Skip to main content
SpringerLink
Log in
Book cover

Hepatocytes pp 83–105Cite as

Optimisation of the Cryopreservation of Primary Hepatocytes

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 640))

Abstract

The use of cryopreserved hepatocytes has increased in the last decade due to the improvement of the freezing and thawing methods and has even achieved acceptance by the U.S. Food and Drug Administration for use in drug-metabolising enzyme induction studies. This chapter provides an overview of the theories behind the process of cryopreservation and some of the most important advances which have led to the ability to cryopreserve hepatocytes, which when thawed retain functions similar to fresh cells. Parameters such as cell density, cryoprotectants and freezing media should be considered as well as storage conditions and thawing techniques. Special emphasis is placed on human hepatocytes but information for the cryopreservation of animal hepatocytes is also described. Finally, a suggested method for optimising cryopreservation method is outlined.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Nutt, L.H., Attenburrow, V.D., and Fuller, B.J. (1980) Investigations into repair of freeze/thaw damage in isolated rat hepatocytes. Cryo-Letters 1, 513–518.

    Google Scholar 

  2. Innes, G.K., Fuller, B.J., and Hobbs, K.E. (1988) Functional testing of hepatocytes following their recovery from cryopreservation. Cryobiology 25, 23–30.

    Article  PubMed  CAS  Google Scholar 

  3. Diener, B., Utesch, D., Beer, N., Dürk, H., and Oesch, F. (1993) A method for the cryopreservation of liver parenchymal cells for studies of xenobiotics. Cryobiology 30, 116–127.

    Article  PubMed  CAS  Google Scholar 

  4. Roymans, D., Annaert, P., Van Houdt, J., Weygers, A., Noukens, J., Sensenhauser, C., Silva, J., Van Looveren, C., Hendrickx, J., Mannens, G., and Meuldermans, W. (2005) Expression and induction potential of cytochromes P450 in human cryopreserved hepatocytes. Drug Metab. Dispos. 33, 1004–1016.

    Article  PubMed  CAS  Google Scholar 

  5. Bi, Y.A., Kazolias, D., and Duignan, D.B. (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab. Dispos. 34, 1658–1665.

    Article  PubMed  CAS  Google Scholar 

  6. Gómez-Lechón, M.J., Lopez, P., and Castell, J.V. (1984) Biochemical functionality and recovery of hepatocytes after deep freezing storage. In Vitro 20, 826–832.

    Article  PubMed  Google Scholar 

  7. Loretz, L.J., Li, A.P., Flye, M.W., and Wilson, A.G. (1989) Optimization of cryopreservation procedures for rat and human hepatocytes. Xenobiotica 19, 489–498.

    Article  PubMed  CAS  Google Scholar 

  8. Chesné, C. and Guillouzo, A. (1988) Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions. Cryobiology 25, 323–330.

    Article  PubMed  Google Scholar 

  9. Meryman, H.T. (1961) Freezing of living cells: biophysical considerations. Natl. Cancer Inst. Monogr. 7, 7–15.

    Google Scholar 

  10. Diller, K.R. (1985) The influence of controlled ice nucleation on regulating the thermal history during freezing. Cryobiology 22, 268–281.

    Article  Google Scholar 

  11. Chesné, C., Guyomard, C., Fautrel, A., Poullain, M.G., Frémond, 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.

    PubMed  Google Scholar 

  12. Swales, N.J., Luong, C., and Caldwell, J. (1996) Cryopreservation of rat and mouse hepatocytes. I. Comparative viability studies. Drug Metab. Dispos. 24, 1218–1223.

    PubMed  CAS  Google Scholar 

  13. Swales, N.J., Johnson, T., and Caldwell, J. (1996) Cryopreservation of rat and mouse hepatocytes. II. Assessment of metabolic capacity using testosterone metabolism. Drug Metab. Dispos. 24, 1224–1230.

    PubMed  CAS  Google Scholar 

  14. Price, J.A., Caldwell, J., and Hewitt, N.J. (2006) The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes. Cryobiology 53, 182–193.

    Article  PubMed  CAS  Google Scholar 

  15. Gómez-Lechón, M.J., Lahoz, A., Jiménez, N., Vicente Castell, J., and Donato, M.T. (2006) Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing. Xenobiotica 36, 457–472.

