Skip to main content

Advertisement

SpringerLink
Log in

Long-term culture of primary hepatocytes: new matrices and microfluidic devices

  • Review Article
  • Published:
Hepatology International Aims and scope Submit manuscript

Abstract

Prediction of in vivo drug-induced hepatotoxicity by in vitro cell culture systems is still one of the main challenges in drug development. To date, most in vitro approaches are based on monolayer cultures of primary hepatocytes, although it is known that they rapidly lose their morphology and liver-specific functions, such as activities of drug-metabolizing enzymes and transporters. Hepatocyte dedifferentiation can be delayed by culturing cells in a 3D environment. Combination with continuous medium flow, which creates a more physiological situation, further improves the maintenance of hepatic functions. Here, we present recently developed hydrogels and scaffolds for 3D culture of hepatocytes, which aim at preserving hepatic morphology and functionality for up to 4 weeks in culture. Furthermore, major benefits and drawbacks of microfluidic devices for in vitro hepatotoxicity screening are discussed. Although promising advances have been made regarding the preservation of hepatic functions in 3D flow culture, major issues, such as expensive equipment, large cell numbers and low throughput, are still hampering their use in drug toxicity screening. For these devices to be applied and accepted in the drug-developing industry, it is necessary to combine easily accessible matrices that highly preserve the activities of drug-metabolizing enzymes with a user-friendly microfluidic platform, thereby finding the right balance between reflecting the in vivo situation and enabling satisfying throughput for drug candidate screening.

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

Similar content being viewed by others

Abbreviations

3D:

Three-dimensional

CYP:

Cytochrome P450 enzymes

ECM:

Extracellular matrix

PEG:

Polyethylene glycol

PLA:

Polylactic acid

PLGA:

Poly(lactic-co-glycolic)acid

PVA:

Polyvinyl alcohol

PS:

Polystyrene

RGD:

Arginine-glycine-aspartate tripeptide

References

  1. Dambach DM, Andrews BA, Moulin F. New technologies and screening strategies for hepatotoxicity: use of in vitro models. Toxicol Pathol 2005;33:17–26

    Article  CAS  PubMed  Google Scholar 

  2. De Bruyn T, Chatterjee S, Fattah S, Keemink J, Nicolai J, Augustijns P, et al. Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 2013;5:589–616

    Article  Google Scholar 

  3. Knobeloch D, Ehnert S, Schyschka L, Buchler P, Schoenberg M, Kleeff J, et al. Human hepatocytes: isolation, culture, and quality procedures. Methods Mol Biol 2012;806:99–120

    Article  CAS  PubMed  Google Scholar 

  4. Schyschka L, Sanchez JJ, Wang Z, Burkhardt B, Muller-Vieira U, Zeilinger K, et al. Hepatic 3D cultures but not 2D cultures preserve specific transporter activity. Arch Toxicol 2013;87:1581–1593

    Article  CAS  PubMed  Google Scholar 

  5. Prot JM, Briffaut AS, Letourneur F, Chafey P, Merlier F, Grandvalet Y, et al. Integrated proteomic and transcriptomic investigation of the acetaminophen toxicity in liver microfluidic biochip. PLoS One 2011;6:e21268

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Meng Q. Three-dimensional culture of hepatocytes for prediction of drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 2010;6:733–746

    Article  CAS  PubMed  Google Scholar 

  7. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 2013;87:1315–1530

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Vinken M, Elaut G, Henkens T, Papeleu P, Snykers S, Vanhaecke T, et al. Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures. Methods Mol Biol 2006;320:247–254

    CAS  PubMed  Google Scholar 

  9. Gottwald E, Kleintschek T, Giselbrecht S, Truckenmuller R, Altmann B, Worgull M, et al. Characterization of a chip-based bioreactor for three-dimensional cell cultivation via magnetic resonance imaging. Z Med Phys 2013;23:102–110

    Article  PubMed  Google Scholar 

  10. LeCluyse EL, Witek RP, Andersen ME, Powers MJ. Organotypic liver culture models: meeting current challenges in toxicity testing. Crit Rev Toxicol 2012;42:501–548

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Gibas I. Janik H. Chem: Synthetic polymer hydrogels for biomedical applications; 2010

    Google Scholar 

  12. Vinken M, Papeleu P, Snykers S, De Rop E, Henkens T, Chipman JK, et al. Involvement of cell junctions in hepatocyte culture functionality. Crit Rev Toxicol 2006;36:299–318

