Hypothermic storage of isolated human hepatocytes: a comparison between University of Wisconsin solution and a hypothermosol platform

Abstract

Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4 degrees C for 24-72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5'-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

“…Trolox is also known to have a significant iron-chelating capacity [28]; iron chelators have been found to be one of the key elements to enable high attachment efficiency of preserved cells after long term hypothermic storage of rat hepatocytes [27,28]. The importance of Trolox for cold storage of human hepatocytes beyond 48 hours was shown by Ostrowska and co-workers [29] where they compared HTS-FRS against UW and HTS-Base (without Trolox) and found that HTS-FRS was the only solution to prevent damages beyond 48 hours. While ROS generation during short static cold storage at +4 o C has been shown to be low [30] and the lipid peroxidation to be slow [31], similar data does not exist for long storage during cold storage or supercooling.…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…Oxidative stress has been ascribed a significant role in cellular damage because it alters the functional properties of cell membranes through lipid peroxidation (Vara et al 1995; Meng 2003). Therefore, recent preservation solution development has focused on a few critical topics including the maintenance of ionic and osmotic balance, the prevention of cell swelling and blebbing, the control of free radical formation and the development of serum-free media (Ostrowska et al 2009). Consequently, cold storage solutions containing antioxidants, iron chelators and/or membrane stabilizers have been developed to protect cells from such damages (Meng 2003; Pless et al 2012).…”

From production to patient: challenges and approaches for delivering cell therapies

“…• C for up to 72 hours was high (∼70%), the metabolic function of the cells was approximately half that of freshly isolated cells (Ostrowska et al, 2009). It may be that 'paused' cells would also benefit from a period of recovery after rewarming.…”