Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Feb 2;116(4):1043–1053. doi: 10.1083/jcb.116.4.1043

Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation

PMCID: PMC2289333  PMID: 1734019

Abstract

The influence of extracellular matrix configuration on the tissue- specific function of cultured hepatocytes was investigated. Adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single layer of collagen gel for differences in the total RNA content, the level of albumin-specific mRNA, the rate of albumin gene transcription, and the rate of albumin mRNA translation. Adult hepatocytes in the sandwich system maintained the level of albumin mRNA similar to that found in the normal liver for at least six weeks, whereas the level of albumin mRNA declined rapidly in the single gel system. After one week of culture, hepatocytes in the single gel system could be induced to recover the high level of albumin mRNA and albumin production when a second layer of collagen gel was overlaid at that time. Furthermore, sandwiched hepatocytes maintained significantly higher transcriptional activity compared to cells in the single gel system. In addition to transcriptional control, the ultimate rate of albumin production was shown to depend on the rate of translation, which increased with culture time and reached a plateau in one to two weeks. This increase in translational activity over time in culture was observed in both the sandwich and the single gel systems and, thus, appeared to be independent of the configuration of extracellular matrix.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartles J. R., Feracci H. M., Stieger B., Hubbard A. L. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J Cell Biol. 1987 Sep;105(3):1241–1251. doi: 10.1083/jcb.105.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Robinson G. S., Bucher N. L., Farmer S. R. Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2161–2165. doi: 10.1073/pnas.85.7.2161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bissell D. M., Arenson D. M., Maher J. J., Roll F. J. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest. 1987 Mar;79(3):801–812. doi: 10.1172/JCI112887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caron J. M. Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol. 1990 Mar;10(3):1239–1243. doi: 10.1128/mcb.10.3.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter E. A., Wands J. R. Ethanol-induced inhibition of liver cell function: I. Effect of ethanol on hormone stimulated hepatocyte DNA synthesis and the role of ethanol metabolism. Alcohol Clin Exp Res. 1988 Aug;12(4):555–562. doi: 10.1111/j.1530-0277.1988.tb00241.x. [DOI] [PubMed] [Google Scholar]
  7. Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  8. Chambard M., Gabrion J., Mauchamp J. Influence of collagen gel on the orientation of epithelial cell polarity: follicle formation from isolated thyroid cells and from preformed monolayers. J Cell Biol. 1981 Oct;91(1):157–166. doi: 10.1083/jcb.91.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen J., Stuckey E. C., Berry C. L. Three-dimensional culture of rat exocrine pancreatic cells using collagen gels. Br J Exp Pathol. 1985 Oct;66(5):551–559. [PMC free article] [PubMed] [Google Scholar]
  10. Clayton D. F., Darnell J. E., Jr Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol. 1983 Sep;3(9):1552–1561. doi: 10.1128/mcb.3.9.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clayton D. F., Harrelson A. L., Darnell J. E., Jr Dependence of liver-specific transcription on tissue organization. Mol Cell Biol. 1985 Oct;5(10):2623–2632. doi: 10.1128/mcb.5.10.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Demetriou A. A., Reisner A., Sanchez J., Levenson S. M., Moscioni A. D., Chowdhury J. R. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology. 1988 Sep-Oct;8(5):1006–1009. doi: 10.1002/hep.1840080505. [DOI] [PubMed] [Google Scholar]
  13. Derman E., Krauter K., Walling L., Weinberger C., Ray M., Darnell J. E., Jr Transcriptional control in the production of liver-specific mRNAs. Cell. 1981 Mar;23(3):731–739. doi: 10.1016/0092-8674(81)90436-0. [DOI] [PubMed] [Google Scholar]
