title: 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.
authors.affiliation: Second Department of Surgery, Asahikawa Medical College, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Arikura
authors.forename: Naoya
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Teru
authors.initials: T
authors.lastname: Okitsu
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Noguchi
authors.forename: Toshinori
authors.initials: T
authors.lastname: Totsugawa
authors.forename: Takamasa
authors.initials: T
authors.lastname: Watanabe
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Matsumura
authors.forename: Masanobu
authors.initials: M
authors.lastname: Maruyama
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kosaka
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Onodera
authors.forename: Shinichi
authors.initials: S
authors.lastname: Kasai
mesh_terms: D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D015925:Cryopreservation; D005978:Glutathione; D066298:In Vitro Techniques; D007328:Insulin; D007763:Lac Operon; D016086:Lentivirus / Q000235:genetics; D008297:Male; D019188:Organ Preservation Solutions; D011887:Raffinose; D051381:Rats; D017208:Rats, Wistar; D014021:Tissue Preservation; D014161:Transduction, Genetic
chemical_list: D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose