Cryopreservation of primarily isolated porcine hepatocytes with UW solution.
Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.
Division of Gastroenterology I, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan.
Takemi
T
Kunieda
Masanobu
M
Maruyama
Teru
T
Okitsu
Norikuni
N
Shibata
Michihiko
M
Takesue
Toshinori
T
Totsugawa
Yoshikazu
Y
Kosaka
Takashi
T
Arata
Kazuya
K
Kobayashi
Hideaki
H
Ikeda
Mizuko
M
Oshita
Shuhei
S
Nakaji
Kenji
K
Ohmoto
Shinichiro
S
Yamamoto
Yuzuru
Y
Kurabayashi
Makoto
M
Kodama
Noriaki
N
Tanaka
Naoya
N
Kobayashi
D000241:Adenosine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D017690:Cell Transplantation / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005822:Genetic Vectors / Q000235:genetics; D005978:Glutathione / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology / Q000637:transplantation*; D007328:Insulin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D008107:Liver Diseases / Q000628:therapy; D016031:Liver Transplantation / Q000379:methods*; D008297:Male; D019188:Organ Preservation Solutions; D011887:Raffinose / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D034421:Sus scrofa; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous / Q000379:methods
D003451:Cryoprotective Agents; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D000641:Ammonia; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose