A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

“…Hepatocytes are always exposed to a cryoprotectant prior to freezing; hepatocytes frozen without a cryoprotectant are not viable (Koebe et al, 1990;Loretz et al, 1989), and it is therefore a necessary evil of current cryopreservation protocols. By far the most common cryoprotectant is DMSO.…”

“…Paradoxically, DMSO can also promote protein denaturation, depending on the conditions of exposure (Arakawa et al, 1990). Low concentrations of DMSO are cytotoxic, and healthy hepatocytes exposed to it will rapidly lose viability (Beecherl et al, 1987;Watts and Grant, 1996), and the ability to synthesise albumin (Koebe et al, 1990), urea, and protein (Fuller et al, 1980). DMSO has an osmolarity of 1.7 osmol (Diener et al, 1993), which is about five times higher than physiological solutions (Hengstler et al, 2000).…”

“…Typical suspension cryopreservation protocols call for hepatocytes to be frozen immediately upon isolation, although cells are sometimes preincubated with carbogen prior to freezing (Silva et al, 1999). For freezing protocols, cells are suspended in one of many media, such as Modified Eagle's medium, Dulbecco's Modified Eagle's medium, Leibovitz L-15 medium, Williams' E medium, Eagle's basal medium, Waymouth's 752/1 medium, or foetal calf serum (FCS), (Beecherl et al, 1987;Chesné and Guillouzo, 1988;Coundouris et al, 1990;Diener et al, 1993;Koebe et al, 1990;Le Cam et al, 1976;Loretz et al, 1989), though none offer particular advantage in terms of viability, membrane integrity, recovery, or function post thaw. FCS is often included in the cryopreservation media.…”

“…To reduce the osmotic shock experienced by cells exposed to DMSO, a "stepwise addition" of DMSO is sometimes performed, i.e. cells are gradually exposed to increasing concentrations of DMSO (Diener et al, 1993;Koebe et al, 1990). We have found this practice to be quite beneficial to cells cryopreserved as monolayers.…”

“…The pioneering work on hepatocytes cultured between two layers of collagen gel (sandwich system) was carried out by Dunn and co-workers (Dunn et al, 1989;. Their work showed that the collagen gel sandwich culture system supported the secretion of albumin, transferrin, fibrinogen, bile acids and urea for at least six weeks, and (Borel Rinkes et al, 1992a;Hubel et al, 1991;Koebe et al, 1990). The method was developed for freezing rat hepatocytes and showed a survival rate (in terms of morphology) on thawing of only 30%, signs of severe cytoplasmic damage in all cells, and a decrease in albumin secretion down to 20% of control cultures.…”