Cell cryopreservation and resuscitation experiments - Database & Sql Blog Articles

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Cell cryopreservation and resuscitation can be: (1) for biological conservation; (2) for medical stem cell research; (3) for subculture.

The basic principle of cell cryopreservation and resuscitation is slow freezing and rapid melting, which has been shown to maximize cell viability. At present, the cell cryopreservation uses glycerol or dimethyl sulfoxide as a protective agent. These two substances can improve the permeability of the cell membrane to water, and the slow freezing can cause the water inside the cell to ooze out of the cell and reduce the intracellular ice crystal. Formation, thereby reducing cell damage caused by ice crystal formation. Resuscitation cells should be rapidly melted to ensure that extracellular crystallization melts in a short period of time.
Avoid infiltration of water into cells by slow thawing to form intracellular recrystallization to damage cells.

Principle of experimental method

cell D-Hanks liquid calf serum culture solution penicillin streptomycin trypsin HClNaHCO3 DMSO glycerol Micro sample gun tip centrifuge tube cryotube tube glue plug pipette glass bottle red blood cell count plate marker pen medical adhesive paste pipette ultra clean table centrifuge thermostat water bath refrigerator inverted phase contrast microscope incubator liquid nitrogen refrigerator First, material preparation

1. Instrument: Purification workbench, centrifuge, constant temperature water bath, refrigerator (4 ° C, -20 ° C, -70 ° C), inverted phase contrast microscope, incubator, liquid nitrogen refrigerator. 2. Glassware: straw (elbow, straight), culture bottle, glass bottle (250ml, 100ml), waste tank. 3. Plastic utensils: tip, tip, rubber stopper, pipette (10ml), 15ml centrifuge tube, cryotube (1~2ml). 4. Other items: micro-sample gun, red blood cell counting board, marker pen, medical adhesive plaster, pipetting gun. 5. Reagents: D-Hanks solution, calf serum, culture medium, double antibody (penicillin, streptomycin), trypsin (0.08%), 1NHCl, 7.4% NaHCO3, DMSO (analytical grade) or colorless fresh glycerol.

2. Cryopreservation of cells 1. Prepare a frozen culture medium containing 10% DMSO or glycerol and 10-20% calf serum. 2. Take cells in the logarithmic growth phase, digest the cells grown in the monolayer with trypsin, and directly grow the cells into a 15 ml centrifuge tube. 3. Centrifuge at 1 000 rpm for 5 min. 4. Remove trypsin and the old culture solution, add appropriate amount of frozen culture medium, gently pipette with a pipette to make the cells uniform, count, and adjust the final density of cells in the frozen solution to 5×10 ⁶ /ml～1 ×10 ⁷ /ml. 5. Dispense the cells into a cryotube, 1 to 1.5 ml per tube. 6. Mark the cell name, freeze time and operator on the cryotube. 7. Freezing: The standard freezing procedure is the cooling rate of -1 to -2 °C / min; when the temperature is below -25 °C, it can be increased to -5 °C ~ -10 °C / min; to -100 °C, It can be quickly immersed in liquid nitrogen. The cell-filled cryotube can also be placed in a -20 ° C refrigerator for 2 h, then placed in a -70 ° C refrigerator overnight, and the cryotube is removed and transferred to a liquid nitrogen container. Third, cell recovery 1. Remove the cryotube from the liquid nitrogen container, directly immersed in warm water at 37 ° C, and occasionally shake it to melt as soon as possible. 2. Remove the cryotube from the 37 ° C water bath, open the lid, aspirate the cell suspension with a pipette, add to the centrifuge tube and add 10 times more culture medium and mix. 3. Centrifuge, 1 000 rpm, 5 min. 4. Discard the supernatant, resuspend the cells in 10% fetal bovine serum, count, adjust the cell density, inoculate the flask, and incubate in a 37 °C incubator. 5. Change the culture solution the next day and continue the culture.

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1. 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. 2. When placing or removing the cryotube from the liquid nitrogen container, be sure to protect it from frostbite.
3. It is best to use freshly prepared culture fluid for cryopreservation and resuscitation. First, the principle of cryopreservation and resuscitation: slow freezing and rapid melting When the cells are cooled below zero, the following changes can be made: dehydration of the organelles, increased concentration of soluble substances in the cells, and formation of ice crystals in 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. 2. Slow freezing procedure 1. Standard procedure: use cell cryostat when the temperature is above -25 °C, 1~2 °C/min; when the temperature is below -25 °C, 5~10 °C/min; when the temperature reaches At -100 ° C, it can be quickly put into liquid nitrogen. 2. Simple procedure: Put the cryotube (the nozzle is facing up) into the gauze bag, and the gauze bag is tied with a rope. The gauze bag is fixed to the liquid nitrogen tank can by the wire rope, and the temperature drops by 1~2 per minute. The speed of °C dropped to the surface of liquid nitrogen overnight in 40 min, and it was injected into liquid nitrogen in the next morning. 3. Traditional procedure: The cryotube is placed at 4 °C for 10 minutes → -20 °C for 30 minutes → -80 °C for 16 to 18 hours (or overnight) → Long-term storage of liquid nitrogen tank. Third, the application of low temperature protective agent in the cell cryopreservation when adding a warm protective agent, can greatly improve the frozen effect. The commonly used cryoprotectant is DMSO, which is a osmotic protective agent that can quickly penetrate into cells, improve the permeability of the membrane to water, lower the freezing point, delay the freezing process, and enable the intracellular water to leak out before freezing. Extracellular, ice crystals are formed extracellularly, reducing intracellular ice crystals, thereby reducing damage to the cells by ice crystals. 4. Cell cryopreservation method 1. Pre-formulated cryopreservation solution (1) 10% DMSO + cell growth solution (20% serum + basal medium) (2) 10% glycerol + cell growth solution (20% serum + basal medium) 2. Take the logarithmic growth phase cells, after trypsinization, add appropriate amount of frozen solution, and pipette to make a cell suspension (1 × 10 ⁶ ~ 5 × 10 ⁶ cells / ml). 3. Add 1 ml of cells to the cryotube and seal and label the frozen cell name and freezing date. Liquid nitrogen is stored for a long time. V. Method of resuscitating the preserved cells 1. Quickly thaw the frozen cells and remove them from the liquid nitrogen. Immediately put them into a 37 ° C water bath and gently shake the cryotubes to melt them in 1 minute (not more than 3 minutes). 2. The thawed cells can be directly inoculated directly into a cell culture flask containing the fully grown culture medium, and the old culture solution is replaced with fresh complete culture solution to remove DMSO 24 hours later. 3. If the cells are particularly sensitive to cryoprotectants, the thawed cells should be centrifuged to remove the cryoprotectant and then inoculated into a flask containing the fully grown medium.

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