Cold Storage of Rat Hepatocyte Spheroids

Abstract

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 mM cyclosporin A (CsA); SFM + 1 mM Def + 1 mM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

An Examination of Packing Methods for Grafts to Prevent Freezing Injury during Transportation for Liver Transplantation

“…However, no marked differences in findings were noted between groups C and D on examining HE-stained high-magnification images. Therefore, an additional examination with electron microscopy was performed, which revealed that while the structure in the hepatocytes was maintained in group D, there was apparent denaturing of the hepatocyte structure in group C. The low-temperature damage observed on electron microscopy has also been previously reported [15], showing findings compatible with our own. Tullius et al [16] reported that the accumulation of intravascular and interstitial crystals and the formation of extracellular and intracellular ice in the organ tissue were the main causes of freezing injury in organs and were particularly harmful to the vascular system.…”

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver

“…Based on a previous study, a similar effect of cold preservation of lower density and total amount (1 × 10 6 cells/mL, 2 × 10 7 cells in total) of rat hepatocyte spheroids in a custom serum‐free medium and UW solution was observed . The conclusion in that paper was based on viability and albumin production.…”

“…Based on a previous study, a similar effect of cold preservation of lower density and total amount (1 × 10 6 cells/mL, 2 × 10 7 cells in total) of rat hepatocyte spheroids in a custom serum-free medium and UW solution was observed. 29 The conclusion in that paper was based on viability and albumin production. The experiments were performed in serum-free medium supplemented with additives, but ammonia clearance ability after cold storage decreased quickly.…”

A Standardized Collagen-Based Scaffold Improves Human Hepatocyte Shipment and Allows Metabolic Studies over 10 Days

“…Therefore it is conceivable, that a high cellular loss of ATP during liver cell transportation in suspension [14] is responsible for the lack and/or delayed urea formation. In the same line of evidence Liu et al clearly showed that cold storage of cells reduces the metabolic testing capacity, including the ammonia detoxification capacity [64]. Based on these data, it seemed that hepatocytes recover faster in 3D than in 2D cultures, suggesting a lack of additional cofactors necessary to fully transform ammonia into urea.…”