A method of thawing cryopreserved human hepatocytes and cryopreserving the hepatocytes a second time without losing viability is described. It allows the preparation of cryopreserved human hepatocytes pooled from multiple donors by thawing the hepatocytes from the individual donors, combining the cells to form pooled hepatocytes, and recryopreserving the pooled hepatocytes. The method involves the thawing of the hepatocytes from the individual donors, maintaining the thawed hepatocytes at a low temperature that is above freezing temperature (e.g., 4° C.), and refreezing the thawed cells without further manipulation. The method allows the cryopreserving the human hepatocytes a second time after thawing, with viability similar to that after one single cryopreservation. This high efficiency method can be used for the preparation of highly viable pooled human hepatocytes for experimentation to minimize individual variations.
