Experimental principle:
The principle of cryopreservation and resuscitation: slow freezing and rapid melting.
When the cells are cooled below zero, the following changes can occur: dehydration of the organelles, an increase in the concentration of soluble substances in the cells, and formation of ice crystals within the cells. If it is slowly frozen, the cells can be gradually dehydrated, and large ice crystals will not be produced in the cells; on the contrary, the crystals will be large, and large crystals will cause damage and rupture of the cell membrane and the organelle. The recovery process should be fast-melting, in order to prevent small ice crystals from forming large ice crystals, that is, recrystallization of ice crystals. Resuscitation cells should be rapidly melted to ensure that extracellular crystallization is melted in a short period of time, avoiding the infiltration of water into cells to form intracellular recrystallization due to slow melting.
Experimental Materials
15ml centrifuge tube, culture dish, dropper, alcohol lamp, culture flask, culture solution, PBS, 75% alcohol, 0.25% trypsin, ultra clean bench, carbon dioxide incubator, inverted microscope, microscope, counting plate, centrifuge, Constant temperature water bath, refrigerator (4 ° C, -20 ° C, -70 ° C), liquid nitrogen tank, cryotube, cryopreservation, waste tank, etc.
Experimental steps:
First, cell cryopreservation
1. Pipette the cell suspension after passage, centrifuge, remove the culture solution, add the frozen solution, and dispense the frozen tube (the number of cells in the cryotube is generally (5 ~ 10) × 106 / ml, 2ml frozen tube Usually put 1 ~ 1.5ml cells).
2. Freeze by step
Cryopreservation method 1: The standard cryopreservation procedure is a cooling rate of -1 to -2 °C/min; when the temperature is below -25 °C, it can be increased to -5 to -10 °C / min; to -100 °C, then It can be quickly immersed in liquid nitrogen.
Cryopreservation method 2: The cryotube is placed in a programmed cooling machine with a temperature of 1 to 3 ° C to -80 ° C per minute, and then stored in liquid nitrogen for a long time.
Second, cell recovery
1. Remove the cryotube and immediately thaw it in a 37 ° C water bath. Gently shake the cryotube to melt it in 1 minute and transfer it into the aseptic table.
2. Open the cryotube and pipette the cell suspension into the centrifuge tube.
Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.
4. After adding the appropriate culture solution, the cells were transferred to a culture flask, cultured at 37 ° C, and the growth was observed the next day.
Precautions
1. After sucking the nutrient solution, the straw can no longer be burned with flame. Because the nutrient solution remaining in the pipette head can be charred to form a carbon film, when it is used, the harmful substances will be brought into the nutrient solution.
2. Do not touch the mouth of the sterilized vessel, the inside of the stopper, the front part of the straw, etc., which may not be in contact with the cells. If it has been touched, use flame to burn or replace the spare parts.
3. When the culture bottle with cells is opened or closed, the flame sterilization time should be short. Prevent cells from burning out due to excessive temperature.
4. Dump the waste liquid when changing the liquid. The bottle mouth should not touch the waste liquid tank. The speed should not be too fast to prevent the waste liquid from splashing.
5. When blowing the cells, pay attention to whether the cells in the corners are blown down.
6. Hand or relatively dirty items can pass through the open bottle opening, ie it cannot be operated above the open container.
7. Only one cell is treated per operation to avoid cross-contamination of cells.
8. Pay attention to your own safety and be especially careful about cell lines from human or viral infections. During the operation, avoid the generation of aerosols, beware of toxic reagents such as DMSO, and avoid sharp objects from injuring people.
9. The cultured cells from the proliferative phase to the formation of dense monolayer cells can be used for cryopreservation, but preferably logarithmic growth phase cells. It is best to change the culture solution one day before cryopreservation.
10. When placing the frozen tube in or out of the liquid nitrogen container, it is necessary to do a good job of protection against frostbite.
11. It is best to use freshly prepared culture fluid for cryopreservation and resuscitation.
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