    Article  CAS  PubMed  Google Scholar 

  13. Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng 2009;103:655–663

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. De Colli M, Massimi M, Barbetta A, Di Rosario BL, Nardecchia S, Conti Devirgiliis L, et al. A biomimetic porous hydrogel of gelatin and glycosaminoglycans cross-linked with transglutaminase and its application in the culture of hepatocytes. Biomed Mater 2012;7:055005

    Article  PubMed  Google Scholar 

  15. Moghe PV, Berthiaume F, Ezzell RM, Toner M, Tompkins RG, Yarmush ML. Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. Biomaterials 1996;17:373–385

    Article  CAS  PubMed  Google Scholar 

  16. LeCluyse EL. Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur J Pharm Sci 2001;13:343–368

    Article  CAS  PubMed  Google Scholar 

  17. Khalil M, Shariat-Panahi A, Tootle R, Ryder T, McCloskey P, Roberts E, et al. Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function. J Hepatol 2001;34:68–77

    Article  CAS  PubMed  Google Scholar 

  18. Shen C, Zhang G, Qiu H, Meng Q. Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers. Chem Biol Interact 2006;162:53–61

    Article  CAS  PubMed  Google Scholar 

  19. Meng Q, Zhang G, Shen C, Qiu H. Sensitivities of gel entrapped hepatocytes in hollow fibers to hepatotoxic drug. Toxicol Lett 2006;166:19–26

    Article  CAS  PubMed  Google Scholar 

  20. Lee W, Cho NJ, Xiong A, Glenn JS, Frank CW. Hydrophobic nanoparticles improve permeability of cell-encapsulating poly(ethylene glycol) hydrogels while maintaining patternability. Proc Natl Acad Sci U S A 2010;107:20709–20714

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Kim M, Lee JY, Jones CN, Revzin A, Tae G. Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes. Biomaterials 2010;31:3596–3603

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Lin CC, Anseth KS. PEG hydrogels for the controlled release of biomolecules in regenerative medicine. Pharm Res 2008;26:631–643

    Article  PubMed  Google Scholar 

  23. Genove E, Schmitmeier S, Sala A, Borros S, Bader A, Griffith LG, et al. Functionalized self-assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro. J Cell Mol Med 2009;13:3387–3397

    Article  PubMed  Google Scholar 

  24. Wang S, Nagrath D, Chen PC, Berthiaume F, Yarmush ML. Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel. Tissue Eng Part A 2008;14:227–236

    Article  CAS  PubMed  Google Scholar 

  25. Li J, Pan J, Zhang L, Guo X, Yu Y. Culture of primary rat hepatocytes within porous chitosan scaffolds. J Biomed Mater Res A 2003;67:938–943

    Article  PubMed  Google Scholar 

  26. Dvir-Ginzberg M, Gamlieli-Bonshtein I, Agbaria R, Cohen S. Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability, morphology, and function. Tissue Eng 2003;9:757–766

    Article  CAS  PubMed  Google Scholar 

  27. Li K, Wang Y, Miao Z, Xu D, Tang Y, Feng M. Chitosan/gelatin composite microcarrier for hepatocyte culture. Biotechnol Lett 2004;26:879–883

    Article  CAS  PubMed  Google Scholar 

  28. Li Y, Yang S-T. Effects of three-dimensional scaffolds on cell organization and tissue development. Biotech Bioprocess Eng 2001;6:311–325

    Article  CAS  Google Scholar 

  29. Saavedra YGL, Mateescu MA, Averill-Bates DA, Denizeau F. Polyvinylalcohol three-dimensional matrices for improved long-term dynamic cultureof hepatocytes. J Biomed Mater Res A 2002;66:562–570

    Google Scholar 

  30. Torok E, Lutgehetmann M, Bierwolf J, Melbeck S, Dullmann J, Nashan B, et al. Primary human hepatocytes on biodegradable poly(l-lactic acid) matrices: a promising model for improving transplantation efficiency with tissue engineering. Liver Transpl 2011;17:104–114

    Article  PubMed  Google Scholar 

  31. Wang T, Feng Z, Leach M, Wu J, Jiang Q. Nanoporous fibers of type-I collagen coated poly(l-lactic acid) for enhancing primary hepatocyte growth and function. J Mat Chem B 2013;1:339–346

    Article  CAS  Google Scholar 

  32. Schutte M, Fox B, Baradez MO, Devonshire A, Minguez J, Bokhari M, et al. Rat primary hepatocytes show enhanced performance and sensitivity to acetaminophen during three-dimensional culture on a polystyrene scaffold designed for routine use. Assay Drug Dev Technol 2011;9:475–486

