Yi Li1,2,3, Harvey S Chen2,3, Mohammed Shaheen2,3, Dong Jin Joo2,3, Bruce P Amiot2, Piero Rinaldo4, Scott L Nyberg2,3. 1. Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China. 2. Department of Surgery, Mayo Clinic, Rochester, Minnesota. 3. William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota. 4. Department Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
Abstract
BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.
BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroidscold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.
Authors: Jaime M Glorioso; Shennen A Mao; Brian Rodysill; Taufic Mounajjed; Walter K Kremers; Faysal Elgilani; Raymond D Hickey; Hakon Haugaa; Christopher F Rose; Bruce Amiot; Scott L Nyberg Journal: J Hepatol Date: 2015-07-02 Impact factor: 25.083
Authors: Harvey S Chen; Dong Jin Joo; Mohammed Shaheen; Yi Li; Yujia Wang; Jian Yang; Clara T Nicolas; Kelly Predmore; Bruce Amiot; Gregory Michalak; Taofic Mounajjed; Jeff Fidler; Walter K Kremers; Scott L Nyberg Journal: Hepatology Date: 2018-12-27 Impact factor: 17.425
Authors: Filippos G Sagias; Ragai R Mitry; Robin D Hughes; Sharon C Lehec; Ameet G Patel; Mohamed Rela; Giorgina Mieli-Vergani; Nigel D Heaton; Anil Dhawan Journal: Cell Transplant Date: 2010-06-29 Impact factor: 4.064
Authors: Achilles A Demetriou; Robert S Brown; Ronald W Busuttil; Jeffrey Fair; Brendan M McGuire; Philip Rosenthal; Jan Schulte Am Esch; Jan Lerut; Scott L Nyberg; Mauro Salizzoni; Elizabeth A Fagan; Bernard de Hemptinne; Christoph E Broelsch; Maurizio Muraca; Joan Manuel Salmeron; John M Rabkin; Herold J Metselaar; Daniel Pratt; Manuel De La Mata; Lawrence P McChesney; Gregory T Everson; Philip T Lavin; Anthony C Stevens; Zorina Pitkin; Barry A Solomon Journal: Ann Surg Date: 2004-05 Impact factor: 12.969