Cryopreservation of human embryonic stem cells: A protocol by programmed cooling

Human embryonic stem (hES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the hES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES,cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0 degrees C to -35 degrees C at a cooling rate of 0.5 degrees C /min, with seeding was set at -10 degrees C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of I I colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.