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.