Cryopreservation of rhesus macaque embryonic stem cells

Studies using nonhuman primate embryonic stem (ES) cells will facilitate the translation of basic ES cell research to clinical use by providing a large animal model for in vivo testing. Unfortunately, nonhuman primate ES cells do not survive well following cryopreservation, a problem that limits the quantity and quality of these cells for research. More lines have to be established to fulfill demand, and thawed aliquots must go through more passages to generate adequate numbers. In addition, suboptimal cryopreservation can induce epigenetic changes and impose a selection bias for their outgrowth. Therefore, defining the optimal cryopreservation technique for nonhuman primate ES cells is critical for the further development of this research. To address this problem, we tested various cryoprotectants as well as cryopreservation procedures in an attempt to define a protocol that yields high viability with retention of ES cell phenotype and function. Here, we report a freezing protocol that preserves the intercellular attachments that are vital to primate ES cell function. We describe a slow, controlled-rate cooling protocol with ice crystal induction that increased the survival rate of ES cells from <22% to >90%. Preserved cells retained a normal karyotype and did not lose their ability to express markers of undifferentiated ES cells.