Human hepatocytes: Isolation, cryopreservation and applications in drug development

https://doi.org/10.1016/j.cbi.2007.01.001Get rights and content

Abstract

The recent developments in the isolation, culturing, and cryopreservation of human hepatocytes, and the application of the cells in drug development are reviewed. Recent advances include the improvement of cryopreservation procedures to allow cell attachment, thereby extending the use of the cells to assays that requires prolong culturing such as enzyme induction studies. Applications of human hepatocytes in drug development include the evaluation of metabolic stability, metabolite profiling and identification, drug–drug interaction potential, and hepatotoxic potential. The use of intact human hepatocytes, because of the complete, undisrupted metabolic pathways and cofactors, allows the development of data more relevant to humans in vivo than tissue fractions such as human liver microsomes. Incorporation of key in vivo factors with the intact hepatocytes in vitro may help predictive human in vivo drug properties. For instance, evaluation of drug metabolism and drug–drug interactions with intact human hepatocytes in 100% human serum may eliminate the need to determine in vivo intracellular concentrations for the extrapolation of in vitro data to in vivo. Co-culturing of hepatocytes and nonhepatic primary cells from other organs in the integrated discrete multiple organ co-culture (IdMOC) may allow the evaluation of multiple organ interactions in drug metabolism and drug toxicity. In conclusion, human hepatocytes represent a critical experimental model for drug development, allowing early evaluation of human drug properties to guide the design and selection of drug candidates with a high probability of clinical success.

Introduction

Primary cultures of hepatocytes represent an experimental tool that has been used extensively in biomedical research, both in academia as well as for commercial purposes such as drug development. The most exciting advances are the successful isolation, culturing, and cryopreservation of hepatocytes from human livers. Human hepatocytes are used routinely in drug development as an experimental model for the evaluation of key human-specific drug properties such as metabolic fate, drug–drug interactions, and drug toxicity. The applications range from the early screening the most appropriate new chemical entities for further development, to the determination of key drug properties for New Drug Applications (e.g. drug–drug interactions) to U.S. FDA.

Successful application of human hepatocytes in drug development requires a thorough understanding of the strengths and weaknesses of this valuable experimental system. This review is an effort to present a comprehensive review on the state-of-the-art of the isolation, cryopreservation, and applications of human hepatocytes in drug development, with emphasis on the research performed in our laboratories in the past 20 years.

Section snippets

Human hepatocyte isolation

One of the major advances in human hepatocyte technology is the availability of human livers for research. In the United States, livers procured but not used for transplantation are allowed to be used in research. The major reasons that procured livers are not used for transplantation are as follows:

  • 1.

    unavailability of a matched recipient;

  • 2.

    physical damage to the liver;

  • 3.

    pre-existing liver diseases;

  • 4.

    breach of sterility during the procurement process;

  • 5.

    high liver fat content;

  • 6.

    inappropriate age (too young

Cryopreservation

Hepatocytes, especially human hepatocytes, are now routinely used after they are cryopreserved [7], [8]. The general procedures for hepatocyte cryopreservation have not deviated extensively from the original procedures [9]. Via the use of equipments to control freezing rates (e.g. programmable control-rate freezer) and appropriate cryopreservation agents (e.g. dimethyl sulfoxide), hepatocytes now can be stored in liquid nitrogen (lower than −150 °C) for an extensively time period (years) with

Application of human hepatocytes in drug development

The key areas in drug development that can benefit from the use of human hepatocytes are drug metabolism, drug–drug interaction, and drug toxicity evaluation.

Conclusions

In the past decade, human hepatocytes have been used increasingly in drug development. The experience as of now is that relevant information can be obtained with this experimental system, including human-specific drug properties such as metabolic stability and fate, drug–drug metabolizing enzyme interactions, drug–transporter interactions, and drug toxicity. The availability of human hepatocytes for research has been dramatically increased due to the increased availability of commercial sources

References (50)

  • J.A. DiMasi et al.

    The price of innovation: new estimates of drug development costs

    J. Health Econ.

    (2003)
  • L.D. Kier et al.

    Applications of microarrays with toxicologically relevant genes (tox genes) for the evaluation of chemical toxicants in Sprague Dawley rats in vivo and human hepatocytes in vitro

    Mutat. Res.

    (2004)
  • A.P. Li

    A review of the common properties of drugs with idiosyncratic hepatotoxicity and the “multiple determinant hypothesis” for the manifestation of idiosyncratic drug toxicity

    Chem. Biol. Interact.

    (2002)
  • A.P. Li et al.

    A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

    Chem. Biol. Interact.

    (2004)
  • D. Jouin et al.

    Cryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance

    Eur. J. Pharm. Biopharm.

    (2006)
  • A.P. Li

    An integrated, multidisciplinary approach for drug safety assessment

    Drug. Discov. Today

    (2004)
  • X.N. Feng et al.

    Current status and perspective of liver preservation solutions

    Hepatobiliary Pancreat. Dis. Int.

    (2006)
  • M.N. Berry et al.

    High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study

    J. Cell Biol.

    (1969)
  • A.P. Li et al.

    Isolation and culturing of hepatocytes from human livers

    J. Tissue Cult. Meth.

    (1992)
  • L. Pichard et al.

    Human hepatocyte culture

    Meth. Mol. Biol.

    (2006)
  • K. Alexandrova et al.

    Large-scale isolation of human hepatocytes for therapeutic application

    Cell Transplant.

    (2005)
  • J.G. Hengstler et al.

    Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction

    Drug Metab. Rev.

    (2000)
  • L.J. Loretz et al.

    Optimization of cryopreservation procedures for rat and human hepatocytes

    Xenobiotica

    (1989)
  • M.J. Gomez-Lechon et al.

    Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing

    Xenobiotica

    (2006)
  • C. Terry et al.

    Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing

    Liver Transplant.

    (2006)
  • Cited by (0)

    View full text