Skip to main content
SpringerLink
Log in

A stereotypic, transplantable liver tissue-culture system

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

A method of coculturing adult rat hepatic parenchymal cells (PC) and stromal cells in a three-dimensional framework of nylon filtration screens or biodegradable polymer meshes was developed in our laboratory. Rat liver stroma, which includes vascular and bile duct endothelial cells, fat-storing cells, fibroblasts, and Kupffer cells, were isolated by gradient centrifugation afterin situ liver perfusion and expanded in monolayer culture prior to seeding onto nylon screens or bioresorbable polyglycolic acid (PGA) polymers oriented into a felt-like construct. A second inoculum of freshly isolated PC was applied after the stromal cells became established. Histological analyses revealed that PC proliferation occurred until all available space for expansion within the template was exhausted. These cells retained their rounded morphology, and after 4–5 wk 7–9 “layers” of PC filled the 140-μm deep template. Dioxin-inducible cytochrome P450 activity was detected for up to 58 d in culture, and albumin, fibrinogen, transferrin, and soluble fibronectin were detected in the medium by enzyme-linked immunosorbent assay (ELISA) for 48 d in vitro. Immunohistochemical analysis of sections through the cultures confirmed the presence of these proteins as well as cytokeratin at the cellular level; the extracellular matrix stained for both collagen type III and laminin. Long-term PC proliferation and function were enhanced by the presence of stromal cells as well as by a meshwork template whose geometry allows the interaction of PC with stroma and matrix on several different planes. To permit transplantation, co-cultures of hepatic PC and stromal cells were established on PGA felt constructs instead of nylon screens. After 24 d in vitro, these constructs were grafted into sites in the mesentery, omentum, and sub-cutaneous tissues of adult Long-Evans rats. The growth of hepatocytes after 30 din situ was evident by histological analysis; grafts of co-cultures regenerated a liver-like architecture consisting of sinusoids and putative biliary structures. In addition, PC at these extrahepatic graft sites were positive for albumin, transferrin, and fibrinogen synthesis by immunohistochemistry. Graft survival was enhanced when recipients were subjected to 40% hepatectomy. Hepatic PC:stromal cell cocultures may prove useful in the restoration of liver function either by direct transplantation using PGA or similar templates, or as extracorporeal devices, using nylon screens.

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

Abbreviations

cP450:

cytochrome P450

ECM:

extracellular matrix EFEE, ethoxyfluorescein ethyl ester

ELISA:

enzyme-linked immunosorbent assay

FBS:

fetal bovine serum

FLS:

forward-angle light scatter

PBS:

phosphate-buffered saline

PC:

parenchymal cells

PGA:

polyglycolic acid

PTFE:

polytetrafluoroethylene

SEM:

standard error of the mean

SFM:

serum-free medium

SS:

side or 90° light scatter

TCDD:

2,3,7,8-tetrachlorodibenzo-p-dioxin.

References

  1. Michalopoulos, G. and Pitot, H. C. (1975),Exp. Cell Res. 94, 70–78.

    Article  CAS  Google Scholar 

  2. Dunn, J. C. Y., Yarmush, M L., Koebe, H. G., and Tompkins, R. G. (1989),FASEB J. 3, 174–177.

    CAS  Google Scholar 

  3. Reid, L. M., Gaitmaitan, Z., Arias, I., Ponce, P., and Rojkind, M. (1980),Ann. NY Acad. Sci. 349, 70–76.

    Article  CAS  Google Scholar 

  4. Bissell, D. M., Arenson, D. M., Maher, J. J., and Roll, J. (1987),J. Clin. Invest. 79, 801–812.

    Article  CAS  Google Scholar 

  5. Deschenes, J., Valet, J. P., and Mareceau, N. (1980),In Vitro 16, 722–730.

    Article  CAS  Google Scholar 

  6. Guguen-Guillouzo, C., Clement, B., Baffet, G., Beaumont, C., Morel-Chany, E., Glaise, D., and Guillouzo, A. (1983),Exp. Cell Res. 143, 47–54.

    Article  CAS  Google Scholar 

  7. Begue, J. M., LeBigot, J. F., Guguen-Guillouzo, C., Kiechel, J. R., and Guillouzo, A. (1983),Biochem. Pharmacol. 32, 1643–1646.