  14. Dunn J. C., Tompkins R. G., Yarmush M. L. Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog. 1991 May-Jun;7(3):237–245. doi: 10.1021/bp00009a007. [DOI] [PubMed] [Google Scholar]
  15. Dunn J. C., Yarmush M. L., Koebe H. G., Tompkins R. G. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 1989 Feb;3(2):174–177. doi: 10.1096/fasebj.3.2.2914628. [DOI] [PubMed] [Google Scholar]
  16. Elsdale T., Bard J. Collagen substrata for studies on cell behavior. J Cell Biol. 1972 Sep;54(3):626–637. doi: 10.1083/jcb.54.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fagan R. J., Lazaris-Karatzas A., Sonenberg N., Rozen R. Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E. J Biol Chem. 1991 Sep 5;266(25):16518–16523. [PubMed] [Google Scholar]
  18. Fausto N. New perspectives on liver regeneration. Hepatology. 1986 Mar-Apr;6(2):326–327. doi: 10.1002/hep.1840060229. [DOI] [PubMed] [Google Scholar]
  19. Fioretti W. C., Davis D. F., Ledford B. E. Polyribosome size analysis. Measurement of number-average polyribosome sizes. Biochim Biophys Acta. 1979 Aug 29;564(1):79–89. doi: 10.1016/0005-2787(79)90190-4. [DOI] [PubMed] [Google Scholar]
  20. Fraslin J. M., Kneip B., Vaulont S., Glaise D., Munnich A., Guguen-Guillouzo C. Dependence of hepatocyte-specific gene expression on cell-cell interactions in primary culture. EMBO J. 1985 Oct;4(10):2487–2491. doi: 10.1002/j.1460-2075.1985.tb03960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garrett J. E., Collard M. W., Douglass J. O. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis. Mol Cell Biol. 1989 Oct;9(10):4381–4389. doi: 10.1128/mcb.9.10.4381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guguen-Guillouzo C., Clément B., Baffet G., Beaumont C., Morel-Chany E., Glaise D., Guillouzo A. Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp Cell Res. 1983 Jan;143(1):47–54. doi: 10.1016/0014-4827(83)90107-6. [DOI] [PubMed] [Google Scholar]
  23. Hall H. G., Farson D. A., Bissell M. J. Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4672–4676. doi: 10.1073/pnas.79.15.4672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hay E. D. Extracellular matrix. J Cell Biol. 1981 Dec;91(3 Pt 2):205s–223s. doi: 10.1083/jcb.91.3.205s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ingber D. E., Madri J. A., Jamieson J. D. Basement membrane as a spatial organizer of polarized epithelia. Exogenous basement membrane reorients pancreatic epithelial tumor cells in vitro. Am J Pathol. 1986 Jan;122(1):129–139. [PMC free article] [PubMed] [Google Scholar]
  26. Isom H. C., Secott T., Georgoff I., Woodworth C., Mummaw J. Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci U S A. 1985 May;82(10):3252–3256. doi: 10.1073/pnas.82.10.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Isom I., Georgoff I., Salditt-Georgieff M., Darnell J. E., Jr Persistence of liver-specific messenger RNA in cultured hepatocytes: different regulatory events for different genes. J Cell Biol. 1987 Dec;105(6 Pt 2):2877–2885. doi: 10.1083/jcb.105.6.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jeejeebhoy K. N., Bruce-Robertson A., Ho J., Sodtke U. The comparative effects of nutritional and hormonal factors on the synthesis of albumin, fibrinogen and transferrin. Ciba Found Symp. 1972;9:217–247. doi: 10.1002/9780470719923.ch12. [DOI] [PubMed] [Google Scholar]
  29. Jefferson D. M., Clayton D. F., Darnell J. E., Jr, Reid L. M. Posttranscriptional modulation of gene expression in cultured rat hepatocytes. Mol Cell Biol. 1984 Sep;4(9):1929–1934. doi: 10.1128/mcb.4.9.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kleinman H. K., Klebe R. J., Martin G. R. Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol. 1981 Mar;88(3):473–485. doi: 10.1083/jcb.88.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Koebe H. G., Dunn J. C., Toner M., Sterling L. M., Hubel A., Cravalho E. G., Yarmush M. L., Tompkins R. G. A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration. Cryobiology. 1990 Oct;27(5):576–584. doi: 10.1016/0011-2240(90)90045-6. [DOI] [PubMed] [Google Scholar]
  32. Kreamer B. L., Staecker J. L., Sawada N., Sattler G. L., Hsia M. T., Pitot H. C. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev Biol. 1986 Apr;22(4):201–211. doi: 10.1007/BF02623304. [DOI] [PubMed] [Google Scholar]
