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Optimization of culture conditions for human corneal endothelial cells

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Summary

Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.

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References

  1. Baird, A.; Ling, N. Fibroblast growth factors are present in the extracellular matrix produced by endothelial cellsin vitro: implication for a role of heparinase-like enzymes in the neovascular response. Biochem. Biophys. Res. Comm. 142:428–435; 1987.

    Article  PubMed  CAS  Google Scholar 

  2. Barnes, D.; Sato, G. Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102:255–270; 1980.

    Article  PubMed  CAS  Google Scholar 

  3. Baum, J. L.; Niedra, R.; Davis, C., et al. Mass culture of human corneal endothelial cells. Arch. Opthalmol. 97:1136–1140; 1979.

    CAS  Google Scholar 

  4. Bourne, W. M.; Lindstrom, R. L.; Doughman, D. J. Endothelial cell survival on transplanted human corneas preserved by organ culture with 1.35% chondroitin sulfate. Am. J. Ophthalmol. 100:789–793; 1985.

    PubMed  CAS  Google Scholar 

  5. Burgess, W. H.; Mehlman, T.; Marshak, D. R., et al. Structural evidence that endothelial cell growth factor β is the precursor of both endothelial growth factor α and acidic fibroblast growth factor. Proc. Natl. Acad. Sci. USA 83:7216–7220; 1986.

    Article  PubMed  CAS  Google Scholar 

  6. Carley, W. W.; Milici, A. J.; Madri, J. A. Extracellular matrix specificity for the differentiation of capillary endothelial cells. Exp. Cell Res. 178:426–434; 1988.

    Article  PubMed  CAS  Google Scholar 

  7. Engelmann, K.; Böhnke, M.; Friedl, P. Isolation and long-term cultivation of human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 29:1656–1662; 1988.

    PubMed  CAS  Google Scholar 

  8. Engelmann, K.; Böhnke, M.; Friedl, P. Life span of human corneal endothelial cells in long-term cultures. Ophthalmol. Res. 21:303–308; 1989.

    Article  CAS  Google Scholar 

  9. Fabricant, R. N.; Alpar, A. J.; Centifanto, Y. M., et al. Epidermal growth factor receptors on corneal endothelium. Arch. Ophthalmol. 99:305–308; 1981.

    PubMed  CAS  Google Scholar 

  10. Friedl, P.; Tatje, D.; Czapla, R. An optimized culture medium for human vascular endothelial cells from umbilical cord veins. Cytotechnology 2:171–172; 1989.

    Article  Google Scholar 

  11. Giguere, L.; Cheng, J.; Gospodarowicz, D. Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium. J. Cell. Physiol. 110:72–80; 1982.

    Article  PubMed  CAS  Google Scholar 

  12. Gospodarowicz, D.; Mescher, A. L.; Birdwell, Ch. R. Stimulation of corneal endothelial cell proliferationin vitro by fibroblast and epidermal growth factors. Exp. Eye Res. 25:75–89; 1977.

    Article  PubMed  CAS  Google Scholar 

  13. Gospodarowicz, D.; Bialecki, H.; Greenburg, G. Purification of the fibroblast growth factor activity from bovine brain. J. Biol. Chem. 253:3736–3743; 1978.

    PubMed  CAS  Google Scholar 

  14. Gospodarowicz, D.; Vlodavsky, I.; Savion, N. IV. Factors for growth or differentiation: the role of fibroblast growth factor and the extracellular matrix in the control of proliferation and differentiation of corneal endothelial cells. Vision Res. 21:87–103; 1981.

    Article  PubMed  CAS  Google Scholar 

  15. Gospodarowicz, D.; Lui, G.-M. Effect of substrata and fibroblast growth factor on the proliferationin vitro of bovine aortic endothelial cells. J. Cell. Physiol. 109:69–81; 1981.

    Article  PubMed  CAS  Google Scholar 

  16. Gospodarowicz, D.; Ferrara, N.; Schweigerer, L., et al. Structural characterization and biological functions of fibroblast growth factor. Endocrinol. Rev. 8:95–114; 1987.

    CAS  Google Scholar 

  17. Ham, R. G. Survival and growth requirements of nontransformed cells. Hdbk. Exp. Pharmacol. 57:13–88; 1981.

    Google Scholar 

  18. Ham, R. G.; St. Clair, J. A.; Webster, C., et al. Improved media for normal human muscle satellite cells: serum-free clonal growth and enhanced growth with low serum. In Vitro 24:833–844; 1988.

    CAS  Google Scholar 

  19. Hammond, S. L.; Ham, R. G.; Stampfer, M. R. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc. Natl. Acad. Sci. USA 81:5435–5439; 1984.

    Article  PubMed  CAS  Google Scholar 

  20. Harper, R. A. Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts. Exp. Cell Res. 178:254–263; 1988.

    Article  PubMed  CAS  Google Scholar 

  21. Hayashi, K.; Sueishi, K.; Tanaka, K., et al. Immunohistochemical evidence of the origin of human corneal endothelial cells and keratocytes. Graefes Arch. Clin. Exp. Ophthalmol. 224:452–456; 1986.

