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

Abstract

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.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an “off-the-shelf” bioartificial liver system

“…Solutions containing 3 × 10 10 cryopreserved hepatocyte spheroids would have a volume of 1L, and an additional 30 L of thawing medium (at a 1:30 ratio) would be required to properly dilute the cryopreservation reagent (dimethyl sulfoxide [DMSO]). Moreover, 3 × 10 10 hepatocyte spheroid beads would constitute a volume of 2L, and 60 L of thawing medium would be required for resuscitation 17 .…”

“…Hepatocyte spheroids or immobilized hepatocyte spheroid beads were centrifuged at 50× g for 5 min at 4 °C. The supernatant was discarded, and the hepatocyte spheroids and immobilized hepatocyte spheroid beads were resuspended in ice-cold cryopreservation solution based on the protocol given by Lee et al 17 using modified histidine-tryptophan-ketoglutarate (mHTK; containing 4% human serum albumin, 3000 U/L insulin, 50 mg/L dexamethasone, and 0.04% amino acids) with N-acetylcysteine (15 mM NAC). The cryopreservation solution contained 15% dimethyl sulfoxide (DMSO) as a cryoprotectant.…”

Hepatocyte transplantation: The progress and the challenges

“… 19 PHH are typically frozen in University of Wisconsin solution with 10%–15% DMSO and 5% glucose. 19 Recently, modifications have been reported to improve the quality of cryopreserved PHH, such as encapsulation of hepatocytes, 39 alternative cryopreservation media to University of Wisconsin solution, 40 the addition of membrane stabilizer 41 and antioxidative chemicals (myricetin), 42 and bulk droplet vitrification. 43 Despite these improvements, the variability between different batches of PHH remains a major challenge.…”