1. Cell recovery
After the cryopreserved cells were removed from the liquid nitrogen, they were thawed with constant shaking in a 37°C water bath. Transfer the thawed cells into a centrifuge tube, add pre-warmed DMEM complete medium at 37°C (of which fetal bovine serum is about 10%), gently blow evenly, centrifuge at 500 g for 2 min, and discard the supernatant.
Add DMEM complete medium to wash and discard the supernatant. Add DMEM complete medium, mix gently by pipetting, make a cell suspension, inoculate it in a petri dish/flask, and culture in a cell incubator containing 5% CO2.
2. Cell passage
When the cell density reaches 80%~90% (premature yield is insufficient, too late cells are in poor condition, subculture at a ratio of 1:2 to 1:10 or more, generally 1:3 to 1:5 cell generation, that is, from cell inoculation At the time of separation and re-cultivation, the complete medium was removed and washed twice with 1X PBS.
Add trypsin (note: the amount of digestion solution is best to cover the cells, and the optimal digestion temperature is 37°C. Observe the cells under a microscope: observe the digested cells under an inverted microscope, if the cytoplasm is retracted, the cells are no longer connected together. Slices, indicating that the cells are properly digested at this time), digest them, and place them in a cell incubator for about 2-3 minutes.
Add an appropriate amount of DMEM complete medium to stop trypsinization, transfer to a centrifuge tube and centrifuge at 500 g for 2 min, discard the supernatant, add DMEM complete medium to wash, and discard the supernatant.
Add DMEM complete medium, mix by gently pipetting, pipette 10 microliters for counting, and then continue to culture in a cell incubator containing 5% CO2 according to the required cell volume.
3. Cell cryopreservation
When the cell density reaches 80%~90%, remove the complete medium and wash 2 times with 1X PBS. Add trypsin for digestion and place in a cell incubator for about 2-3 min. Add DMEM complete medium to stop trypsin digestion, transfer to a centrifuge tube and centrifuge at 500 g for 2 min, discard the supernatant, add DMEM complete medium to wash, and discard the supernatant. Add 1ml of freezing solution (90% fetal bovine serum, 10% DMSO. Generally speaking, the serum content can be adjusted between 10% and 90%. Adding serum to the freezing solution can provide nutrients for cells on the one hand, and can Provide non-permeable protective substances during cell cryopreservation, such as sucrose, albumin, etc. to better protect cells), put it into a cryopreservation tube (there is isopropyl alcohol in the tube to ensure the speed of temperature reduction), put it immediately Freeze in a 4°C refrigerator for 30 minutes, then place it in a -20°C refrigerator for 30 minutes, and then place it in a -80°C refrigerator overnight.
Put it in liquid nitrogen the next day, it can be stored for at least two years, and if you don't put it in liquid nitrogen, it can be stored for three months.
The principle of cell cryopreservation and recovery is: slow freezing and thawing, which is more conducive to maintaining cell viability. Cryopreservation of cells without any protective agent will lead to the production of intracellular ice crystals, which will cause endogenous mechanical damage to cells, cause changes in intracellular environment osmotic pressure, pH, electrolytes, etc., and then promote cell death.
4. Matters needing attention
(1) Preheating the culture solution: put the prepared bottle containing the culture solution, PBS and trypsin into a 37°C water bath to preheat;
(2) Use 75% alcohol to wipe the ultra-clean workbench and hands that have been irradiated with ultraviolet rays;
(3) Correct placement of used instruments: ensure sufficient operating space, which is not only easy to operate but also reduces pollution;
(4) Light the alcohol lamp: note that the flame should not be too small;
(5) Strict aseptic operation;
(6) Moderate digestion of adherent cells: The digestion time is affected by many factors such as the type of digestion solution, preparation time, and the amount added to the culture flask. During the digestion process, attention should be paid to the changes in the shape of the cultured cells. Once the cytoplasm shrinks, If the connection becomes loose, or there are signs of floating in pieces, the digestion should be terminated immediately;
(7) All operations on passaged cells should be as close to the flame of an alcohol lamp as possible. It is best to work with only one cell at a time, using one set of equipment for each cell. avoid cross infection;
(8) The bottle mouth of the passaged cell needs to be sterilized on an alcohol lamp every time it is opened or closed.
