20 Questions and Answers on Cell Culture

1. How should the freezing tube be defrosted?

        After taking out the freezing tube, immediately put it in a 37°C water tank to thaw it quickly, gently shake the freezing tube to melt it in 1 minute, and pay attention that the water surface should not exceed the edge of the freezing tube lid, otherwise contamination will easily occur. In addition, when the freezing tube is taken out of the liquid nitrogen tank and thawed, safety must be paid to prevent the freezing tube from bursting.

2. When the cell cryotube is thawed and cultured, should the DMSO be removed immediately?

        Except for a few cells that are specifically noted to be sensitive to DMSO, most cell lines (including suspension cells) should be directly put into a culture flask containing 10-15ml of fresh medium after thawing, and then replaced with fresh medium the next day Just remove DMSO, which can avoid most of the problems that cells cannot grow or attach after thawing.

3. Can I use a different culture medium from the original culture condition?

        Can't. Each cell line has its own specific cell culture medium. If you suddenly use a culture medium that is different from the original culture conditions, most of the cells cannot adapt immediately, causing the cells to fail to survive.

4. Is it possible to use serum types that are different from the original culture conditions?

        Can't. Serum is an extremely important source of nutrients for cell culture. 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 causes cells to fail to survive.

5. What is FBS, FCS, CS, HS?

        FBS (fetal bovine serum) and FCS (fetal calf serum) have the same meaning. Both refer to fetal bovine serum. FCS is a wrong term. Please do not use it anymore. CS (calf serum) refers to calf serum. HS (horse serum) refers to horse serum.

6. Should I use 5% or 10% CO2 when culturing cells ? Or no effect at all?

        Most generally used medium HC03 . 3 - / CO.'S . 3 2- / H +  as a pH buffer system, whereas the medium NaHC03 . 3 CO.'S content should be determined in cell culture using 2 concentration. When the content of NaHCO3 in the medium is 3.7 g per liter, 10% CO2 should be used for cell culture ; when the NaHCO3 in the medium is 1.5 g per liter, 5% CO2 should be used to culture the cells.

7. When should the medium be changed?

        Depending on the cell growth density, or follow the replacement time on the basic data of the cell line, just change the medium on time.

8. Is it necessary to add antibiotics to the culture medium?

        Except for special screening systems, under normal culture conditions, no antibiotics should be added to the culture medium.

9. What concentration of trypsin-EDTA should be used when the adherent cells are subcultured? How should it be handled?

        The generally used trypsin-EDTA concentration is 0.05% trypsin-0.53mM EDTA-4Na. After opening the bottle for the first time, aliquot a small amount into a sterile test tube and store it at -20°C to avoid repeated freezing and thawing to reduce the activity of trypsin and reduce the chance of contamination.

10. How should suspension cells be passaged?

        Generally, it is only necessary to continuously add fresh medium to the original culture flask to dilute the cell concentration. If there is too much culture medium, the mouth of the culture bottle can be slightly raised until it cannot be accommodated. When dividing the bottle, take out a part of the cell-containing culture solution to another new culture bottle, add fresh culture medium to dilute it to an appropriate concentration, and repeat the above steps.

11. If you want to centrifuge general animal cells, what is the speed of the centrifugation?

        If you want to recover animal cells, the centrifugation rate is generally 300xg (about 1,000 rpm), 5-10 minutes, too high rotation speed will cause cell death.

12. How to determine the cell seeding density?

        Inoculate according to the inoculation density or dilution ratio on the basic data of the cell line. Too few cells or too much dilution are the reasons why cells cannot grow.

13. What are the ingredients of the cell freezing medium?

        The most commonly used freezing medium for animal cell cryopreservation is a uniform mixture of 5-10% DMSO (dimethyl sulfoxide) and 90-95% of the original cell growth medium. Note: Dilution by DMSO will release a lot of heat energy, so DMSO cannot be added directly to the cell sap, it must be prepared before use.

14. What is the grade of DMSO and the method of sterile filtration?

        The DMSO grade used for cryopreservation must be cell culture grade DMSO (such as Sigma D2650), which itself is sterile. Immediately after opening the bottle for the first time, aliquot a small amount into a sterile test tube and store it at 4°C to avoid repeated freezing and thawing causing the lysis of DMSO and releasing harmful substances, and reducing the chance of contamination. If you want to filter DMSO, you must use a DMSO-resistant nylon filter membrane.

15. How to cryopreserve cells?

        Cryopreservation method 1: Place the cryotube at 4°C for 30-60 minutes (-20°C for 30 minutes), -80°C for 16-18 hours (or overnight), liquid nitrogen can be stored for a long time.

        Cryopreservation method 2: The cryotube is placed in a programmable cooling machine with a programmed program to reduce 1-3°C to below -80°C every minute, and then put in liquid nitrogen for long-term storage. Do not exceed 1 hour at -20°C to prevent the ice crystals from becoming too large and causing a large number of cell deaths. You can also skip this step and put it in the refrigerator at -80°C, but the survival rate will be slightly reduced.

16. When the cells want to be stored frozen, what should be the cell concentration in the cell cryotube?

        The number of cells in the cryotube is generally 1x10 6 cells/ml per tube, and 5x10 6 cells/ml per tube is appropriate for fusion tumor cells .

17. How to avoid cell contamination?

        The types of cell contamination can be divided into bacteria, yeasts, molds, viruses and mycoplasma. The main causes of contamination are improper aseptic operation techniques, poor operating room environment, contaminated serum and contaminated cells. Strict aseptic operation techniques, clean environment, and good-quality cell sources and medium preparation are the best ways to reduce pollution.

18. What should I do if the cells are contaminated by microorganisms?

        Discard after sterilization.

19. Can mycoplasma contaminated cells be observed with the naked eye?

        Can't. Except for highly experienced experts, most cell lines contaminated by mycoplasma cannot be distinguished by their appearance.

20. How does mycoplasma contamination affect cell culture?

        Mycoplasma contamination can affect the growth and metabolism of almost all cells. Therefore, before the experiment, the cells must be confirmed to be mycoplasma-free, so that the data of the experimental results are meaningful.