    Article  CAS  Google Scholar 

  8. Kuri-Harcuch, W. and Mendoza-Figueroa, T. (1989),Differentiation 41, 148–157.

    Article  CAS  Google Scholar 

  9. Michalopoulos, G., Russell, F., and Biles, C. (1979),In Vitro 15, 796–806.

    Article  CAS  Google Scholar 

  10. Sudhakaran, P. R., Stomatoglou, S. C., and Hughes, R. C. (1986),Exp. Cell Res. 167, 505–516.

    Article  CAS  Google Scholar 

  11. Grisham, J. W. (1980),Ann. NY Acad. Sci. 349, 128–137.

    Article  CAS  Google Scholar 

  12. Bucher, N. L. R. (1987), inThe Isolated Hepatocyte: Use in Toxicology and Xenobiotic Biotransformations, Rauckman, E. J. and Padilla, G. M. eds., Academic, New York, pp. 1–19.

    Google Scholar 

  13. Naughton, B. A., Kaplan, S. M., Roy, M., Gordon, A. S., and Piliero, S. J. (1977),Science 196, 301–302.

    Article  CAS  Google Scholar 

  14. Pertoft, H. and Smedsrod, B. (1987), inCell Separation: Methods and Selected Applications, vol. 4, Pretlow, T. G., II, and Pretlow, T. P., eds., Academic, New York, pp. 1–24.

    Google Scholar 

  15. Naughton, B. A., San Roman, J., Sibanda, B., Weintraub, J. P. (1994)Biotechnol. Bioeng. 43, 810–825.

    Article  CAS  Google Scholar 

  16. Enat, R., Jefferson, D. M., Ruiz-Opazo, N., Gatmaitan, Z., Leinwand, L. A., and Reid, L. M. (1984),Proc. Nat. Acad. Sci. USA 81, 1411–1415.

    Article  CAS  Google Scholar 

  17. Miller, A. G. (1983),Anal. Chem. 133, 46–57.

    CAS  Google Scholar 

  18. White, I. N. H., Green, M. L., and Legg, R. F. (1987),Biochem. J. 247, 23–28.

    CAS  Google Scholar 

  19. Naughton, B. A., Birnbach, D. J., Liu, P., Kolks, G. A., Tung, M., Piliero, S. J., and Gordon, A. S. (1979),Proc. Soc. Exp. Biol. Med. 160, 170–174.

    CAS  Google Scholar 

  20. Naughton, B. A., Sibanda, B., Triglia, D., and Naughton, G. K. (1991),Toxicol. In Vitro 5, 389–394.

    Article  CAS  Google Scholar 

  21. Hayner, N. T., Braun, L., Yaswen, P., Brooks, M., and Fausto, N. (1988),Cancer Res. 48, 368–378.

    Google Scholar 

  22. Ben-Ze’ev, A., Robinson, G. S., Bucher, N. L., and Farmer, S. R. (1988),Proc. Natl. Acad. Sci. USA 85, 1–6.

    Article  Google Scholar 

  23. Clayton, D. F., Harrelson, A. L., and Darnell, J. E. (1985),Mol. Cell Biol. 5, 2623–2632.

    CAS  Google Scholar 

  24. Landry, J., Bernier, D., Ouellet, C., Goyette, R., and Marceau, N. (1985),J. Cell Biol. 101, 914–923.

    Article  CAS  Google Scholar 

  25. Bernuau, D., Feldman, G., and Rogier, E. (1981),Biol. Cell. 40, 17–22.

    Google Scholar 

  26. Guillouzo, A., Beaumont, C., LeRumeur, M., Rissel, Latinier, M-F., Guguen-Guillouzo, C., and Bourel, M. (1982),Biol. Cell 43, 163–171.

    CAS  Google Scholar 

  27. Schreiber, G., Lesch, R., Weinssen, U., and Zahringer, J. (1970),J. Cell Biol. 47, 285–290.

    Article  CAS  Google Scholar 

  28. Araki, H., Ueda, H., and Fujimoto, S. (1992),Acta Anat. 143, 169–177.

    CAS  Google Scholar 

  29. Maurice, M., Feldmann, G., Druet, P., Laliberte, F., and Bouige, D. (1979),Lab. Invest. 40, 39–45.

    CAS  Google Scholar 

  30. Tonomura, A., Sawada, N., Sattler, G. L., Kleinman, H. K., and Pitot, H. C. (1987),J. Cell Physiol. 130, 221–227.

    Article  Google Scholar 

  31. Matsumara, T. and Thurman, R. G. (1983),A. J. Physiol. 244, G656-G659.

    Google Scholar 

  32. Martinez-Hernandez, A. and Amenta, P. S. (1993), inExtracellular Matrix: Chemistry, Biology, Pathology, Zern, M. and Reid, L., eds., Marcel-Dekker, New York, pp. 255–327.

