Why do cells freeze?
Cell cryopreservation is one of the main methods for cell preservation.
Technology puts cells - 196 ℃ using cryopreserved in liquid nitrogen cryopreservation, can temporarily out of the cell growth state and to save the cell characteristics, so in need of recovery cells used in the experiment.
In addition, a certain amount of cells can be kept in order to prevent cell loss from being contaminated or other unexpected events.
In addition, cells can be purchased, donated, exchanged and transported in the form of cell freeze.
To add protective agent into the medium - cells cryopreserved final concentration of 5% 15% of glycerol or dimethyl sulfoxide (DMSO), can make the solution to lower the freezing point, and under the condition of slow freezing, moisture passes inside the cell, reduced the ice crystals form, so as to avoid cell damage.
1.The procedure of cell freezing
(1) choose cells that are in logarithmic growth, preferably before freezing.
The cell culture medium in multiple culture bottles was removed and 0.25% trypsin digestion was used.
Remove trypsin and add a small amount of new medium.
The cells were repeatedly blown on the wall of the bottle with a straw, making it a uniformly dispersed cell suspension.
Suspended production cells do not digest processing.
The cells were then collected in a centrifuge tube (1000r/min, 10 minutes).
(2) to go to clear liquid, add the complete culture medium containing 20% calf serum, precooling at 4 ℃ for 15 minutes later, one by one drop join is sterile DMSO or glycerin, gently blowing through a straw even the cell, cell concentration of 5 x between 106 ~ 107 / mL.
(do you need to use calf serum for the storage of frozen medium?
The answer is not necessarily, depending on the type of cell that is cultured, like some scientists use corning fetal bovine serum and others use sigma's calf serum.
(3) the above cell suspension is divided into 2ml frozen storage tube, 0.25ml per tube.
The cold storage tube shall cover the lid and mark the name of the cell and the date of freeze, while making the registration (date, type of cell and number of subunits and cryoplast counts).
What does a cold storage tube look like?
Let's look at the picture below: Corning cell frozen storage tube 430659
The method of "slow freeze-thaw" can ensure cell survival.
Standard freezing rate began to to 1-2 ℃ / min, when the temperature below 25 ℃ can accelerate, in the after - 80 ℃ can be directly put into liquid nitrogen (196 ℃).
(4) will be packed cells cryopreserved tubes into the frozen storage box, - 80 ℃ refrigerator overnight.
If there is a program to cool (in - 80 ℃ refrigerator overnight, into the liquid nitrogen tank) the best;
Or can be in 4 ℃, 2 h;
Then go to - 20 ℃, 2 h;
- 80 ℃, 2 h;
Put in the liquid nitrogen tank.
(cold storage box and program cooling box, look at the picture below)
(5) cells frozen in liquid nitrogen can be saved for a long time, but, to be properly, cryopreserved after six months, it is best to remove the tube a cryopreserved cells recovery training, to observe the growth situation, and then continue to freeze.
2.How to do cell recovery
(1) The pipe from the liquid nitrogen container cryopreserved, directly into 37 ℃ warm water, do not shake to the melt as soon as possible.
(2) from the 37 ℃ water bath cryopreserved tubes, open the lid and out of the cell suspension through a straw, add to the centrifuge tube and add more than 10 times of the culture, blending;
(3) Centrifugal, 1000rpm, 5min;
(4) Leave to go to clear liquid, add heavy suspension cells containing 10% calf serum broth, count, adjust the cell density, inoculation culture bottle, 37 ℃ cultivation box let stand;
(5) Change the culture medium once the next day and continue to develop.
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