Cell Preparation Method
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, 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.
1 . A method for preparing cryopreserved human hepatocytes pooled from multiple individuals without centrifugation and without diluting or changing the hepatocyte suspension, the method comprising:
thawing separate vessels of hepatocytes from multiple individuals in a bath;
pooling the thawed hepatocytes from the separate vessels of the multiple individuals in a container;
distributing the pooled hepatocytes into multiple vessels; and
cryopreserving the multiple vessels of pooled hepatocytes.
2 . The method of claim 1 , wherein thawing the vessels of the hepatocytes from multiple individuals includes thawing the vessels of the hepatocytes from multiple individuals in a 37° C. water bath.
3 . The method of claim 1 , wherein thawing the vessels of the hepatocytes from multiple individuals in a bath includes thawing until the hepatocyte suspension media reaches a temperature of approximately 4° C.
4 . The method of claim 1 , wherein the hepatocyte suspension media comprises a cryoprotectant.
5 . The method of claim 4 , wherein the cryoprotectant comprises Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium supplemented with 10% fetal calf serum and 10% dimethyl sulfoxide (DMSO).
6 . The method of claim 1 , wherein cryopreserving the hepatocytes comprises cryopreserving the hepatocytes to a temperature of about −70° C. at a rate of about −1° C./min.
7 . The method of claim 1 , further comprising storing the cryopreserved pooled hepatocytes in a cryogenic storage system.
8 . The method of claim 7 , wherein storing the hepatocytes comprises storing at a temperature of about −150° C. or cooler.
9 . The method of claim 1 , further comprising isolating the hepatocytes from livers of multiple individuals.
10 . The method of claim 1 , further comprising thawing of the cryopreserved pooled hepatocytes for experimentation.
11 . The method of claim 1 , wherein thawing the vessels of the hepatocytes includes thawing the vessels of the hepatocytes until the hepatocyte suspension media reaches a temperature of approximately 4° C.
12 . The method of claim 1 , wherein thawing the vessels of the hepatocytes includes thawing the vessels of the hepatocytes until ice crystals in the hepatocyte suspension just disappear.
13 . The method of claim 1 , wherein pooling the thawed hepatocytes includes pooling the thawed hepatocytes while maintaining the temperature of the hepatocyte suspension media at approximately 4° C.
14 . The method of claim 1 , further comprising:
cryopreserving human hepatocytes from livers of multiple individuals in hepatocyte suspension media in separate vessels for each of the multiple individuals.