    Article  CAS  PubMed  Google Scholar 

  33. Messner S, Agarkova I, Moritz W, Kelm JM. Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol 2013;87:209–213

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Selimovic S, Piraino F, Bae H, Rasponi M, Redaelli A, Khademhosseini A. Microfabricated polyester conical microwells for cell culture applications. Lab Chip 2011;11:2325–2332

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Vinci B, Duret C, Klieber S, Gerbal-Chaloin S, Sa-Cunha A, Laporte S, et al. Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes. Biotechnol J 2011;6:554–564

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Miranda JP, Rodrigues A, Tostoes RM, Leite S, Zimmerman H, Carrondo MJ, et al. Extending hepatocyte functionality for drug-testing applications using high-viscosity alginate-encapsulated three-dimensional cultures in bioreactors. Tissue Eng Part C Methods 2010;16:1223–1232

    Article  CAS  PubMed  Google Scholar 

  37. Hoffmann SA, Muller-Vieira U, Biemel K, Knobeloch D, Heydel S, Lubberstedt M, et al. Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies. Biotechnol Bioeng 2012;109:3172–3181

    Article  CAS  PubMed  Google Scholar 

  38. Lubberstedt M, Muller-Vieira U, Biemel KM, Darnell M, Hoffmann SA, Knospel F, et al. Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies. J Tissue Eng Regen Med 2012. doi:10.1002/term.1652

  39. Mueller D, Tascher G, Muller-Vieira U, Knobeloch D, Nuessler AK, Zeilinger K, et al. In-depth physiological characterization of primary human hepatocytes in a 3D hollow-fiber bioreactor. J Tissue Eng Regen Med 2011;5:e207–e218

    Article  CAS  PubMed  Google Scholar 

  40. Unger JK, Kuehlein G, Schroers A, Gerlach JC, Rossaint R. Adsorption of xenobiotics to plastic tubing incorporated into dynamic in vitro systems used in pharmacological research—limits and progress. Biomaterials 2001;22:2031–2037

    Article  CAS  PubMed  Google Scholar 

  41. Xu Q, Sun X, Qiu Y, Zhang H, Ding Y. The optimal hepatocyte density for a hollow-fiber bioartificial liver. Ann Clin Lab Sci 2004;34:87–93

    CAS  PubMed  Google Scholar 

  42. Sivaraman A, Leach JK, Townsend S, Iida T, Hogan BJ, Stolz DB, et al. A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr Drug Metab 2005;6:569–691

    Article  CAS  PubMed  Google Scholar 

  43. Eschbach E, Chatterjee SS, Noldner M, Gottwald E, Dertinger H, Weibezahn KF, et al. Microstructured scaffolds for liver tissue cultures of high cell density: morphological and biochemical characterization of tissue aggregates. J Cell Biochem 2005;95:243–255

    Article  CAS  PubMed  Google Scholar 

  44. Schutte J, Hagmeyer B, Holzner F, Kubon M, Werner S, Freudigmann C, et al. “Artificial micro organs”—a microfluidic device for dielectrophoretic assembly of liver sinusoids. Biomed Microdevices 2011;13:493–501

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The present work has been partially supported by BMBF-0316058A and the Set Foundation.

Compliance with ethical requirements and conflict of interest

In the studies with human subjects carried out by the authors that are mentioned in this review, all procedures met the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for inclusion in the study. Britta Burkhardt, Juan José Martinez-Sanchez, Anastasia Bachmann, Ruth Ladurner and Andreas K. Nüssler declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, Schnarrenbergstr. 95, 72076, Tübingen, Germany

    Britta Burkhardt, Juan José Martinez-Sanchez, Anastasia Bachmann & Andreas K. Nüssler

  2. Clinic for General, Visceral and Transplantation Surgery, Eberhard Karls University Tübingen, Hoppe-Seyler-Str. 3, 72076, Tübingen, Germany

    Ruth Ladurner

Authors
  1. Britta Burkhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan José Martinez-Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anastasia Bachmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruth Ladurner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas K. Nüssler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreas K. Nüssler.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Burkhardt, B., Martinez-Sanchez, J.J., Bachmann, A. et al. Long-term culture of primary hepatocytes: new matrices and microfluidic devices. Hepatol Int 8, 14–22 (2014). https://doi.org/10.1007/s12072-013-9487-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12072-013-9487-3

Keywords

Search

Navigation