Skip to main content
SpringerLink
Log in

Investigation of Functional and Morphological Integrity of Freshly Isolated and Cryopreserved Human Hepatocytes

  • Published:
Cell and Tissue Banking Aims and scope Submit manuscript

Abstract

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7×107 cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

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

Similar content being viewed by others

References cited

  • Adams RM, Wang M, Crane AM, Brown B, Darlington GJ and Ledley FD (1995) Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential. Cell Trasplant 4: 579-586

    Google Scholar 

  • Barr J, Weir AJ, Brendel K and Sipes IG (1991) Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue. Xenobiotica 21: 341-350

    Google Scholar 

  • Berry MN, Edwards AM and Barritt GJ (1991) Isolated hepatocytes: preparation, properties and applications. In: Burdon RH and Knippenberg PH (eds) Laboratory Techniques in Biochemistry and Molecular Biology, Vol 21, pp 1-460. Elsevier, Amsterdam

    Google Scholar 

  • Bilir B, Ostrowska A, Hanks T and Karrer FM (1995) Controlled rate freezing of hepatocytes: a step closer to human hepatocyte banking. Hepatology 22(pt 2): 211A

    Google Scholar 

  • Bode DC (1997) Organization and activities of human tissue banks in the U.S. with special reference to the International Institute for the Advancement of Medicine. Tiss Cult Res Commun 16: 157-163

    Google Scholar 

  • Brandhorst D, Brandhorst H, Hering BJ, Federlin K and Bretzel RG (1999) Large variability of the intracellular ATP content of human islets isolated from different donors. J Mol Med 77: 93-95

    Google Scholar 

  • Chesne C, Guyomard C, Fautrel A, Poullain MG, Fremond B, De Jong H and Guillouzo A (1993) Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation. Hepatology 18: 406-414

    Google Scholar 

  • Coundouris JA, Grant MH, Engeset J, Petrie JC and Hawksworth GM (1993) Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica 23: 1399-1409

    Google Scholar 

  • De Loecker R, Fuller BJ, Gruwez J and De Loecker W(1990) The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes. Cryobiology 27: 143-152

    Google Scholar 

  • De Loecker P, Fuller BJ, Koptelov VA and De Loecker W (1993) Metabolic activity of freshly prepared and cryopreserved hepatocytes in monolayer culture. Cryobiology 30: 12-18

    Google Scholar 

  • Denizot F and Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271-277

    Google Scholar 

  • Dorko K, Freeswick PD, Bartoli F, Cicalese L, Bardsley BA, Tzakis A and Nussler AK (1994) A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation. Cell Transplant 3: 387-395

    Google Scholar 

  • Fox IJ, Chowdhury JR, Kaufmann SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV and Strom SC (1998) Treatment of the Criggler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 14: 1422-1426

    Google Scholar 

  • Fujii T, Ha H, Yokoyama H, Hamamoto H, Yoon SH and Hori H (1995) Applications of MTT assay to primary cultured hepatocytes. Biol Pharm Bull 18: 1446-1449

    Google Scholar 

  • Guillouzo A, Morel F, Langouët, Maheo K and Rissel M (1997) Use of hepatocyte cultures for the study of hepatotoxic compounds. J Hepatol 26 (Suppl 2): 73-80

    Google Scholar 

  • Hewitt WR, Corno V, Eguchi S, Kamlot A, Middleton Y, Beeker T, Demetriou AA and Rozga J (1997) Isolation of human hepatocytes from livers rejected for whole organ transplantation. Transplant Proc 29: 1945-1947

    Google Scholar 

  • Hasset C, Laurenzana EM, Sidhu JS and Omiecinski C (1998) Effects of chemical inducers on human microsomal epoxide hydrolase in primary hepatocyte cultures. Biochem Pharmacol 55: 1059-1069

    Google Scholar 

  • Howard RB, Christensen AK, Gibbs FA and Pesch LA (1967) The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol 35: 675-684

    Google Scholar 

  • de Kanter R, Olinga P, Hof I, de Jager M, Verwillegen WA, Slooff MJ, Koster HJ, Meijer DKF and Groothius GMM (1998) A rapid and simple method for cryopreservation of human liver slices. Xenobiotica 28: 225-234

    Google Scholar 

  • Karrer FM, Bilir B, Gill A, Ostrowska A, Stegall M, Wachs M and Kam I (1997) Cryopreserved hepatocytes are poor stimulators of in vitro cytotoxicity. Transplant Proc 29: 1111-1112

    Google Scholar 

  • Li AP, Reith MK, Rasmussen A, Gorski JC, Hall SD, Xu L, Kaminski D and Cheng LK (1997) Primary human hepatocytes as a tool for the evaluation of structure-activity relationship in cytochrome P450 induction potential of xenobiotics: evaluation of rifampin, rifapentine and rifabutin. Chem Biol Interact 107: 17-30

