Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside.
Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 microg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 +/- 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 microg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 microg/ml) would be a useful cold preservation means for the development of cell therapies.
Takesue
Michihiko
M
Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan.
Maruyama
Masanobu
M
Shibata
Norikuni
N
Kunieda
Takemi
T
Okitsu
Teru
T
Sakaguchi
Masakiyo
M
Totsugawa
Toshinori
T
Kosaka
Yoshikazu
Y
Arata
Akira
A
Ikeda
Hideaki
H
Matsuoka
Junji
J
Oyama
Toshie
T
Kodama
Makoto
M
Ohmoto
Kenji
K
Yamamoto
Shinichiro
S
Kurabayashi
Yuzuru
Y
Yamamoto
Itaru
I
Tanaka
Noriaki
N
Kobayashi
Naoya
N
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000493:Allopurinol / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D017690:Cell Transplantation / Q000379:methods* / Q000639:trends; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods* / Q000639:trends; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000637:transplantation*; D007328:Insulin / Q000494:pharmacology; D008107:Liver Diseases / Q000628:therapy; D016031:Liver Transplantation / Q000379:methods* / Q000639:trends; D008297:Male; D019188:Organ Preservation Solutions; D011887:Raffinose / Q000494:pharmacology; D034421:Sus scrofa; D014183:Transplantation, Heterologous / Q000379:methods / Q000639:trends
D061945:Caspase Inhibitors; D003451:Cryoprotective Agents; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; C065378:ascorbic acid 2-O-glucoside; D000493:Allopurinol; D000641:Ammonia; D000255:Adenosine Triphosphate; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose; D001205:Ascorbic Acid