BioFiles Volume 5, Number 4 — Cryopreservation

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Table of Contents

 


Introduction

Don Finley
Don Finley

Freezing living cells with the hope of full recovery began with the freezing of sperm in glycerol in 1949.1 Later DMSO was shown to be a better preservative as it can better permeate cells like red blood cells.2 Cryopreservation for all practical purposes is limited to individual cells or small clumps of cells. So preserving mammalian cells, plant cells, blood, sperm, and embryos is routine practice for clinicians and researchers. DMSO has proven to be the most robust cryopreservative agent and the most widely used. DMSO has some drawbacks in that it is cytotoxic and given that it permeates cells, it is difficult to completely eliminate. There are a number of non-permeating cryoprotectants that have also proven effective such as hydroxyethyl starch, PVP and CMC. Typically non-permeating cryoprotectants are not as efficient at preserving cell viability and are often used in concert with DMSO to enhance the efficiency of cryoprotection.

There are two common methods of cryopreservation, controlled rate freezing and vitrification. Controlled rate freezing typically involves placing the cells in a cryoprotectant media and cooling at a rate of –1 to –3 °C per minute, to about –80 °C and finally storing at –196 °C in liquid nitrogen. In this method, ice crystals form outside of cells first, and the higher concentrations of solutes that are excluded from ice crystals draw water from the cells and prevent or minimize freezing inside the cells. Controlled rate freezing is the most commonly practiced method, as it has proven to be robust and widely applicable. However, the osmotic changes and ice crystals formed using this technique can lead to reduced recovery and cryopreservation-induced delayed-onset cell death. Vitrification involves rapid freezing in higher concentration of cryoprotectants. The higher concentrations of cryoprotectants and rapid freezing prevent the formation of crystalline ice and promote the formation of an amorphous ice or glass. Unfortunately, vitrification is not without some problems as near toxic concentrations of DMSO are often required to promote the formation of a glass. Furthermore at this time the conditions required to achieve vitrification vary from application to application.

Sigma® offers a wide selection of equipment and reagents for your cryopreservation needs that include DMSO, prepared freezing media and Mr. Frosty Freezers. In addition, we recenty introduced CryoStor™ a completely optimizable serum free cryopreservation system, HypoThermosol®, a highly protective media for storage of cells and tissues at 2–8 °C and will soon offer sericin, a protein from silkworm that has proved to be a valuable FBS replacement in cryopreservation.

References

  1. Revival of spermatozoa after vitrification and dehydration at low temperatures. C. Polge, et al., Nature, 164 (1949) 666–667.
  2. Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide. Lovelock, J. E. and Bishop, M. W. H. Nature, 183: 1394–1395 (1959).

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