    Article  PubMed  Google Scholar 

  16. Alexandre, E., Viollon-Abadie, C., David, P., Gandillet, A., Coassolo, P., Heyd, B., Mantion, G., Wolf, P., Bachellier, P., Jaeck, D., and Richert, L. (2002) Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiology 44, 103–113.

    Article  PubMed  CAS  Google Scholar 

  17. Loven, A.D., Olsen, A.K., Friis, C., and Andersen, B. (2005) Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes. Chem. Biol. Interact. 155, 21–30.

    Article  PubMed  Google Scholar 

  18. Li, A.P., Lu, C., Brent, J.A., Pham, C., Fackett, A., Ruegg, C.E., and Silber, P.M. (1999) Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chem. Biol. Interact. 121, 17–35.

    Article  PubMed  CAS  Google Scholar 

  19. Mazur, P. (1984) Freezing of living cells: mechanisms and implications. Am. J. Physiol. Cell Physiol. 247, C125–C142.

    CAS  Google Scholar 

  20. Swales, N.J. and Utesch, D. (1998) Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions. Xenobiotica 28, 937–948.

    Article  PubMed  CAS  Google Scholar 

  21. Hewitt, N.J. and Utesch, D. (2004) Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures. Hum. Exp. Toxicol. 23, 307–316.

    Article  PubMed  CAS  Google Scholar 

  22. Swales, N.J. and Caldwell, J. (1997) Phase 1 and 2 metabolism in freshly isolated hepatocytes and subcellular fractions from rat, mouse, chicken and ox livers. Pest. Sci. 49, 291–299.

    Article  CAS  Google Scholar 

  23. Swales, N.J. (1993) Mechanistic studies of the cytotoxicity of trans-cinnamaldehyde. PhD Thesis. University of London.

    Google Scholar 

  24. McKay, G.C., Henderson, C., Goldie, E., Connel, G., Westmoreland, C., and Grant, M.H. (2002) Cryopreservation of rat hepatocyte monolayers: cell viability and cytochrome P450 content in post-thaw cultures. Toxicol. In Vitro 16, 71–79.

    Article  PubMed  CAS  Google Scholar 

  25. Stevenson, D.J., Morgan, C., Goldie, E., Connel, G., and Grant, M.H. (2004) Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation. Cryobiology 49, 97–113.

    Article  PubMed  CAS  Google Scholar 

  26. Koebe, H.G., Mühling, B., Deglmann, C.J., and Schildberg, F.W. (1999) Cryopreserved porcine hepatocyte cultures. Chem. Biol. Interact. 121, 99–115.

    Article  PubMed  CAS  Google Scholar 

  27. Kafert-Kasting, S., Alexandrova, K., Barthold, M., Laube, B., Friedrich, G., Arseniev, L., and Hengstler, J.G. (2006) Enzyme induction in cryopreserved human hepatocyte cultures. Toxicology 220, 117–125.

    Article  PubMed  CAS  Google Scholar 

  28. Terry, C., Dhawan, A., Mitry, R.R., Lehec, S.C., and Hughes, R.D. (2006) Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing. Liver Transplant. 12, 165–177.

    Article  Google Scholar 

  29. Guillouzo, A., Rialland, L., Fautrel, A., and Guyomard, C. (1999) Survival and function of isolated hepatocytes after cryopreservation. Chem. Biol. Interact. 121, 7–16.

    Article  PubMed  CAS  Google Scholar 

  30. Silva, J.M., Day, S.H., and Nicoll-Griffith, D.A. (1999) Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes. Chem. Biol. Interact. 121, 49–63.

    Article  PubMed  CAS  Google Scholar 

  31. Orrenius, S. and Moldeus, P. (1984) The multiple roles of glutathione in drug metabolism. Trends Pharmacol. Sci. 5, 432–435.

    Article  CAS  Google Scholar 

  32. Stevenson, D.J., Morgan, C., McLellan, L.I., and Helen Grant, M. (2007) Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures. Toxicol. In Vitro 21, 527–532.

    Article  PubMed  CAS  Google Scholar 

  33. Vairetti, M., Griffini, P., Pietrocola, G., Richelmi, P., and Freitas, I. (2001) Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione. Free Radic. Biol. Med. 31, 954–961.

    Article  PubMed  CAS  Google Scholar 

  34. Griffith, O.W., Bridges, R.J., and Meister, A. (1979) Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 76, 6319–6322.

