UW solution: a promising tool for cryopreservation of primarily isolated rat hepatocytes

Abstract

Cryopreserved hepatocytes are a ready source of metabolic and synthetic functions for hepatocyte transplantation and bioartificial livers. In this study, we evaluated a cytoprotective effect of University of Wisconsin (UW) solution during cryopreservation of rat hepatocytes. We also investigated the feasibility of lentivirus-based gene transfer into thawed hepatocytes after cryopreservation. Primary rat hepatocytes were isolated using a two-step collagenase perfusion technique, and the resulting hepatocytes with more than 85% viability assessed by a trypan blue exclusion test were subjected to the present study. These cells were subjected to the present study. Cells were cryopreserved with UW solution containing 10% fetal bovine serum (FBS) with 12% dimethylsulfoxide (DMSO) (group 1, G1), Cellbanker solution (group 2, G2), and 10% FBS-containing Dulbecco modified Eagle medium (DMEM) with 12% DMSO (group 3, G3). After thawing the cryopreserved hepatocytes, cell viability, plating efficiency, morphological appearance, and ammonia clearance activity were determined for each group. The efficacy of lentivirus-mediated Escherichia coli LacZ gene delivery was evaluated. Hepatocyte viabilities after 3- and 7-day cryopreservation were 73.2% and 62.5% for G1, 57.5% and 46.5% for G2, and 57.3% and 41.5% for G3, respectively. Plating efficiency and ammonia clearance activity were improved in G1 hepatocytes compared to G2 and G3 cells. Lentiviral transfer of a LacZ gene was confirmed in the thawed hepatocytes after cryopreservation by an X-gal stain assay.

Albumin-Producing Hepatocytes Derived from Cryopreserved F344 Rat Bone Marrow Cells Transplanted in the Livers of Congenic Nagase's Analbuminemic Rats

“…In a 13 previous study, hepatocytes cryopreserved with UW solution were significantly functional in terms of plating efficiency, ammonia metabolism, hepatic enzyme leakage, and in vivo transplantation [26]. The present study also demonstrated that the UW solution is also useful for cryopreservation of bone marrow cells.…”

“…The UW solution was originally developed for hypothermic preservation of organs and is now widely used in cardiac, hepatic, and renal transplantation [25,26]. In a 13 previous study, hepatocytes cryopreserved with UW solution were significantly functional in terms of plating efficiency, ammonia metabolism, hepatic enzyme leakage, and in vivo transplantation [26].…”

“…Because University of Wisconsin (UW) solution is now currently used for the cryopreservation of various tissues and cells [25,26], F344 BMCs were cryopreserved in UW solution containing 10% fetal bovine serum (FBS) and 12% dimethylsulfoxide (DMSO). Thawed cells were infused into the portal vein of F344alb immediately after 70% hepatectomy (PH), so we could evaluate whether they increased the numbers of clusters of albumin positive (alb+) hepatocytes within the recipient livers.…”

Serum-free cryopreservation of porcine hepatocytes

“…For the experimental application of cryopreserved hepatocytes, the loss of function can, at least in part, be prohibited by inclusion of serum in the freezing and thawing media. Accordingly, most studies with cryopreserved hepatocytes destined for basic research purposes have employed protocols with serum to improve hepatocyte functional maintenance (Alexandre et al 2002;Arikura et al 2002;Chesne et al 1993;Dandri et al 2001;Houle et al 2003;Ikeda et al 2002;Jamal et al 2000;Swales et al 1996;Vian et al 2002). Nevertheless, for the clinical use of cryopreserved hepatocytes, cells processed under serum-free conditions are required.…”

Von der Gewebezüchtung zur individuellen Geweberegeneration – Der Mensch als Bioreaktor

“…Diese können durch eine standardisierte Technik kryokonserviert und wieder aufgetaut werden (Abb. 9); sie sind also ohne große Probleme zum Einsatzort transportierbar und haben in ihrer Wirkung keinen gravierenden Funktionsverlust (Arikura et al, 2002;Aoki et al, 2005).…”