Cell freezing and resuscitation experiments

Summary

Cell freezing and resuscitation can be

(1) used for biological preservation

(2) used for stem cell research in medicine, and

(3) used for passaging culture.

Operation method

Cell freezing and resuscitation

Principle

The basic principle of cell freezing and resuscitation is slow freezing and fast thawing, and experiments have proved that this can maximize the preservation of cell viability. At present, cell freezing and storage of glycerol or dimethyl sulfoxide as a protective agent, these two substances can improve the permeability of the cell membrane to water, coupled with slow freezing can make the water inside the cell to seep out of the cell, reducing the formation of intracellular ice crystals, thereby reducing the formation of ice crystals due to cell damage. Resuscitation of cells should be used to thaw quickly, so as to ensure that the extracellular crystals in a very short period of time that is melted, to avoid slow melting so that the water seeped into the cells to form intracellular recrystallization of cell damage.

Materials and Instruments

Cells
D-Hanks solution Calf serum Culture medium Penicillin Streptomycin Trypsin HCl NaHCO3 DMSO Glycerin
Micro Dosing Guns Pipette Tips Tubes Freezing Tubes Gun Heads Gum Plugs Pipette Glass Bottles Red Blood Cell Counting Plates Markers Medical Eraser Pipette Guns Ultra-clean Benches Centrifuges Constant Temperature Baths Refrigerators Refrigerators Inverted Phase Comparison Microscopes Culture Boxes Liquid Nitrogen Refrigerators

Move

I. Preparation of materials
1. Instruments: purification bench, centrifuge, constant temperature water bath, refrigerator (4℃, -20℃, -70℃), inverted phase contrast microscope, incubator, liquid nitrogen refrigerator.

2. Glassware: Pipettes (elbow, straight), culture flasks, glass bottles (250 ml, 100 ml), waste tanks.

3. Plasticware: pipette tips, gun tips, rubber stoppers, pipettes (10 ml), 15 ml centrifuge tubes, freezing tubes (1-2 ml).

4. Other items: micro-volume spiking guns, red blood cell counting plates, marking pens, medical ointment, pipette guns.

5. Reagents: D-Hanks solution, calf serum, culture medium, double antibiotic (penicillin, streptomycin), trypsin (0.08%), 1NHCl, 7.4% NaHCO3, DMSO (analytically pure) or colorless fresh glycerol.

II. Cell freezing

1. Prepare a frozen culture solution containing 10% DMSO or glycerol and 10-20% calf serum.

2. Take cells in the logarithmic growth phase and trypsinize the monolayer growth cells to digest them, while the suspension growth cells are directly transferred to a 15 ml centrifuge tube.

3. Centrifuge at 1,000 rpm for 5 min.

4. Remove trypsin and old culture medium, add appropriate amount of prepared frozen culture medium, gently blow with a pipette to make the cells homogeneous, count and adjust the final density of cells in the frozen culture medium to ^{5×106/ml～1×107/ml}.

5. Dispense cells into freezing tubes of 1 to 1.5 ml each.

6. Label the cryopreservation tubes with the name of the cells, the time of freezing and the operator.

7. Freezing: The standard freezing procedure is a cooling rate of -1 to -2°C/min; when the temperature reaches below -25°C, it can be increased to -5°C to -10°C/min; and when it reaches -100°C, it can be rapidly immersed in liquid nitrogen. The cryopreservation tube with cells can also be put into the -20℃ refrigerator for 2 h, and then put into the -70℃ refrigerator overnight, take out the cryopreservation tube and move it into the liquid nitrogen container.

III. Cell recovery

1. Remove the cryotubes from the liquid nitrogen container and immerse them directly in warm water at 37°C, shaking them from time to time so that they thaw as quickly 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 solution dropwise and mix well.

3. Centrifuge, 1 000 rpm, 5 min.

4. Discard the supernatant, add 10% calf serum culture medium to resuspend the cells, count, adjust the cell density, inoculate the culture flask and incubate at 37℃.

5. Replace the culture medium once on the following day and continue the incubation.

Caveat

1. Cultured cells from the proliferative phase until the formation of a dense monolayer can be used for freezing, but logarithmic growth phase cells are preferred. It is advisable to change the culture medium once a day before freezing.

2. Protect cryopreservation tubes from frostbite when placing them in or removing them from liquid nitrogen containers.

3. It is best to use freshly prepared culture medium for freezing and resuscitation.

Common Problems

I. Principles of freezing and resuscitation: slow freezing and fast thawing

When cells are chilled below freezing, the following changes can occur: dehydration of organelles, increased concentration of soluble substances in the cell, and formation of ice crystals within the cell.

If frozen slowly, the cells can be dehydrated gradually, and large ice crystals are not produced inside the cells; on the contrary, the crystallization will be large, and the large crystals will cause damage and rupture of the cell membranes and outline organelles. The recovery process should be fast thawing, the purpose is to prevent the formation of small ice crystals into large ice crystals, that is, the recrystallization of ice crystals.

I I . slow freezing procedures

1. Standard procedure: the use of cell freezer

When the temperature is above -25 ℃, 1~2 ℃/min;

When the temperature reaches below -25 ℃, 5~10 ℃/min;

When the temperature reaches -100 ℃, it can be quickly put into liquid nitrogen.

2. Simple procedure: put the freezing tube (the mouth of the tube should be facing up) into a gauze bag, tie the gauze bag with a string, fix the gauze bag to the mouth of the liquid nitrogen tank through the string, and then the temperature will be decreased by 1~2 ℃ per minute, and then it will be lowered to the surface of the liquid nitrogen in 40 min overnight, and then put into liquid nitrogen in the next morning.

3. Traditional procedure: the freezing tube is placed in 4 ℃ for 10 minutes → -20 ℃ for 30 minutes → -80 ℃ for 16~18 hours (or overnight) → liquid nitrogen tank for long-term storage.

Application of cryoprotectant

Adding temperature protection agent in cell freezing can greatly improve the effect of freezing.

Commonly used cryoprotectant is DMSO, which is an osmotic protective agent, can rapidly penetrate into the cell, improve the permeability of the cell membrane to water, reduce the freezing point, delay the freezing process, can make the intracellular water in the freezing of water before the cell is permeable to the cell, the formation of ice crystals outside the cell, to reduce intracellular ice crystals, so as to reduce the damage to the cell of ice crystals.

Cell freezing method

1. Pre-formulation of freezing liquid

(1) 10% DMSO + cell growth solution (20% serum + basal culture)

(2) 10% glycerol + cell growth solution (20% serum + basal medium)

2. Take logarithmic growth phase cells, digested by trypsin, add appropriate amount of freezing solution, and blow with a pipette to make cell suspension (1 × ¹⁰⁶ ~ 5 × ¹⁰⁶ cells/ml).

3. Add 1 ml of cells into a freezing tube, seal and label with the name of the frozen cells and the date of freezing. Liquid nitrogen for long-term preservation.

V. Resuscitation Methods for Preserved Cells

1. Quick thawing

Immediately after removing frozen cells from liquid nitrogen, place them in a 37°C water bath and gently shake the cryotubes so that they all thaw within 1 minute (do not exceed 3 minutes).

2. Thawed cells can be directly inoculated into cell culture flasks containing complete growth medium for direct culture, and then replace the old culture medium with fresh complete culture medium after 24 hours to remove DMSO.

3. If the cells are particularly sensitive to cryoprotectants

Thawed cells should be centrifuged to remove the cryoprotectant before inoculation into culture flasks containing full growth medium.

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