UW solution: a promising tool for cryopreservation of primarily isolated rat hepatocytes.
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.
Arikura
Jun
J
Second Department of Surgery, Asahikawa Medical College, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan.
Kobayashi
Naoya
N
Okitsu
Teru
T
Noguchi
Hirofumi
H
Totsugawa
Toshinori
T
Watanabe
Takamasa
T
Matsumura
Toshihisa
T
Maruyama
Masanobu
M
Kosaka
Yoshikazu
Y
Tanaka
Noriaki
N
Onodera
Kazuhiko
K
Kasai
Shinichi
S
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
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose