Specific steps, requirements and precautions of cell culture

After the frozen cells are taken out of liquid nitrogen, they are constantly shaken in a water bath at 37℃ to promote their melting. Transfer to 15ml centrifuge tube, add 10ml preheated complete DMEM medium, gently blow evenly, centrifuge at 2000 rpm for 2 min, and discard the supernatant.

Add 10mlDMEM medium for cleaning, and discard the supernatant. Add 10mlDMEM complete medium, gently blow, inoculate on 10cm plate, and culture in a cell incubator containing 5%CO2.

Second, cell passage

When the cell density reached 80%~90%, the culture medium was taken out and washed twice with 10mlPBS. Add 3ml tryps3min containing 0.25%EDTA for 3 minutes and put it in a cell incubator for 3 minutes. Add 1mlDMEM complete culture medium to stop pancreatin digestion and transfer to 15ml centrifuge tube.

Add 10mlPBS to clean the cell culture plate, transfer to 15ml centrifuge tube, run at 2000rpm for 2min, and discard the supernatant. Add 10mlPBS (autoclaved and stored at 40℃), blow evenly, suck10μ l, inoculate at 1× 106 cells per plate, and continue to cultivate in a cell incubator containing 5%CO2.

Thirdly, cell cryopreservation.

When the cell density reached 80% ~ 90%, the medium was removed and washed twice with 10mlPBS. Add 3ml tryps3min containing 0.25%EDTA for 3 minutes and put it in a cell incubator for 3 minutes. Complete medium lmlDMEM was added to terminate pancreatin digestion and transferred to 15ml centrifuge tube. Add 10mlPBS to clean the cell culture plate, transfer to 15ml centrifuge tube, run at 2000rpm for 2min, and discard the supernatant.

Add lml freezing liquid (90% serum, 10%DMSO), put it in a freezer (isopropanol is contained in the freezer to ensure the cooling speed), put it in a refrigerator at 80℃ immediately, stay overnight, and put it in liquid nitrogen the next day, which can be stored for at least two years, and if the liquid nitrogen is not released, it can be stored for three months.

Preparation of frozen storage solution: 70% complete medium +20% FBS+ 10% DMSO should be slowly added and shaken at the same time.

Matters needing attention

(1) When entering the intercellular space to start cell culture, the following steps must be strictly followed:

① Ensure that all solutions and consumables used for cell operation have been disinfected and tested, and there is no problem. Do not use uncertain solutions and consumables, and do not borrow other people's solutions unless there are special circumstances.

(2) Make sure that the cuffs of clothes have been rolled up or the cuffs of white coats have been fastened.

③ Determine the amount of alcohol in the alcohol lamp and replenish it in time if necessary.

(4) Ensure that all required solutions and consumables are at hand. In order to open the bottle caps with one hand, all the bottle caps can be unscrewed before the experiment begins.

⑤ Unless the bottle mouth is not burnt, try not to pour the solution directly. If the pouring fails and the solution sticks to the bottle mouth, please carefully clean around the bottle mouth with a paper towel sprayed with 75% alcohol (don't touch the bottle mouth), and then simply burn it on the flame.

6. If you are not sure whether the consumables used are clean during the operation, you must replace them in time.

All landowners clean up in time after the experiment, keep the work area clean and tidy, and finally clean the table with 75% alcohol.

(2) Prevent cell pollution

(1) experimental supplies to prevent pollution. The reagents, consumables and equipment used in cell culture should be thoroughly cleaned and disinfected, and all kinds of solutions should be carefully sterilized before use. Operating room and remaining sterile equipment should be cleaned, disinfected and sterilized regularly.

(2) Prevent pollution during operation.

③ Clothes that are easy to generate static electricity or absorb dust must be put on a white coat before entering the cell.

(4) Before the experiment, make sure that there is no problem with the gloves. Gloves must be disinfected in time as long as they come into contact with items outside the biosafety cabinet.

⑤ Close the door after entering the cell culture room, and sit and walk as little as possible, so as not to affect the air curtain of the biosafety cabinet. Wipe your hands and bottle caps with 75% alcohol cotton balls before starting work. Strictly check the equipment, solutions and cells used in advance, and do not bring contaminated or unsterilized items into the sterile room, let alone use them casually, so as not to cause large-scale pollution.

⑥ Cell operation should be light, the bottle mouth must be opened in the sterile area around the flame, and the bottle mouth should be simply rotated and cauterized around the flame. Be careful not to let the flame melt the plastic bottle mouth.

⑦ During the experimental operation, the partition of the biosafety cabinet should be as low as possible, and the conversation should be reduced as much as possible. Never point to the work area when sneezing or coughing to avoid unnecessary pollution.

⑧ Keep the bottle cap away from yourself to avoid being polluted by misoperation.

Pet-name ruby don't pass above the open container mouth, so as to avoid unknown objects falling from clothes and polluting cells.

Attending experimental operation should pay attention to timely replacement of Pasteur pipette, pipette gun tip and pipette, and never go all the way. Once an unclean or uncertain item is found, it must be discarded directly. After the experiment, clean it in time to keep the laboratory clean and tidy, and finally clean the table with 75% alcohol.

(3) to prevent cross-contamination of cells

① When performing various cell culture operations, the instruments used should be strictly distinguished, and it is best to mark them for easy identification. When operating in sequence, only one cell is processed at a time, and multiple cells and operations are carried out together, which is prone to T- chaos.

(2) During liquid exchange or operation, the pipette head and the pipette containing cells should not touch the bottle mouth of reagent bottle, so as to avoid bringing cells into the culture medium and polluting other cells.

(3) Once all cells are purchased, introduced from other places or established by themselves, they must be preserved and frozen in time, and once they are polluted, they can be revived and cultured.

Extended data:

Cell culture is a method to simulate the internal environment (aseptic, suitable temperature, pH and certain nutritional conditions) in vitro, so that it can survive, grow, reproduce and maintain its main structure and function. Cell culture is also called cell cloning technology, and the formal term in biology is cell culture technology.

Cell culture is an indispensable process for the whole bioengineering technology or one of the biological cloning technologies, and cell culture itself is a large-scale cloning of cells. Cell culture technology can transform a cell into a simple single cell or multi-cell with little differentiation after a large number of cultures, which is an essential link of cloning technology, and cell culture itself is cell cloning.

Cell culture technology is an important and commonly used technology in cell biology research methods. Through cell culture, a large number of cells can be obtained to study cell signal transduction, cell anabolism, cell growth and proliferation.

References:

Baidu Encyclopedia: Cell Culture