Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an "Off-the-Shelf" Bioartificial Liver System

To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.