Cryopreservation of living cells: principles and practice

Abstract

Increasingly, the cryopreservation of living cells is being attempted by researchers whose primary interest and experience is with the medical applications of those cells or tissues and whose prior experience with cryobiology may be negligible. It is therefore generally necessary to imitate some regimen used by others, perhaps with some other cell type and attempt to optimize the recovery empirically. This article makes no attempt to provide specific protocols for the many individual cell types. Rather it is a primer that may help to give such investigators an insight into the basic principles of cell freezing, cryoprotectants, and, particularly, their addition and removal. Finally, the article summarizes the five different approaches to applied cryopreservation: ultrarapid freezing and thawing, controlled-rate freezing, freezing with nonpenetrating polymers, vitrification, and equilibrium freezing.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

“…Since most cells survive freezing and thawing after proper anti-freeze treatment, this widely used method allows keeping single cell suspensions, small organisms, tissue pieces, even embryos viable for storage. Due to their dehydrating activity, however, anti-freeze agents introduce various alterations in cellular structure, such as severe shrinking and speciWc responses to osmotic stress (Meryman 2007). Although these types of changes are not lethal and reversible, the ultrastructure of such cryoprotected samples is changed.…”

“…In electron micrographs, so-called segregation patterns become visible (Allison et al 1987;Dubochet 2007;Escaig 1982). In the worst case, growing ice crystals poke holes into cellular membranes and destroy organelles (Meryman 2007). When thawed again, such badly frozen tissues or cells are nothing but dead mush.…”

Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing

“…Although the reasons for activity of PVA remain unclear at present, the ongoing search for other polymeric IRIs has not identified many active candidates 31,36 . Other non-penetrative polymers, poly (vinylpyrrolidone) and dextran, have also been explored as cryopreservatives but have minimal IRI activity 37,38 .…”

Prevention of Lethal Osmotic Injury to Cells During Addition and Removal of Cryoprotective Agents: Theory and Technology

“…However, the glycerol in RBCs must be reduced to final concentration below 1% before infusion to prevent hemolysis (Valeri et al, 2001). The step of removing CPAs may cause serious cell loss due to the cell volume excursion induced by osmotic disequilibria (Meryman, 2007). In the past decades, many efforts have been made to improve the process (Rowe et al, 1968;Meryman et al, 1972Meryman et al, , 1977Valeri et al, 1975Valeri et al, , 2001Castino et al,1996;Arnaud et al 2003).…”

“…In the USA, the FDA has approved the storage of frozen RBCs at -80°C for as long as 10 years (Meryman, 2007). However, the glycerol in RBCs must be reduced to final concentration below 1% before infusion to prevent hemolysis (Valeri et al, 2001).…”