How to select the appropriate cell culture cryoprotectant?

2017-09-19 16:07:50 GMT+0800

Cryoprotectants are substances that protect cells from freezing damage.

The cryoprotectant is often made into a solution.

Generally speaking, a few red blood cells, most of the microorganisms and nuclear of mammalian cells suspended in water without cryoprotectants or simple saline solution in and with the optimal rate of frozen frozen, can get live cryopreserved.

But for most nuclear mammalian cells, there is no optimum freezing rate, and no living frozen substances, in the absence of cryoprotectant.

Such as mice bone marrow cells suspended in a balanced salt solution without cryoprotectants, and freezing rate by 0.3 ~ 600 ℃ / min cooling refrigeration, more than 98% of the cells will die;

When the cryopreservation was frozen, more than 98% of the cells survived.

Cryoprotectants can be classified into two types: permeability and non-permeability.

  • Permeable cryoprotectant

The permeable cryoprotectant can penetrate into the cell, generally a small molecular substance, mainly including glycerol, DMSO, ethylene glycol, propylene glycol, acetamide, methanol etc.

The protection mechanism is in cell suspension before solidification completely frozen, infiltrate into the cell, both inside and outside the cells produce certain molar concentration, reduce the cells inside and outside the freeze concentration of electrolytes in the solution, not to protect cells from damage by high concentration of electrolyte, at the same time, the moisture inside the cell is not particularly permeability, avoids the cell excessive dehydration shrinkage.

Glycerol and DMSO does not prevent freezes inside the cell, when using the class cryoprotectants, need a certain amount of time for precooling, allow the composition such as glycerin or DMSO to infiltrate inside the cell, both inside and outside the cell balance to a sufficient protection.

Now DMSO used more widely than glycerol, but note that the toxic effects of DMSO to cells at room temperature is bigger, and in 4 ℃, its toxic effects greatly abate, and still to faster penetration into the cells.

So, cryopreserved DMSO balance under 4 ℃, usually need to 40 ~ 60 minutes.

  • Non-permeable cryoprotectant

Impermeable cryoprotectants, can't penetrate the cell is generally some macromolecular substances, including polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, glucan, albumin and hydroxyethyl starch, etc.

The hypothesis of a lot of the protection mechanism, one possibility is that macromolecular material such as polyvinylpyrrolidone can give priority to with water molecules in solution, reduce the ratio of free water in the solution, lower the freezing point, reduce the formation of ice crystals;

At the same time, due to its high molecular weight, the electrolyte concentration in the solution decreases, thus reducing the solute damage.

  • epilogue

Different cryoprotectants have different advantages and disadvantages.

At present, more than two kinds of cryoprotectants are used to form protective liquid.

Because many cryoprotectants (such as DMSO) can protect cells at low temperature, they are harmful to cells at normal temperature, so they should be washed in time after the cells are reheated.

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