  33. Lee E. Y., Lee W. H., Kaetzel C. S., Parry G., Bissell M. J. Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1419–1423. doi: 10.1073/pnas.82.5.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Leffert H. L., Paul D. Studies on primary cultures of differentiated fetal liver cells. J Cell Biol. 1972 Mar;52(3):559–568. doi: 10.1083/jcb.52.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lodish H. F., Froshauer S. Rates of initiation of protein synthesis by two purified species of vesicular stomatitis virus messenger RNA. J Biol Chem. 1977 Dec 25;252(24):8804–8811. [PubMed] [Google Scholar]
  36. Maher J. J. Primary hepatocyte culture: is it home away from home? Hepatology. 1988 Sep-Oct;8(5):1162–1166. doi: 10.1002/hep.1840080534. [DOI] [PubMed] [Google Scholar]
  37. Martin G. R., Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57–85. doi: 10.1146/annurev.cb.03.110187.000421. [DOI] [PubMed] [Google Scholar]
  38. Mesnil M., Fraslin J. M., Piccoli C., Yamasaki H., Guguen-Guillouzo C. Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions. Exp Cell Res. 1987 Dec;173(2):524–533. doi: 10.1016/0014-4827(87)90292-8. [DOI] [PubMed] [Google Scholar]
  39. Michalopoulos G., Pitot H. C. Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Exp Cell Res. 1975 Aug;94(1):70–78. doi: 10.1016/0014-4827(75)90532-7. [DOI] [PubMed] [Google Scholar]
  40. Montesano R., Mouron P., Amherdt M., Orci L. Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers into islet-like organoids. J Cell Biol. 1983 Sep;97(3):935–939. doi: 10.1083/jcb.97.3.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peters T., Jr Serum albumin. Adv Protein Chem. 1985;37:161–245. doi: 10.1016/s0065-3233(08)60065-0. [DOI] [PubMed] [Google Scholar]
  42. Powell D. J., Friedman J. M., Oulette A. J., Krauter K. S., Darnell J. E., Jr Transcriptional and post-transcriptional control of specific messenger RNAs in adult and embryonic liver. J Mol Biol. 1984 Oct 15;179(1):21–35. doi: 10.1016/0022-2836(84)90304-8. [DOI] [PubMed] [Google Scholar]
  43. Reid L. M., Gaitmaitan Z., Arias I., Ponce P., Rojkind M. Long-term cultures of normal rat hepatocytes on liver biomatrix. Ann N Y Acad Sci. 1980;349:70–76. doi: 10.1111/j.1749-6632.1980.tb29516.x. [DOI] [PubMed] [Google Scholar]
  44. Reid L. M., Jefferson D. M. Culturing hepatocytes and other differentiated cells. Hepatology. 1984 May-Jun;4(3):548–559. doi: 10.1002/hep.1840040332. [DOI] [PubMed] [Google Scholar]
  45. Schuetz E. G., Li D., Omiecinski C. J., Muller-Eberhard U., Kleinman H. K., Elswick B., Guzelian P. S. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J Cell Physiol. 1988 Mar;134(3):309–323. doi: 10.1002/jcp.1041340302. [DOI] [PubMed] [Google Scholar]
  46. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  47. Spray D. C., Fujita M., Saez J. C., Choi H., Watanabe T., Hertzberg E., Rosenberg L. C., Reid L. M. Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures. J Cell Biol. 1987 Jul;105(1):541–551. doi: 10.1083/jcb.105.1.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thompson J. A., Haudenschild C. C., Anderson K. D., DiPietro J. M., Anderson W. F., Maciag T. Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7928–7932. doi: 10.1073/pnas.86.20.7928. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  49. Uchino J., Tsuburaya T., Kumagai F., Hase T., Hamada T., Komai T., Funatsu A., Hashimura E., Nakamura K., Kon T. A hybrid bioartificial liver composed of multiplated hepatocyte monolayers. ASAIO Trans. 1988 Oct-Dec;34(4):972–977. [PubMed] [Google Scholar]
  50. Weinberg A. D., Swain S. L. IL-2 receptor (Tac antigen) protein expression is down-regulated by the 5'-untranslated region of the mRNA. J Immunol. 1990 Jun 15;144(12):4712–4720. [PubMed] [Google Scholar]
  51. Yamada K. M. Cell surface interactions with extracellular materials. Annu Rev Biochem. 1983;52:761–799. doi: 10.1146/annurev.bi.52.070183.003553. [DOI] [PubMed] [Google Scholar]
  52. Yang J., Flynn D., Larson L., Hamamoto S. Growth in primary culture of mouse submandibular epithelial cells embedded in collagen gels. In Vitro. 1982 May;18(5):435–442. doi: 10.1007/BF02796470. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES