Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

Abstract

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VSbasic (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VSbasic and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5days. Results indicated Ficoll as the least toxic additive. Within 60min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10-3M Ficoll+0.6M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VSbasic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10-3M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison. © 2012.