Postthaw viability of precultured hepatocytes

Hepatocytes are bring studied for a wide variety of applications, including drug metabolism studies. gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordin ation of the cell supply with patient care regimes and the completion of sa fety and quality-control testing. The objective of this investigation was t o develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different ap plications, Freshly isolated hepatocytes were cultured in a spinner flask f or different periods of time. up to 48 h. The cells were cryopreserved by u se of a range of solution concentrations and cooling rates. For fresh, nonf rozen hepatocytes precultured for 24 h prior to being plated on collagen, t he albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were pr ecultured for 24 h. frozen in a solution containing 10% Me2SO with a coolin g rate of 1 degreesC/min, thawed, plated on collagen, and cultured, the alb umin secretion rate was 0.21 +/- 0.14 mug/ml/h. In contrast, freshly isolat ed hepatocytes cryopreserved without preculture and cultured on collagen ha d an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of dif ferent solution compositions and cooling rates on postthaw function of prec ultured hepatocytes were also determined. These results indicate that the u se of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes. (C) 2001 Academic Press.