Cell resuscitation experiments

Summary

Cell recovery assay is a process in which frozen cells are removed from - 196 °C liquid nitrogen and thawed to restore their viability.

Principle

The basic principle of cell resuscitation experiments is to ensure that the extracellular crystals melt in a very short time by means of rapid melting, avoiding damage to the cells due to recrystallization of water penetrating into the cells as a result of slow melting. Successfully resuscitated cells maintain a high level of viability.

Operation method

Cell resuscitation experiments

Principle

The basic principle of cell resuscitation experiments is to ensure that the extracellular crystals are melted in a very short time by means of rapid melting, avoiding damage to the cells due to recrystallization of water infiltrated into the cells as a result of slow melting. Successfully resuscitated cells maintain a high level of viability.

Materials and Instruments

Equipment:
① Hemostat
② liquid nitrogen tank
③ cell freezing tube
④ Water bath
⑤ Alcohol cotton balls
⑥ Ultra-clean bench
⑦ Centrifuge tube
⑧ Centrifuge
⑧ Centrifuge ⑨ Culture bottle
⑩ Incubator
Reagents: ① DMEM medium (with 10% calf serum)
① DMEM medium (containing 10% calf serum)
② D-Hanks liquid
D-Hanks liquid ③ Freezing solution

Move

The basic process of cell recovery experiment can be divided into the following steps:
A. Use hemostat to take out 1 cell cryopreservation tube from the liquid nitrogen tank, quickly put it into a 37 ℃ water bath, and shake it constantly to make the frozen cell suspension melt as soon as possible.
B. Wipe the cryopreservation tube with an alcohol cotton ball, and put it into an ultra-clean bench.
C. Transfer the melted cell suspension into a centrifugal tube with a pipette and add 10 times the volume of DMEM medium, blowing and mixing. D. 1500 r/min centrifugation for 3 min, discard the supernatant. E. Add culture medium to blow the precipitated cells to make them suspended, and count the cells. Mix well.
D. Centrifuge at 1500 r/min for 3 min, and discard the supernatant.
E. Add culture medium and blow the precipitated cells to make them suspended, and count the cells.
F. Inoculate the cells in culture flasks at a concentration of 5 x ¹⁰⁵ cells/ml and place them in an incubator.
G. Remove the flasks after 24 h, and observe the growth status of the cells.

Caveat

1. During resuscitation, the process of removing the cryopreserved tubes from the liquid nitrogen to the water bath for thawing should be quick, otherwise it will lead to the formation of ice crystals, which will harm the cells. At the same time, the number of cryopreserved tubes in one resuscitation should not be too large, otherwise it will cause poor heat transfer in the water bath and delay the melting time of the stored cell suspension.

2. To prevent liquid nitrogen frostbite, cotton gloves should be worn during resuscitation.

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