    Google Scholar 

  • Maganto P, Cienfuegos JA, Santamaria L, Rodriguez V, Eroles G, Andres S, Castillo-Olivares JL and Municio AM (1990) Auxiliary liver by transplanted frozen-thawed hepatocyte. J Surg Res 48: 24-32

    Google Scholar 

  • Matsuda H, Yagi T, Matsuoka J, Yamamura H and Tanaka N (1999) Subzero nonfreezing storage of isolated rat hepatocytes in University of Wisconsin solution. Transplantation 67: 186-191

    Google Scholar 

  • Mito M and Kusano M (1993) Hepatocyte transplantation in man. Cell Transplant 2: 65-74

    Google Scholar 

  • Nyberg SL, Shatford RA, Payne WD, Hu WS and Cerra FB (1993) Staining with fluorescein diacetate correlates with hepatocyte function. Biotech Histochem 68: 56-63

    Google Scholar 

  • Olinga P, Merema M, Hof IH, de Jong KP, Slooff MJH, Meijer DKF and Groothuis GMM (1998) Effect of human liver source on the functionality of isolaited hepatocytes and liver slices. Drug Metab Dispos 26: 1-5

    Google Scholar 

  • Ostrowska A, Karrer FM and Bilir B (1999) Histological identification of purified and cryopreserved allogeneic hepatocytes following transplantation in a murine model without host immunosuppression. Transplant Int 12: 188-194

    Google Scholar 

  • Pang JM, Zaleski J and Kauffman FC (1997) Toxicity of allyl alcohol in primary cultures of freshly isolated and cryopreserved hepatocytes maintained on hydrated collagen gels. Toxicol Appl Pharmacol 142: 87-94

    Google Scholar 

  • Powis G, Melder DC and Wilke TJ (1989) Human and dog, but not rat, isolated hepatocytes have decreased foreign compound metabolizing activity compared to liver slices. Drug Metab Dispos 17: 526-531

    Google Scholar 

  • Ruegg CE, Silber PM, Mughal RA, Ismail J, Lu C, Bode DC and Li AP (1997) Cytochrome-P450 induction and conjugated metabolism in primary human hepatocytes after cryopreservation. In Vitro Toxicol 10: 217-222

    Google Scholar 

  • Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13: 29-83

    Google Scholar 

  • Soriano HE, Lewis D, Legner M, Brandt M, Baley P, Darlington G, Finegold M and Ledley FD (1992) The use of Dil-marked hepatocytes to demonstrate orthotopic, intrahepatic engraftment following hepatocellular transplantation. Transplantation 54: 717-723

    Google Scholar 

  • Strom SC, Fisher RA, Thompson MT, Sanyal AJ, Cole PE, Ham JM and Posner MP (1997) Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 63: 559-569

    Google Scholar 

  • Strom SC, Chowdhury JR and Fox IJ (1999) Hepatocyte transplantation for the treatment of human disease. Semin Liv Dis 19: 39-48

    Google Scholar 

  • Sun J, Wang L, Waring MA, Wang C, Woodman KK and Sheil AG (1997) Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices. Artif Organs 21: 408-413

    Google Scholar 

  • Vreugdenhil PK, Marsh DC and Southard JH (1996) Comparison of isolated hepatocytes and tissue slices for study of liver hypothermic preservation/reperfusion injury. Cryobiology 33: 430-435

    Google Scholar 

  • Walsh JS, Patanella JE, Halm KA and Facchine KL (1995) An improved assay for the assessment of liver slice metabolic viability using 7-Ethoxycoumarin. Drug Metab Dispos 23: 869-874

    Google Scholar 

  • Wroblewski F and LaDue JS (1955) Lactic dehydrogenase activity in blood. Proc Soc Exp Biol Med 90: 210

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. International Institute for the Advancement of Medicine, Scranton, PA, USA; IIAM/T3

    Alina Ostrowska

  2. 125 May St., Suite 300, Edison, N7 08837, USA

    Alina Ostrowska

  3. Rhône-Poulenc Rorer, Collegeville, PA, USA

    Donald C. Bode

  4. International Institute for the Advancement of Medicine, Scranton, PA, USA

    Jennifer Pruss, Gina D. Smith & Stephen Zeisloft

  5. Centura Health Porter Adventist Hospital, Denver, CO, USA

    Bahri Bilir

Authors
  1. Alina Ostrowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Donald C. Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer Pruss
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bahri Bilir
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gina D. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephen Zeisloft
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ostrowska, A., Bode, D.C., Pruss, J. et al. Investigation of Functional and Morphological Integrity of Freshly Isolated and Cryopreserved Human Hepatocytes. Cell Tissue Banking 1, 55–68 (2000). https://doi.org/10.1023/A:1010175906791

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1010175906791

Search

Navigation