|ognizant Communication Corporation|
VOLUME 12, NUMBER 2, 2003
Cell Transplantation, Vol. 12, pp. 101-107, 2003
0963-6897/03 $20.00 + 00
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Yo-ichi Yamashita,1 Mitsuo Shimada,1 Eiji Tsujita,1 Ken Shirabe,2 Hiroyuki Ijima,1 Kohji Nakazawa,2 Ryoichi Sakiyama,2 Junji Fukuda,2 Kazumori Funatsu,2 and Keizo Sugimachi1
1Department of Surgery and Science, Graduate School of Medical Sciences, and 2Department of Chemical Engineering, Graduate School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
We have reported the usefulness of a polyurethane foam packed-bed culture system of hepatocyte spheroids as a hybrid artificial liver support system (PUF- HALSS). The aim of this study was to evaluate in detail the efficacy in serum parameters regarding the liver function of a larger version of the PUF-HALSS containing 2 x 1010 porcine hepatocytes for clinical use in warm ischemic liver failure pigs. Warm ischemic liver failure pigs weighing 25 kg were divided into two groups: (1) a control group (n = 3), in which each pig was attached to a PUF-HALSS without hepatocytes, and (2) a HALSS group (n = 3), in which each pig was attached to a PUF-HALSS. In the HALSS group, the increase of blood ammonia was completely suppressed and blood lactate levels were significantly suppressed. The Fisher's ratio was better maintained, and the increase of total bile acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid was significantly suppressed in the HAILSS group. Serum creatinine levels were significantly lower, and blood glucose levels were significantly higher in the HALSS group. Serum levels of tumor necrosis factor- a were not elevated in either group. In conclusion, the larger version of the PUF-HALSS demonstrated many advantages as a liver support system in warm ischemic liver failure pigs.
Key words: Hybrid artificial liver support system; Polyurethane foam; Porcine hepatocytes; Spheroid; Fulminant hepatic failure
Address correspondence to Yo-ichi Yamashita, M.D., Ph.D., Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: 81-92-642-5469; Fax: 81-92-642-5482; E-mail: firstname.lastname@example.org
Apoptotic Cell Death and Function of Cryopreserved Porcine Hepatocytes in a Bioartificial Liver
Takakazu Matsushita,1 Toshikazu Yagi,1 Joseph A. Hardin,1 Jennifer D. Cragun,2 Frank W. Crow,2 H. Robert Bergen, III,2 Gregory J. Gores,3 and Scott L. Nyberg1
1Division of Transplantation Surgery, 2Division of Biochemistry and Molecular Biology, and 3Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN
We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 mmol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 mmol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.
Key words: Bioartificial liver; Hepatocyte; Apoptosis; Cryopreservation
Address correspondence to Scott L. Nyberg, M.D., Ph.D., Liver Transplantation Unit, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905. Tel: (507) 266-6772; Fax: (507) 266-1856; E-mail: email@example.com
Dynamics of Cryoprotectant Permeation in Porcine Heart Valve Leaflets
Jonathan R. T. Lakey,1,2 Lisa M. H. Helms,1 Gabrielle Moser,3 Bruce Lix,4 Carolyn M. Slupsky,4 Ivan M. Rebeyka,2 Brian D. Sykes,4 and Locksley E. McGann2,3
1Surgical-Medical Research Institute, 2Department of Surgery, 3Department of Laboratory Medicine and Pathology, and 4Protein Engineering Network Centres of Excellence, University of Alberta, Edmonton, Canada
Valve replacement is a common cardiovascular procedure for the treatment of a variety of congenital and acquired defects. Many surgical programs rely on cryopreserved heart valves from regional tissue bank programs to meet clinical demands. Current cryopreservation strategies for heart valves are empirically derived. The aim of this study was to use proton nuclear magnetic resonance spectroscopy (NMR) to monitor changes in cryoprotectant concentration in isolated heart valve leaflets. Porcine aortic valves were locally obtained, freshly isolated, and allowed to equilibrate at various experimental temperatures (22°C, 10°C, 4°C) for 1 h prior to immersion in 1 M Me2SO solution. At defined intervals (0, 0.25, 0.5, 1, 2, 6, and 24 h) the valves were removed from the Me2SO and the leaflets were rapidly dissected and equilibrated in deuterium oxide (D2O). Using previously described techniques the Me2SO concentration in the heart valve leaflets was determined by NMR and the diffusion coefficient was calculated as a function of time and temperature. Heart valve leaflets were fully equilibrated with Me2SO after approximately 2 h of exposure at 22°C while equilibrium was not reached >6 h or more at 10°C and 4°C. These results indicate that that permeation of Me2SO in heart valves is strongly temperature dependent. Furthermore, this study provides a quantitative measure of Me2SO permeation and cryoprotectant at equilibration in heart valve leaflets. The clinical applications of these findings may help to optimize the balance between the protective and toxic effects of cryoprotectants and lead to improved methods of preservation of heart valves.
Key words: Cryopreservation; Heart valve; Me2SO; Cryoprotectant permeation; Proton NMR spectroscopy
Address correspondence to Jonathan R. T. Lakey, Ph.D., Assistant Professor of Surgery, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: firstname.lastname@example.org
Digital Imaging as a Possible Approach in Evaluation of Islet Yield
Peter Girman,1 Jan Kríz,1 Jozef Friedmanský,2 and Frantisek Saudek1
1Diabetes Center, Laboratory of Pancreatic Islets, Institute
for Clinical and Experimental Medicine, Prague, Czech Republic
2Technical University, Kosice, Slovak Republic
Digital image analysis (DIA) is a new method in assessment of islet amount, which is expected to provide reliable and consistent results. We compared this method with conventional counting of small numbers of rat islets. Islets were isolated from 8 pancreases and counted in 24 samples in duplicate, first routinely by sizing according to estimated diameters under a calibrated reticule and then by processing of islets pictures taken by camera. As presumed, no significant difference was found in absolute numbers of islets per sample between DIA and conventional assessment. Volumes of islets per sample measured by DIA were on average more than 10% higher than amounts evaluated conventionally, which was statistically significant. DIA has been shown to be an important method to remove operator bias and provide consistent results. Evaluation of only two dimensions of three-dimensional objects still represents a certain limitation of this technique. With lowering of computer prices the system could become easily available for islet laboratories.
Key words: Digital imaging; Islets; Quantification; Rat
Address correspondence to Peter Girman, Institute for Clinical and Experimental Medicine, Laboratory of Pancreatic Islets, Diabetes Center, Vídenská 19858/9, Praha-Krc, Czech Republic. Tel: 00420 2 6136 4107/00420 2 6136 2288; E-mail: email@example.com