Storage of cells for many researchers simply involves addition of 10% DMSO or similar cryoprotectants to cells in suspension, putting them in cryovials and slow cooling to subzero temperaturesf and storage in a mechanical or nitrogen cooled freezer or dewar. As long as viable cells are present upon thawing, cell yield may be a secondary consideration due to the high proliferative potential of the cells. However, there are significant cell types that are difficult to preserve with existing cryopreservation techniques including many primary cells, such as cardiac myocytes, and hepatocytes, plus embryonic stem cells and inducible pluripotent stem cells. Others, such as keratinocytes, are easy to cryopreserve in suspension but difficult when attached to a substrate. The study of how organisms survive extreme temperatures has revealed that multiple phenomena may occur in synchrony to promote survival including production of cryoprotectants, antifreeze compounds (peptides and lipids), dehydration and nucleators. Mammalian cell cryopreservation may be possible by mimicking strategies employed by certain insects to survive sub-zero temperatures. These insects combine increases in cryoprotectant content (such as glycerol) with production of antifreeze compounds, at the onset of cold environmental conditions, to survive temperatures as low as -80oC. The presence of these compounds lowers the freezing point of the solution and also changes the shape and formation of ice. In this way, potentially less cryoprotectants, such as DMSO or glycerol, may be used to preserve cells reducing potential cryoprotectant induced cytotoxicity concerns and offering alternative preservation methods for cells and tissues that can be used in clinical applications and cell and tissue-based screening assays for research applications.
The purpose of this proposal is to determine the feasibility of using an insect-derived anti-freeze glycolipid (AFGL) to cryopreserve mammalian cells. AFGLs have recently been isolated from insects and other sources and have been shown to control the formation of ice. These studies will evaluate the ability of the AFGL to control ice and improve the viability of cells during cryopreservation. The glycolipid will be isolated from two natural sources and its ability to control ice formation and improve the viability of cells after cryopreservation will be measured. More specifically, its ability to improve viability either alone in combination with traditional cryoprotectants like DMSO at reduced concentrations or by using less traditional cryoprotectants such as trehalose and sucrose will be examined. At the conclusion of these studies, it is anticipated that optimal concentrations of AFGL for cryopreservation will be determined and combinations of AFGL with other cryoprotectants that produce good viability in cryopreserved cells will also be defined.