Abstract
Until now little is known about the functional integrity of human hepatocytes after hypothermic storage. In order to address this limitation, we evaluated several commercially available hypothermic preservation media for their abilities to protect freshly isolated hepatocytes during prolonged cold storage. Human hepatocytes were isolated from non-transplantable/rejected donor livers and resuspended in ice-cold University of Wisconsin solution (UW), HypoThermosol-Base (HTS-Base), or HypoThermosol-FRS (HTS-FRS) with or without the addition of fetal bovine serum. Cells were stored at 4°C for 24–72 h, and evaluated for hepatocyte viability (trypan blue exclusion, or labeling with fluorochromes), cell attachment, and function. The energy status of hepatocytes was evaluated by measurement of intracellular adenosine 5′-triphosphate. To determine whether the test cells expressed metabolic functions of freshly isolated cells, the activities of major phase I (cytochromes P450, FMO) and phase II (UGT, ST) drug-metabolizing enzymes were examined. Although hepatocytes are shown to be satisfactory after 24 h storage in all of the tested solutions, the cell viability, energy status, and xenobiotic metabolism following cold preservation in HTS-FRS was consistently and, in some cases, markedly higher when compared with other systems. The same metabolites for each of the tested substrates were detected in all groups of cells. Moreover, the use of HTS-FRS eliminates the need for serum in preservation solutions. HTS-FRS represents an improved solution compared to HTS-Base and UW for extending the shipping/storage time of human hepatocytes.
Similar content being viewed by others
References
Abrahamse SL, d Heimel P, Hartman RJ, Chamuleau RA, van Gulik TM (2003) Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW, HTK, and Celsior solutions. Cell Transplant 12:59–68. doi:10.3727/000000003783985160
Adams RM, Wang M, Crane AM, Brown B, Darlington GJ, Ledley FD (1995) Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential. Cell Transplant 4:579–586. doi:10.1016/0963-6897(95)02001-2
Almada L, Bellarosa C, Giraudi P, Mamprin M, Mediavilla M, Guibert E, Tiribelli C, Rodriguez J (2005) The urea cycle activity and its gene expression in rat hepatocytes are not affected by cold storage in University of Wisconsin solution. Ann Hepatol 4:224–227
Almada L, Bellarosa C, Giraudi P, Mamprin M, Mediavilla M, Guibert E, Tiribelli C, Rodriguez J (2006) Gene expression and activity of urea cycle enzymes of rat hepatocytes cold stored up to 120 h in University of Wisconsin solution. Cryobiology 52:393–400. doi:10.1016/j.cryobiol.2006.02.001
Altman SA, Randers L, Rao G (1993) Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog 9:671–674. doi:10.1021/bp00024a017
Arikura J, Kobayashi N, Okitsu T, Noguchi H, Totsugawa T, Watanabe T, Matsumura T, Maruyama M, Kosaka Y, Tanaka N, Onodera K, Kasai S (2002) UW solution: a promising tool for cryopreservation of primarily isolated rat cells. J Hepatobiliary Pancreat Surg 9:742–749. doi:10.1007/s005340200103
Baust JM, Van Buskirk RG, Baust JG (2000) Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell Dev Biol Anim 36:262–270. doi:10.1290/1071-2690(2000)036<0262:CVIFIO>2.0.CO;2
Baust JM, Van Buskirk RG, Baust JG (2002a) Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulfoxide concentration. Cryobiology 45:97–108. doi:10.1016/S0011-2240(02)00100-1
Baust JM, Van Buskirk RG, Baust JG (2002b) Gene activation of the apoptotic caspase cascade following cryogenic storage. Cell Preserv Technol 1:63–80. doi:10.1089/15383440260073301
Bessems M, Doorschodt BM, van Vliet AK, van Gulik TM (2004) Preservation of rat livers by cold storage: a comparison between University of Wisconsin solution and Hypothermosol. Ann Transplant 9:35–37
Dahdah NS, Taylor MJ, Russo P, Wagerle LC (1999) Effects of hypothermosol, an experimental acellular solution fdor tissue preservation and cardiopulmonary bypass, on isolated newborn lamb coronary vessels subjected to ultra profound hypothermia and anoxia. Cryobiology 39:58–68. doi:10.1006/cryo.1999.2185