    Article  PubMed  CAS  Google Scholar 

  35. Zaleski, J., Richburg, J., and Kauffman, F.C. (1993) Preservation of the rate and profile of xenobiotics metabolism in rat hepatocytes stored in liquid nitrogen. Biochem. Pharmacol. 46, 111–116.

    Article  PubMed  CAS  Google Scholar 

  36. Nishimura, M., Koeda, A., Suzuki, E., Kawano, Y., Nakayama, M., Satoh, T., Narimatsu, S., and Naito, S. (2006) Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes. Drug Metab. Pharmacokinet. 21, 297–307.

    Article  PubMed  CAS  Google Scholar 

  37. Nishimura, M., Koeda, A., Suganuma, Y., Suzuki, E., Shimizu, T., Nakayama, M., Satoh, T., Narimatsu, S., and Naito, S. (2007) Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes. Drug Metab. Pharmacokinet. 22, 178–186.

    Article  PubMed  CAS  Google Scholar 

  38. Nishimura, M., Koeda, A., Shimizu, T., Nakayama, M., Satoh, T., Narimatsu, S., and Naito, S. (2008) Comparison of inducibility of sulfotransferase and UDP-glucuronosyltransferase mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes. Drug Metab. Pharmacokinet. 23, 45–53.

    Article  PubMed  CAS  Google Scholar 

  39. Martin, D. and Hauthal, H.G. (1971) Dimethyl-Sulphoxide Van Nostrand Reinhold, New York.

    Google Scholar 

  40. Anchordoguy, T.J., Cecchini, C.A., Crowe, J.H., and Crowe, L.M. (1991) Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers. Cryobiology 28, 467–473.

    Article  PubMed  CAS  Google Scholar 

  41. Sum, A.K. and de Pablo, J.J. (2003) Molecular simulation study on the influence of dimethylsulfoxide on the structure of phospholipid bilayers. Biophys. J. 85, 3636–3645.

    Article  PubMed  CAS  Google Scholar 

  42. Arakawa, T., Carpenter, J.F., Kita, Y.A., and Crowe, J.H. (1990) The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27, 401–415.

    Article  CAS  Google Scholar 

  43. Anchordoguy, T.J., Carpenter, J.F., Crowe, J.H., and Crowe, L.M. (1992) Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide. Biochim. Biophys. Acta 1104, 117–122.

    Article  PubMed  CAS  Google Scholar 

  44. Poullain, M.G., Fautrel, A., Guyomard, C., Chesné, C., Grislain, L., and Guillouzo, A. (1992) Viability and primary culture of rat hepatocytes after hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage. Hepatology 15, 97–106.

    Article  PubMed  CAS  Google Scholar 

  45. Doeppner, T.R., Grune, T., de Groot, H., and Rauen, U. (2003) Cold-induced apoptosis of rat liver endothelial cells: involvement of the proteasome. Transplantation 75, 1946–1953.

    Article  PubMed  Google Scholar 

  46. Gutteridge, J.M. and Quinlan, G.J. (1993) Antioxidant protection against organic and inorganic oxygen radicals by normal human plasma: the important primary role for iron-binding and iron-oxidising proteins. Biochim. Biophys. Acta 1156, 144–150.

    PubMed  CAS  Google Scholar 

  47. Hengstler, J.G., Ringel, M., Biefang, K., Hammel, S., Milbert, U., Gerl, M., Klebach, M., Diener, B., Platt, K.L., Böttger, T., Steinberg, P., and Oesch, F. (2000) Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction. Chem. Biol. Interact. 125, 51–73.

    Article  PubMed  CAS  Google Scholar 

  48. Katenz, E., Vondran, F.W., Schwartlander, R., Pless, G., Gong, X., Cheng, X., Neuhaus, P., and Sauer, I.M. (2007) Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent. Liver Transpl. 13, 38–45.

    Article  PubMed  Google Scholar 

  49. Limaye, L.S. and Kale, V.P. (2001) Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium. J. Hematother. Stem Cell Res. 10, 709–718.

    Article  PubMed  CAS  Google Scholar 

  50. Leekumjorn, S., Wu, Y., Sum, A.K., and Chan, C. (2008) Experimental and computational studies investigating trehalose protection of HepG2 cells from palmitate-induced toxicity. Biophys. J. 94, 2869–2883.

    Article  PubMed  CAS  Google Scholar 

  51. Sasnoor, L.M., Kale, V.P., and Limaye, L.S. (2003) Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells. J. Hematother. Stem Cell Res. 12, 553–564.

