What is subculture?
Refers to the transfer of cells from a culture bottle to 1:2 or more than 1:2 and inoculated to another culture bottle.
Passage cells can be passaged by different methods according to different cells. Adherent cells by digestion method were part of adherent cells by direct pipetting or silica gel soft scraping scraping. Cell suspension can be used with an equal volume of fresh medium and dispersed directly after passage, or by sedimentation after adding new medium and dispersed passage.
This article mainly introduces the method of subculture of adherent cells.
1. material preparation and experiment procedure
Instrument: Shanghai Kang CO2 incubator, inverted microscope, Shanghai Kang safety cabinet
Materials: Corning cell culture flasks, test tubes, pipettes, buses, straws, waste liquid cylinders, 75% alcohol cotton balls, alcohol lamps, cultured cells.
Reagents: medium RPMI1640 (Corning10-040-CVR), fetal bovine serum, 0.25% trypsin, and Hanks (Corning, 21-020-CVR).
(1) wash your hands with soap before entering the asepsis room, then use 75% alcohol to wipe and disinfect your hands;
(2) the morphology of cells was observed under inverted microscope to determine whether the cells were passaged and the number of cells needed to be diluted. The culture solution is preheated at 37 DEG C.
(3) super clean table should be neat and clean with 75% alcohol cotton ball;
(4) turn on the ultraviolet lamp of the ultra clean worktable, illuminate the mesa 20min or so, turn off the ultraviolet lamp, turn on the fan, clean the air, and remove the ozone;
(5) ignite the alcohol lamp; take out the sterile test tube; the bus straw and the calibration straw; place the rubber head; pass the alcohol lamp; the flame is slightly burned; then inserted in the sterile test tube.
(6) use the 75% alcohol to disinfect the bottle mouth of the culture liquid, and put it on the shelf next to the alcohol lamp after the flame of the alcohol lamp.
(7) discard old culture medium of culture cell. Appropriate medium available 2-3ml Hanks liquid to wash away the residue, or with a small amount of trypsin shuanxi.
(8) each flask add 1ml pancreatin, small bottle dosage reduction, cap were observed under inverted microscope. When the cells retract and become rounded, they flip the culture bottle immediately, leaving the cell free of trypsin, and then pour out the pancreatin. Be careful not to let the cells prematurely fall into the digestive juices.
(9) adding fresh medium containing serum medium small, repeated pipetting good digestive cells to take off the wall and dispersed, according to the distribution of number of bottles up fresh medium containing serum medium with a certain amount of (7-10ml/ bottle; 3-5ml/ vial) into cell suspension, then put into a new culture bottle. Cover the cap tightly and turn slightly after proper tightening so as to facilitate the entry of CO2 and put the culture bottle back into the CO2 incubator.
(10) for suspension cultures, step 7-9 is not done. The cell suspension was centrifuged to remove the supernatant of the old medium. The fresh medium was added and then packed into each bottle.
2. matters needing attention
(1) pay attention to aseptic operation and prevent cross contamination among cells during subculture. All operate as close as possible to the alcohol lamp flame. It is best to perform only one cell operation at a time. Each cell uses a set of equipment.
(2) observe the morphology of cells every day and master the standard of whether the cells are healthy: the shape of healthy cells is full, the refractive index is good, and the passage can be passaged when the density is tight.
(3) if the cells are discovered signs of pollution, should take immediate measures, should generally be disposal of contaminated cells, if must be saved, and the BBS culture medium containing antibiotics or repeated washing, and then added to the culture medium is a large number of antibiotics, and often replace the medium.
3. Establishment and maintenance of passage cells
The maintenance of cell line was realized by exchanging liquid, passage, changing fluid, re - passage and cell seed cryopreservation.
For each cell line, it has its own characteristics. In order to maintain the system well, it is necessary to record the cell records, change the passages, and identify and manage the cell lines (or strains).