Jinma focuses on analyzing cell cryopreservation and resuscitation experimental steps - Database & Sql Blog Articles

Cryopreservation of cells is a routine work of the cell compartment. Cell cryopreservation and cell passage

Compared with saving, you can reduce manpower,

Funding

,

Reduce pollution,

Reduce changes in cell biological properties.

Cryopreservation of cells is a routine work of the cell compartment. Cell cryopreservation and cell passage

Compared with saving, you can reduce manpower,

Funding

,

Reduce pollution,

Reduce changes in cell biological properties.

Cryopreservation of cells is a routine work of the cell compartment. Compared with cell subculture, cell cryopreservation can reduce manpower, funding, reduce pollution, and reduce changes in cell biological properties. 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 no large ice crystals are produced in the cells; instead. Crystallization is large, and large crystals can cause damage and rupture of cell membranes and organelles. 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. The following steps were carried out by Shanghai Jinma Experimental Equipment Co., Ltd. to analyze the cell freezing and resuscitation experiments. First, the principle of cell cryopreservation and resuscitation Cell culture can be divided into primary culture and subculture. The tissue cells obtained directly from the body are cultured for the first time as primary culture; when the primary cultured cells reach a certain density, they need to be recultured, and the cells to be cultured are dispersed from a container at a ratio of 1:2 or other ratio. Transfer to another container or several containers to expand the culture. For subculture, the cumulative number of subcultures is the algebra of the cells.
The basic principle of cell cryopreservation and resuscitation is slow freezing and rapid melting, which has been shown to maximize cell viability. At present, chloroglycerin or DMSO is used as a protective agent for cell cryopreservation. 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, thereby reducing the formation of intracellular ice crystals. Reduce cell damage caused by ice crystal formation. 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.
Second, cell cryopreservation and resuscitation experimental methods Materials:
Mouse, saline, 100ml sterilized beaker, 15ml centrifuge tube, culture dish, dropper, sterile tweezers, scissors, mesh, foam board, pin, alcohol lamp, culture flask, culture solution, PBS, 0.25% trypsin , 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 tanks, etc.
method:
(1) Primary culture:
1. Remove the mice, drain the alcohol, place in a clean bench, and fix on the foam board.
2. Lift the skin with tweezers, cut a transverse incision with an anatomical scissors, tear the skin upwards, then cut the muscles, etc., expose the abdominal cavity, find the spleen on the left side, remove the spleen with elbow ophthalmology, and place it in the absence. In the culture dish.
3. Wash the removed spleen several times with saline and remove excess unwanted tissue.
4. Place the sieve in a dish, place the spleen on the sieve, hold it with an elbow, gently grind it on the sieve, and rinse with the serum-free medium.
5. The milled cell suspension was aspirated into a centrifuge tube, centrifuged (1000 rpm, 5 min), and the supernatant was removed (blood removal, etc.).
6. Add 10 ml of the culture solution, mix by pipetting, and sample and count.
7. Dispense the diluted cell suspension into a culture flask, shake gently and mix well on the flask.
Make a mark on the face, indicating the cell, group and date. The flask was then placed in a carbon dioxide incubator for cultivation.
(2) Subculture:
1. Observe the cell morphology under an inverted microscope to determine if the cells need to be passaged.
2. After the culture liquid bottle is opened, pass the alcohol lamp flame and place the alcohol lamp around the flame.
3. Take out the straight dropper, put on the rubber tip, overheat, and put it into the culture bottle.
4. Open the culture flask, over-fire the bottle, gently pour the culture solution in the culture flask into the waste tank, and wash off the residual old medium with 2-3 mL of PBS.
5. Add 0.25% trypsin to the culture flask. It is advisable to cover the layer with a thin layer. Digestion at 37 °C. Immediately under the microscope, the cell is retracted and the flask is turned off, the cells are removed from the trypsin, and then the pancreas is removed. The enzyme was poured off and a small amount of fresh serum-containing medium was added.
6. Take the elbow pipette repeatedly to blow the cells to dissociate and disperse, and then add a certain amount of serum-containing fresh medium according to the number of bottles to make a cell suspension, and then dispense into a new culture bottle. Cover the cap, moderately tighten and then slightly rotate to facilitate the entry of CO2 gas and return the flask to the CO2 incubator.
7. For semi-adherent cultured cells, without pancrease, directly blow, add fresh medium, and then dispense into each bottle.
(3) Frozen storage:
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
o 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, It can be quickly immersed in liquid nitrogen.
o 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.
(4) 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.
Third, cell cryopreservation and recovery experiment results calculation
1. One mouse can obtain (1 to 2.5) x 108 spleen cells.
2. After the cells are inoculated, they can be attached to the wall within a few hours and start to grow. For example, the density of the inoculated cells is appropriate, and a single layer can be formed within 5 days to one week.
3. In general, the cells after passage can be attached to the wall of the culture bottle in about 2 hours. In 2 to 4 days, a single layer can be formed in the bottle, which needs to be subcultured again.
Fourth, cell cryopreservation and resuscitation experiment notes
1. The material requirements are fresh, sterile, and when dissecting the mouse, be careful not to damage the spleen and the organs around it, especially the intestines, to prevent contamination of the spleen.
2. When washing the spleen, wash the blood dirt as much as possible, remove the useless tissue, and prevent the tissue from drying out.
3. Rinse the screen with water after grinding to prevent the cells and cells from blocking the mesh.
4. Before counting, pay attention to the cells in the dish and mix thoroughly to disperse the cells into individual cells.
5. The experimental operation should be carried out in the sterile area in the center of the console, and should not be operated in the non-sterile area at the edge.
6. The metal instrument can't be burned in the flame for too long. The burnt metal crucible needs to be cooled before the tissue can be taken to avoid tissue damage.
7. When the rubber plug is over the flame, it can't be long, so as to avoid burning charred gas and harm the cultured cells.
8. 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, and when used, the harmful substances will be brought into the nutrient solution.
9. Do not touch the mouth of the sterilized vessel, the inside of the stopper, the front part of the straw, etc., which may be in contact with the cells. If it has been touched, use a flame to burn or replace the spare parts.
10. When the cell culture flask is opened or closed, the flame sterilization time is short. Prevent cells from burning out due to excessive temperature.
11. 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.
12. When blowing the cells, pay attention to whether the cells in the corners are blown down.
13. Hands or relatively dirty items cannot pass through the open bottle opening, ie they cannot be operated above the open container.
14. Only one cell is treated per operation to avoid cross-contamination of cells.
15. 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.
16. 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.
17. When placing the frozen tube in or out of the liquid nitrogen container, it is necessary to do a protective work to avoid frostbite.
18. It is best to use freshly prepared culture fluid for cryopreservation and resuscitation.