    Google Scholar 

  33. Arber, N., Zajicek, G., and Ariel, I. (1988),Liver 8, 80–87.

    CAS  Google Scholar 

  34. Sato, Y., Ochiya, T., Yasuda, Y., Matsubara, K. (1994),Hepatology 19, 1023–1028.

    CAS  Google Scholar 

  35. Barnes, D. and Sato, G. (1980),Cell 22, 649–655.

    Article  CAS  Google Scholar 

  36. Tsao, M-S., Smith, J. D., Nelson, K. G., and Grisham, J. W. (1984),Exp. Cell Res. 154, 38–52.

    Article  CAS  Google Scholar 

  37. Farber, E. (1956),Cancer Res. 16, 142–149.

    CAS  Google Scholar 

  38. Firminger, H. I. (1955),J. Natl. Cancer Inst. 15, 1427–1441.

    CAS  Google Scholar 

  39. Fausto, N., Thompson, N. L., and Braun, L. (1987), inCell Separation: Methods and Selected Applications, vol. 4, Pretlow, T. G., II, and Pretlow, T. P., eds., Academic, London, pp. 45–78.

    Google Scholar 

  40. Goulet, F. (1989), Doctoral thesis, Université Laval, Québec.

    Google Scholar 

  41. Parry, E. W. (1978),J. Comp. Pathol. 88, 481–487.

    Article  CAS  Google Scholar 

  42. Naughton, B. A., San Roman, J., Sibanda, B., Weintraub, J. P. (1994),Hematol. Rev. 8, 37–49.

    Google Scholar 

  43. Naughton, B. A., Dai, Y., Sibanda, B., Scharfmann, R., San Roman, J., Zeigler, F., and Verma, I. M. (1992),Somat. Cell Mol. Gen. 18, 451–462.

    Article  CAS  Google Scholar 

  44. Asonuma, K., Gilbert, J. C., Stein, J. E., Takeda, T., Vacanti, J. P. (1992),J. Ped. Res. 27, 298–301.

    CAS  Google Scholar 

  45. Demetriou, A. A., Whiting, J. F., Feldman, D., Levenson, S. M., Chowdhury, N. R., Moscioni, A.D., Kram, M., and Chowdhury, J. R. (1986),Science 233, 1190–1992.

    Article  CAS  Google Scholar 

  46. Dixit, V., Darvasi, R., Arthur, M., Brezina, M., Lewin, K., and Gitnick, G. (1990),Hepatology 12, 1342–1349.

    Article  CAS  Google Scholar 

  47. Matas, A. J., Sutherland, D. E. R. and Steffes, M. W. (1976),Science 192, 892–894.

    Article  CAS  Google Scholar 

  48. Langer, R. and Vacanti, J. P. (1993),Science 260, 920–926.

    Article  CAS  Google Scholar 

  49. Borel-Rinkes, I. H. M., Bijma, A. M., Kappers, W. A., Sinaasappel, M., Hoek, F.J., Jansen, P. L. M., Valerio, D., and Terpstra, O. T. (1992),Transplantation 54, 210–214.

    Article  CAS  Google Scholar 

  50. Uyama, S., Kaufmann, P-M., Takeda, T., Vacanti, J. P. (1993),Transplantation 55, 932–935.

    Article  CAS  Google Scholar 

  51. Vacanti, J. P., Morse, M. A., Saltzman, W. M., Domb, A. J., Perez-Atayde, A., and Langer, R. (1988),J. Pediatr. Surg. 23, 3–9.

    Article  CAS  Google Scholar 

  52. Nyberg, S. L., Shatford, R. A., Hu, W-S., Payne, W. D., and Cerra, F. B. (1992),Crit. Care Med. 20, 1157–1168.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Hematology Laboratory, Advanced Tissue Sciences Inc., 92037, LaJolla, CA

    Brian A. Naughton, Benson Sibanda, Jory P. Weintraub, Julia San Román & Vafa Kamali

Authors
  1. Brian A. Naughton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benson Sibanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jory P. Weintraub
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julia San Román
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vafa Kamali
    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

Naughton, B.A., Sibanda, B., Weintraub, J.P. et al. A stereotypic, transplantable liver tissue-culture system. Appl Biochem Biotechnol 54, 65–91 (1995). https://doi.org/10.1007/BF02787912

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02787912

Index Entries

Search

Navigation