    Article  PubMed  CAS  Google Scholar 

  52. Hewitt, N.J., Lechón, M.J., Houston, J.B., Hallifax, D., Brown, H.S., Maurel, P., Kenna, J.G., Gustavsson, L., Lohmann, C., Skonberg, C., Guillouzo, A., Tuschl, G., Li, A.P., LeCluyse, E., Groothuis, G.M., and Hengstler, J.G. (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.

    Article  PubMed  CAS  Google Scholar 

  53. Powis, G., Santone, K.S., Melder, D.C., Thomas, L., Moore, D.J., and Wilke, T.J. (1987) Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation. Drug Metab. Dispos. 15, 826–832.

    PubMed  CAS  Google Scholar 

  54. Hewitt, N.J. and Utesch, D. (2004) Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures. Hum. Exp. Toxicol. 23, 307–316.

    Article  PubMed  CAS  Google Scholar 

  55. Hewitt, N.J., Fischer, T., Zuehlke, U., Oesch, F., and Utesch, D. (2000) Metabolic activity of fresh and cryopreserved cynomolgus monkey (Macaca fascicularis) hepatocytes. Xenobiotica 30, 665–681.

    Article  PubMed  CAS  Google Scholar 

  56. Dzuba, B.B. and Kopeika, E.F. (2002) Relationship between the changes in cellular volume of fish spermatozoa and their cryoresistance. Cryo Letters 23, 353–360.

    PubMed  CAS  Google Scholar 

  57. Robles, V., Barbosa, V., Herráez, M., Martínez-Páramo, S., and Cancela, M. (2007) The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures. Theriogenology 68, 284–289.

    Article  PubMed  CAS  Google Scholar 

  58. Spencer, J.A. (1999) Cryopreservation of hepatocytes from rodents and food-producing animals and their use for in vitro toxicology. PhD Thesis. University of London.

    Google Scholar 

  59. Skett, P., Roberts, P., and Khan, S. (1999) Maintenance of steroid metabolism and hormone responsiveness in cryopreserved dog, monkey and human hepatocytes. Chem. Biol. Interact. 121, 65–76.

    Article  PubMed  CAS  Google Scholar 

  60. Fautrel, A., Joly, B., Guyomard, C., and Guillouzo, A. (1997) Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation. Toxicol. Appl. Pharmacol. 147, 110–114.

    Article  PubMed  CAS  Google Scholar 

  61. Lawrence, J.N. and Benford, D.J. (1991) Development of an optimal method for the cryopreservation of hepatocytes and their subsequent monolayer culture. Toxicol. In Vitro 5, 39–50.

    Article  PubMed  CAS  Google Scholar 

  62. Jackson, B.A., Davies, J.E., and Chipman, J.K. (1985) Cytochrome P-450 activity in hepatocytes following cryopreservation and monolayer culture. Biochem. Pharmacol. 34, 3389–3391.

    Article  PubMed  CAS  Google Scholar 

  63. Watts, P. and Grant, M.H. (1996) Cryopreservation of rat hepatocyte monolayer cultures. Hum. Exp. Toxicol. 15, 30–37.

    Article  PubMed  CAS  Google Scholar 

  64. De Sousa, G., Dou, M., Barbe, D., Lacarelle, B., Placidi, M., and Rahmani, R. (1991) Freshly isolated or cryopreserved human hepatocytes in primary culture: influence of drug metabolism on hepatotoxicity. Toxicol. In Vitro 5, 483–486.

    Article  PubMed  Google Scholar 

  65. Madan, A., DeHaan, R., Mudra, D., Carroll, K., LeCluyse, E., and Parkinson, A. (1999) Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes. Drug Metab. Dispos. 27, 327–335.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Scientific Writing Services, Erzhausen, Germany

    Nicola J. Hewitt

Authors
  1. Nicola J. Hewitt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicola J. Hewitt .

Editor information

Editors and Affiliations

  1. INSERM U632, Labo. Physiopathologie Hépatique, Université Montpellier I, rte. Mende 1919, Montpellier, 34293, France

    Patrick Maurel

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Hewitt, N.J. (2010). Optimisation of the Cryopreservation of Primary Hepatocytes. In: Maurel, P. (eds) Hepatocytes. Methods in Molecular Biology, vol 640. Humana Press. https://doi.org/10.1007/978-1-60761-688-7_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-688-7_4

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-687-0

  • Online ISBN: 978-1-60761-688-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation