Cell culture considerations

Raise the attention of cells

(1) How should the freezing tube be thawed?

Immediately after taking out the cryotube, it should be quickly thawed in a 37 °C water tank. Gently shake the cryotube to melt it in 1 minute. Note that the water surface should not exceed the edge of the cryotube cover, otherwise it will be prone to contamination. When the chilled tube is removed from the liquid nitrogen drum, it must be safe to prevent the freezing of the freezing tube.

(2) How to treat DMSO when the cell cryotube is thawed

After the cells are thawed, the DMSO can be directly removed by centrifugation, suspended in a fresh medium, and directly placed in a culture flask containing fresh medium for cell culture, thereby preventing cell growth from being affected by DMSO after thawing.

(3) Whether it is possible to use a medium different from the original culture conditions

Each cell strain has its own specific and adapted cell culture medium. If it is used suddenly and the culture medium originally provided with different culture conditions, the cells are mostly unable to adapt immediately, causing the cells to be unable to survive and should not be replaced immediately.

(4) Whether serum types different from the original culture conditions can be used

Serum is an extremely important source of nutrients in cell culture, so the type and quality of serum can have a significant impact on cell growth. Serum from different species varies in the amount or content of some substances or molecules, and errors in serum use often cause 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. CS (calf serum) refers to calf serum. HS (horseserum) refers to horse serum.

(6) 5% or 10% CO ₂ should be used when culturing cells

Most of the general medium uses HCO ₃ ^- /CO ₃ ^2- /H ⁺ as a buffer system for pH, and the content of NaHCO ₃ in the medium will determine the concentration of CO _{2 that} should be used in cell culture. Theory When the NaHCO ₃ content in the medium is 3.7 g per liter, 10% CO ₂ should be used for cell culture; when NaHCO _{3 in the} medium is 1.5 g per liter, the cells should be cultured with 5% CO ₂ .

(7) When to change the medium. Whether antibiotics must be added to the medium.

Depending on the cell growth density, or according to the replacement time on the basic data of the cell line, the medium can be changed on time.

In normal culture, antibiotics can be added to the medium. The double-antibody stock solution (penicillin + streptomycin) was added at a volume fraction of 1% so that the final concentrations of penicillin and streptomycin were 100 U/mL and 100 μ/mL, respectively.

(8) trypsin-EDTA concentration used in the passage of adherent cells and corresponding treatment

The trypsin-EDTA concentration is generally 0.25% trypsin-0.53mMEDTA.4 Na. Immediately after the first opening of the bottle, it should be dispensed in a small amount in a sterile test tube and stored at –20 °C. Avoid repeated freezing and thawing to reduce the activity of trypsin and reduce the chance of contamination.

(9) Centrifuge the general animal cells, how much is the centrifugation rate?

Animal cells are recovered at a rate of 1 000 rpm, 5 to 10 minutes, and excessive speed will cause cell death.

(10) Cell seeding density

Inoculate according to the seeding density on the basic data of the cell line or the ratio of the dilution plate. Too few cells or too much dilution is one of the important reasons why cells cannot grow.

(11) Composition of cell freezing medium and grade of DMSO

The most commonly used freezing medium for cryopreservation of animal cells is a mixture of 5-10% DMSO (dimethyl sulfoxide) and 90-95% of the original medium for cell growth. Note: Due to the large amount of heat released during DMSO dilution, DMSO should not be added directly to the cell fluid and must be prepared before use. The DMSO grade used for cryopreservation must be Tissue culture grade DMSO, which is itself sterile. Immediately after the first opening of the bottle, it should be dispensed in small quantities in sterile tubes in the dark.

(12) Method for cryopreserving cells and concentration of frozen cells

The adherent cells were digested and collected by centrifugation. The suspension cells were directly collected by centrifugation, and the cells were resuspended in a cryopreservation solution containing DMSO complete medium or fetal bovine serum to a final concentration of about 1×10 ⁶ /ml. . Pack 1 to 2 ml per tube into the cryotube. Wrap in a refrigerator at -70 ° C overnight with a heat-insulating material. Save to liquid nitrogen the next day.

The number of cells in the cryotube is generally 1x10 ⁶ cells/ml vial, and the fusion tumor cells are preferably 5x10 ⁶ cells/ml vial.

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