To determine the best and simplest method for cryopreservation of pig hepat
ocytes, we compared immediate cryopreservation with cryopreservation after
short-term culture. Suckling pig hepatocytes were isolated by a modified 2-
step in situ collagenase perfusion method, suspended in serum-free medium,
and preserved for 10 da by two cryopreservation methods. Serial measurement
s were made of cell viability, LDH release, synthesis of protein, urea and
glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transforma
tion after thawing. These measurements were performed on both groups of cul
tured hepatocytes, and on freshly isolated hepatocytes, which served as a c
ontrol. High viability (>95%)of thawed hepatocytes was obtained and maintai
ned in both cryopreservation groups. There were no significant differences
in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity,
or diazepam transformation between the two cryopreservation groups. In the
immediate cryopreservation group, urea synthesis was less than in the grou
p with cryopreservation after short-term culture. Protein synthesis, glucos
e synthesis, and diazepam transformation were lower in both cryopreserved g
roups than in the controls. The results showed that a protocol of immediate
cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, h
ormones, growth factors, and 10% newborn bovine serum, together with rate-c
ontrolled freezing and rapid thawing, provides indices of cell viability an
d function during subsequent serum-free culture that are comparable to hepa
tocytes cryopreserved after short-term culture, except for lower urea. prod
uction. This simple procedure can be used in studies of bioartificial liver
and hepatocyte transplantation.