83ANALYSIS OF RAPID COOLING V. SLOW COOLING COMBINED WITH ICE CRYSTAL SEEDING FOR CRYOPRESERVATION OF PRIMATE EMBRYONIC STEM CELLS

Abstract

Primate embryonic stem (ES) cells have the ability to self-renew indefinitely while maintaining the ability to differentiate. This unique property allows scientists to study the factors necessary for stem cell self-renewal and differentiation in vitro that reflect in vivo processes. Work with primate ES cells is handicapped by the poor survival (1–5%) of rhesus and human ES cells following standard tissue culture methods of rapid cryopreservation. The purpose of this study was to compare and contrast two cryopreservation techniques, slow cooling combined with ice crystal seeding commonly used for mammalian embryos v. rapid cooling commonly used for tissue culture, to find a method for efficient primate ES cell cryopreservation. A combination of trials was run to compare dimethyl sulfoxide (DMSO) v. ethylene glycol as a cryoprotectant, a cooling rate of 0.3°C per minute following ice crystal seeding at −7°C v. placement at −80°C with no seeding, and rapid thaw with step-wise cryoprotectant removal v. one-step sucrose cryoprotectant removal. Cell survival was assessed through a combination of cell surface markers, alkaline phosphatase staining and morphology to look for undifferentiated cells and quantitate survival. All cryopreservations were performed with the same cell density. The survival of the cells with slow embryo-style cooling in DMSO with a step-wise cryoprotectant removal was 64.0% v. 12.8% with rapid cooling.