Duval M, Plin C, Elimadi A, Vallerand D, Tillement JP, Morin D, Haddad PS (2006) Implication of mitochondrial dysfunction and cell death in cold preservation-warm reperfusion-induced hepatocyte injury. Can J Physiol Pharmacol 84:547–554
Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothius GM, Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234. doi:10.1080/03602530601093489
Janssen H, Janssen PH, Broelsch CE (2003) Celsior solution compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia-reperfusion injury. Transpl Int 16:515–522
Kerkweg U, Li T, de Groot H, Rauen U (2002) Cold-induced apoptosis of rat liver cells in University of Wisconsin solution: the central role of chelatable iron. Hepatology 35:560–567. doi:10.1053/jhep.2002.31869
Kim JS, Southard JH (1999) Phospholipid metabolism of hypothermically stored rat hepatocytes. Hepatology 30:1232–1240. doi:10.1002/hep.510300531
Kunieda T, Maruyama M, Okitsu T, Shibata N, Takesue M, Totsugawa T, Kosaka Y, Arata T, Kobayashi K, Ikeda H, Oshita M, Nakaji S, Ohmoto K, Yamamoto Y, Kurabayashi Y, Kodama M, Tanaka N, Kobayashi N (2003) Cryopreservation of primarily isolated porcine hepatocytes with UW solution. Cell Transplant 12:607–616
Lai PH, Sielaff TD, Hu WS (2005) Sustaining a bioartificial liver under hypothermic conditions. Tissue Eng 11:427–437. doi:10.1089/ten.2005.11.427
LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S, Richert L (2005) Isolation and culture of primary human hepatocytes. Methods Mol Biol 290:207–229
Li AP (2007) Human hepatocytes: isolation, cryopreservation and applications in drug development. Chem Biol Interact 168:16–29. doi:10.1016/j.cbi.2007.01.001
Li AP, Kedderis GL (1997) Primary hepatocyte culture as an experimental model for the evaluation of interactions between xenobiotics and drug-metabolizing enzymes. Chem Biol Interact 107:1–3. doi:10.1016/S0009-2797(97)00069-0
Li G, Liu Y, Liang J (2003) Isolation and protective effect in UW solution of human hepatocytes during cold storage. Int Congr Ser 1255:217–218. doi:10.1016/S0531-5131(03)00657-5
Łaba A, Ostrowska A, Patrzałek A, Paradowski D, Lange A (2005) Characterization of human hepatocytes isolated from non-transplantable livers. Arch Immunol Ther Exp (Warsz) 53:442–453
Maccarrone M, Bari M, Battista N, Di Rienzo M, Falciglia K, Finazzi Agro A (2001) Oxidation products of polyamines induce mitochondrial uncoupling and cytochrome c relaease. FEBS Lett 507:30–34. doi:10.1016/S0014-5793(01)02949-0
Mathew AJ, Van Buskirk RG, Baust JG (2002) Improved hypothermic preservation of human renal cells through suppression of both apoptosis and necrosis. Cell Preserv Technol 1:239–253. doi:10.1089/15383440260682071
Mathew AJ, Baust JM, Van Buskirk RG, Baust JG (2004) Cell preservation in reparative medicine: evolution of individualized solution composition. Tissue Eng 10:1662–1671. doi:10.1089/ten.2004.10.1662
Mathew AJ, Baust JM, Ostrowska A, Van Buskirk RG, Baust JG (2006) Extended hypothermic storage of isolated human hepatocytes using HypoThermosol-FRS. Cryobiology 53:437. doi:10.1016/j.cryobiol.2006.10.166
Matsushita T, Yagi T, Hardin JA, Cragun JD, Crow FW, Bergen HRIII, Gores GJ, Nyberg SL (2003) Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver. Cell Transplant 12:109–121
Meneses-Lorente G, Pattison C, Guyomard C, Chesne C, Heavens R, Watt AP, Sohal B (2007) Utility of long-term cultured human hepatocytes as an in vitro model for cytochrome P450 induction. Drug Metab Dispos 35:215–220. doi:10.1124/dmd.106.009423
Miyamoto Y, Suzuki S, Nomura K, Enosawa S (2006) Improvement of hepatocyte viability after cryopreservation by supplementation of long-chain oligosaccharide in the freezing medium in rats and humans. Cell Transplant 15:911–919. doi:10.3727/000000006783981404
Moray G, Sevmis S, Karakayali FY, Gorur SK, Haberal M (2006) Comparison of histidine-tryptophan-ketoglutarate and University of Wisconsin in living-donor liver transplantation. Transplant Proc 38:3572–3575. doi:10.1016/j.transproceed.2006.10.174
