Skip to main content
SpringerLink
Log in

Hypothermic storage of isolated human hepatocytes: a comparison between University of Wisconsin solution and a hypothermosol platform

  • Organ Toxicity and Mechanisms
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

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

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Kim JS, Southard JH (1999) Phospholipid metabolism of hypothermically stored rat hepatocytes. Hepatology 30:1232–1240. doi:10.1002/hep.510300531

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Ostrowska A, Hu X, Bode DC (2002) Pre-clinical characterisation of freshly isolated and cryopreserved human hepatocytes. Adv Tissue Bank 6:233–249

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Pollack M, Leeuwenburgh C (2001) Apoptosis and aging: role of the mitochondria. J Gerontol A Biol Sci Med Sci 56:B475–B482

    PubMed  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Wroclaw University of Environmental and Life Sciences, Kozuchowska 5B, 51-631, Wrocław, Poland

    Alina Ostrowska

  2. Emisphere Technologies, Inc., 765 Old Saw Mill River Road, Tarrytown, NY, 10591, USA

    Kenan Gu

  3. Absorption Systems LP, 440 Creamery Way, Suite 300, Exton, PA, 19341-2554, USA

    Donald C. Bode

  4. Department of Biological Sciences, State University of New York, Vestal Parkway East, Binghamton, NY, 13902, USA

    Robert G. Van Buskirk

Authors
  1. Alina Ostrowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenan Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Donald C. Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert G. Van Buskirk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alina Ostrowska.

Rights and permissions

Reprints 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-009-0419-x

Keywords

Search

Navigation