid can be adjusted to be slightly acidic. When the liquid is filtered through a 0.10um or 0.22um filter membrane, the pH of the solution will also float upwards by about 0.2.
6. The pH of the medium changes rapidly:
The CO2 concentration in the incubator is incorrect. When the concentration range of sodium bicarbonate in the culture medium is 2.0-3.7g / L, the concentration range of CO2 should be 5%-10%, respectively.
7. Problems that should be paid attention to when using medium:
The medium needs to be preheated at 37oC before use. During the cell culture process, the pipette that has drawn the culture fluid can no longer be burned with flames to prevent the medium remaining in the pipette from scorching and bringing harmful substances into the culture fluid. Try to shorten the exposure time of various liquids and cells to reduce the chance of contamination. Avoid cross contamination between liquids and cells. When preparing a complete medium, it is best to use up the amount within 2 weeks.
8. The problem of using trypsin during the cell digestion:
The digestibility of the trypsin solution of the cultured cells is related to the pH, temperature, trypsin concentration of the solution and whether the solution contains Ca ++, Mg + ions and serum. Under normal circumstances, pH 8.0 and temperature 37 oC have the strongest effect. In addition, calcium, magnesium ions and serum can greatly reduce their digestive capacity. Before each passage of digestion, be sure to repeatedly rinse the cell culture flask with calcium-magnesium-free PBS solution or D-Hanks solution in order to rinse the serum-containing medium, which can greatly improve the digestive capacity of the digestive juice. The commonly used digestive juice concentration is: (1) 0.05% trypsin-EDTA; (2) 0.25% trypsin-EDTA. Most cells can be digested with 0.05% trypsin-EDTA.
After opening the bottle for the first time, it should be divided into sterile tubes in small quantities and stored at –20 ° C, to avoid repeated freezing and thawing to reduce trypsin activity and reduce the chance of contamination.
Note: If you choose a serum-free medium (such as the product made by TNC Bio, an agent of Qiwei Yicheng Company-a serum substitute that is completely free of animal ingredients, XerumFreeTM is mixed with other culture medium), during cell passage Do not use trypsin digestion, because trypsin can remove the receptor on the surface of the cell membrane in the absence of serum. In this case, AccutaseTM should be used for digestion.
9. Adherent cells are not easy to separate when digested with protease:
1) Inhibitors of enzymes are present in the culture broth, wash them with Dulbecco's PBS or pancreatin-EDTA pre-heated at 37oC without calcium and magnesium ions, and then digest
2) The concentration of pancreatin is too low, use higher concentration of pancreatin, higher concentration of EDTA or different enzyme digestion, 37oC digestion to improve enzyme activity
3) The cells are in the confluent state for too long, forming a tight connection between the cells, and pay attention to subculture before the cells are confluent.
10. The cells form clumps after separation:
1) The process of cell dispersion is too excessive, such as vigorous pipetting, excessive enzyme concentration, long digestion time or high temperature, the enzyme solution is toxic (such as buffer pH or osmotic pressure is incorrect, etc.), so that the genomic DNA is broken Released. The solution is to add a drop of sterile 1mg / ml DNase dissolved in water to the cell suspension.
2) When removing the supernatant, the intensity of cell centrifugation is too large or the time is too long. 125g should be used and centrifuged lightly for 10 minutes.
11. Suspended cells form clumps:
1) The culture medium contains calcium and magnesium ions
2) Mycoplasma pollution
3) Cells are lysed due to excessive digestion, releasing genomic DNA
12. The cell does not adhere to the wall:
1) The digestion time is too long, the adherent protein on the cell membrane is removed
2) Insufficient serum or adherent factors in the culture medium (commonly found in serum-free media) is insufficient, you can add adherent factors to the culture medium or use collagen, poly-L-lysine, fibronectin, etc. coated culture bottle.
3) Mycoplasma pollution
13. Centrifugal rate of animal cells:
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