Neveux N, De Bandt JP, Chaumeil JC, Cynober L (2002) Hepatic preservation, liposomally entrapped adenosine triphosphate and nitric oxide production: a study of energy state and protein metabolism in the cold-stored rat liver. Scand J Gastroenterol 37:1057–1063. doi:10.1080/003655202320378266
Oleson FB, Berman CL, Li AP (2004) An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes. Chem Biol Interact 150:137–147. doi:10.1016/j.cbi.2004.08.004
Olinga P, Marema M, Slooff MJ, Meijer DK, Groothuis GM (1997) Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes. J Hepatol 27:738–743. doi:10.1016/S0168-8278(97)80091-8
Ostrowska A, Bode DC, Pruss J, Bilir B, Smith GD, Zeisloft S (2000) Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes. Cell Tissue Bank 1:55–68. doi:10.1023/A:1010175906791
Ostrowska A, Hu X, Bode DC (2002) Pre-clinical characterisation of freshly isolated and cryopreserved human hepatocytes. Adv Tissue Bank 6:233–249
Panzera P, Rotelli MT, Salerno AM, Cicco G, Catalano G, D’Elia G, Greco L, Lupo L, Memeo V (2005) Solutions for organ perfusion and storage: haemorheologic aspects. Transplant Proc 37:2456–2458. doi:10.1016/j.transproceed.2005.06.024
Pollack M, Leeuwenburgh C (2001) Apoptosis and aging: role of the mitochondria. J Gerontol A Biol Sci Med Sci 56:B475–B482
Rauen U, Polzar B, Stephan H, Mannherz HG, De Grot H (1999) Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB 13:155–168
Richert L, Liguori MJ, Abadie C, Heyd B, Mantion G, Halkic N, Waring JF (2006) Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab Dispos 34:870–879. doi:10.1124/dmd.105.007708
Riga AT, Fuller BJ, Davidson BR (2000) The effect of human organ preservation and albumin flush solution on in vitro cell metabolic activity. Clin Chem Lab Med 38:1191–1193. doi:10.1515/CCLM.2000.186
Serralta A, Donato MT, Martinez A, Pareja E, Orbis F, Castell JV, Mir J, Gomez-Lechon MJ (2005) Influence of preservation solution on the isolation and culture of human hepatocytes from liver grafts. Cell Transplant 14:837–843. doi:10.3727/000000005783982495
Shanina IV, Kravchenko LP, Fuller BJ, Grischenko VI (2000) A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes. Cryobiology 41:315–318. doi:10.1006/cryo.2000.2286
Snyder KK, Van Buskirk RG, Baust JM, Mathew AJ, Baust JG (2004) Biological packaging for the global cell and tissue therapy markets. Bioprocessing J 3:39–45
Snyder KK, Baust JM, Van Buskirk RG, Baust JG (2005) Enhanced hypothermic storage of neonatal cardiomyocytes. Cell Preserv Technol 3:61–74. doi:10.1089/cpt.2005.3.61
Sosef MN, Baust JM, Sugimachi K, Fowler A, Tompkins RG, Toner M (2005) Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function. Ann Surg 241:125–133
Straatsburg IH, Abrahamse SL, Song SW, Hartman RJ, Van Gulik TM (2002) Evaluation of rat liver apoptotic and necrotic cell death after cold storage using UW, HTK, and Celsior. Transplantation 74:458–464. doi:10.1097/00007890-200208270-00005
Takesue M, Maruyama M, Shibata N, Kunieda T, Okitsu T, Sakaguchi M, Totsugawa T, Kosaka Y, Arata A, Ikeda H, Matsuoka J, Oyama T, Kodama M, Ohmoto K, Yamamoto S, Kurabayashi Y, Yamamoto I, Tanaka N, Kobayashi N (2003) Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside. Cell Transplant 12:599–606
Tanaka K, Soto-Gutierrez A, Navarro-Alvarez N, Carrillo JD, Kobayashi N (2006) Functional hepatocyte culture and its application to cell therapies. Cell Transplant 15:855–864. doi:10.3727/000000006783981332
Ullrich A, Berg C, Hengstler JG, Runge D (2007) Use of standardised and validated long-term human hepatocyte culture system for repetitive analyses of drugs: repeated administrations of acetaminophen reduces albumin and urea secretion. ALTEX 24:35–40
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ostrowska, A., Gu, K., Bode, D.C. et al. Hypothermic storage of isolated human hepatocytes: a comparison between University of Wisconsin solution and a hypothermosol platform. Arch Toxicol 83, 493–502 (2009). https://doi.org/10.1007/s00204-009-0419-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-009-0419-x