Steps and precautions of cell resuscitation

1. Conventional cell culture instruments and equipment

Constant temperature water bath oscillator.

2. Operating procedures of medium (DMEM), fetal bovine serum (CBS) and dimethyl sulfoxide (DMSO)

1. Cells were routinely disinfected in the laboratory and irradiated with ultraviolet rays for more than 40min.

2. Preheat the culture medium (DMEM) and 0.25% trypsin in a constant temperature water bath at 37℃ for 20 minutes.

3. Dimethyl sulfoxide (DMSO) was refrigerated at 40℃ for 30 minutes.

4. Take out the freezing tube from the liquid nitrogen storage tank, immediately put it in a water bath at 400℃ and shake it quickly until the freezing solution is completely melted.

5. Transfer the cell suspension to 15ml centrifuge tube, slowly add 4ml culture medium and centrifuge (1000r/min, 5min).

6. Suspend the precipitated cells with culture solution suspension, adjust the cell concentration, and cultivate them in a square incubator.

7. Record the recovery date. Matters needing attention

1. Pay attention to wear antifreeze gloves and goggles when taking cells. This project is particularly important. Liquid nitrogen may leak into the cell cryopreservation tube, and the temperature in the cryopreservation tube will rise sharply during thawing, which may lead to explosion.

2. The problem of cryopreservation solution: The configuration of cryopreservation solution is common sense, so I won't repeat it here, but dimethyl sulfoxide (DMSO) is not completely non-toxic to cells. At room temperature, dimethyl sulfoxide is toxic to cells.

It is relatively large, so the frozen liquid must be completely melted within 1-2 minutes. Too slow resuscitation temperature will cause cell damage, so dimethyl sulfoxide (DMSO) should be injected.

3. A small amount of culture solution must be added before centrifugation. After thawing, the concentration of dimethyl sulfoxide is high, which can be diluted by adding a small amount of culture solution to reduce the damage to cells.

4. Centrifugal problem: There are two main opinions at present. One is that the thawed cell suspension is directly blown evenly and then put into a culture bottle for culture, and the liquid is changed the next day. Because there are two purposes of centrifugation, removal

DMSO, removing dead cells, is a standard process, but for ordinary people, if you don't grasp the speed and time of centrifugation, if you don't rotate enough, the living cells will be thrown away, and if you turn around, the living cells will bear too much pressure.

Death. In addition, it is easy to pollute during operation and is not recommended. Another view is that cell suspension contains dimethyl sulfoxide (DMSO), which has certain toxic and side effects on cells, so it is necessary to centrifuge the liquid.

Clean the front, be sure to clean it. In my experiment, I resuscitated according to the conventional centrifugal subpackaging method, and the results were normal.

5. The problem of less cell adhesion: The textbook states that 10 ml- 15 m cell fluid should be added with 1m culture medium when frozen cells are thawed, but my experimental experience is that the less culture medium, the easier it is for cells to adhere to the wall.

6. Repackaging of cells after resuscitation: My experience in the experiment shows that 1 tubular cells after resuscitation can generally be repackaged in 1-2 culture bottles. Too much repackaging and low cell concentration are not conducive to cell adhesion.

7. The amount of medium added: This amount is mainly related to the concentration of DMSO, because if you add too little medium, the concentration of DMSO will be larger, which will have an impact.

According to the previous data, the concentration of DMSO below 0.5% has no effect on ordinary cells, and another saying is 1%. So if the concentration of your frozen solution is

10% DMSO, and then add more than 10ml of culture medium, just diluted to harmless concentration.