    Article  PubMed  CAS  Google Scholar 

  22. Hoshi, H.; McKeehan, W. L. Brain- and liver cell-derived factors are required for growth of human endothelial cells in serum-free culture. Proc. Natl. Acad. Sci. USA 81:6413–6417; 1984.

    Article  PubMed  CAS  Google Scholar 

  23. Hyldahl, L. Primary cell cultures from human embryonic corneas. J. Cell. Sci. 66:343–351; 1984.

    PubMed  CAS  Google Scholar 

  24. Hyldahl, L. Factor VIII expression in the human embryonic eye: differences between endothelial cells of different origin. Ophthalmologica 191:184–187; 1985.

    Article  PubMed  CAS  Google Scholar 

  25. Kleinman, H. K.; McGarvey, M. L.; Hassell, J. R., et al. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.

    Article  PubMed  CAS  Google Scholar 

  26. Knedler, A.; Ham, R. G. Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum. In Vitro 23:481–491; 1987.

    CAS  Google Scholar 

  27. Kovacs, G.; Szücs, S.; De Riese, W., et al. Specific chromosome aberration in human renal cell carcinoma. Int. J. Cancer 40:171–178; 1987.

    Article  PubMed  CAS  Google Scholar 

  28. Lechner, J. F.; Haugen, A.; McClendon, I. A., et al. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642; 1982.

    PubMed  CAS  Google Scholar 

  29. MacCallum, D. K.; Lillie, J. H.; Scaletta, L. J., et al. Bovine corneal endotheliumin vitro. Exp. Cell Res. 139:1–13; 1982.

    Article  PubMed  CAS  Google Scholar 

  30. McKeehan, W. L.; McKeehan, K. A.; Hammond, S. L., et al. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13:399–416; 1977.

    PubMed  CAS  Google Scholar 

  31. Nayak, Sh.K.; Binder, P. S. The growth of endothelium from human corneal rims in tissue culture. Invest. Ophthalmol. Vis. Sci. 25:1213–1216; 1984.

    PubMed  CAS  Google Scholar 

  32. Nayak, Sh.K.; Samples, J. R.; Deg, J. K., et al. Growth characteristics of primate (baboon) corneal endotheliumin vitro. Invest. Ophthalmol. Vis. Sci. 27:607–611; 1986.

    PubMed  CAS  Google Scholar 

  33. Olsen, E. G.; Davanger, M. The healing of human corneal endothelium. Acta Ophthalmol. 62:885–892; 1984.

    Article  CAS  Google Scholar 

  34. Panjwani, N.; Moulton, P.; Alroy, J., et al. Localization of lectin binding sites in human, cat, and rabbit corneas. Invest. Ophthalmol. Vis. Sci. 27: 1280–1284; 1986.

    PubMed  CAS  Google Scholar 

  35. Perlmann, M.; Baum, J. L. The mass culture of rabbit corneal endothelial cells. Arch. Ophthalmol. 92:235–237; 1974.

    Google Scholar 

  36. Pistov, M. Yu.; Sandovnikova, E. Yu.; Danilov, S. M. Human corneal endothelial cells: isolation, characterization and longterm cultivation. Exp. Eye Res. 47:403–414; 1988.

    Article  Google Scholar 

  37. Risen, L. A.; Binder, P. S.; Nayak, Sh.K. Intermediate filaments and their organization in human and corneal endothelium. Invest. Ophthalmol. Vis. Sci. 28:1933–1938; 1987.

    PubMed  CAS  Google Scholar 

  38. Rowe, W. P.; Hartley, J. W.; Lander, M. R., et al. Noninfectious AKR mouse embryo cell lines in which each cell has the capacity to be activated to produce infectious murine leukemia virus. Virology 46:866–876; 1971.

    Article  PubMed  CAS  Google Scholar 

  39. Shipley, G. D.; Ham, R. G. Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of swiss 3T3 cells. In Vitro 17:656–670; 1981.

    PubMed  CAS  Google Scholar 

  40. Shipley, G. D.; Childs, C. B.; Volkenant, M. E., et al. Differential effects of epidermal growth factor, transforming growth factor, and insulin on DNA and protein synthesis and morphology in serum-free cultures of AKR-2B cells. Cancer Res. 44:710–716; 1984.

    PubMed  CAS  Google Scholar 

  41. Smith, P. K.; Krohn, R. I.; Hermanson, G. B., et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76–85; 1985.

    Article  PubMed  CAS  Google Scholar 

  42. Stocker, F. W.; Eiring, A.; Georgiade, R., et al. A tissue culture technique for growing corneal epithelial, stromal, and endothelial tissue separately. Am. J. Ophthalmol. 46:294–298; 1958.

    PubMed  CAS  Google Scholar 

  43. Vlodavsky, I.; Folkman, J.; Sullivan, R., et al. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. USA 84:2292–2296; 1987.

    Article  PubMed  CAS  Google Scholar 

  44. Yang, A. H.; Gould-Kostka, J.; Oberley, T. D. In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate. In Vitro 23:34–46; 1987.

    CAS  Google Scholar 

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  1. Department of Cytogenetics, GBF-Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300, Braunschweig, FRG

    Katrin Engelmann & Peter Friedl

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  1. Katrin Engelmann
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Engelmann, K., Friedl, P. Optimization of culture conditions for human corneal endothelial cells. In Vitro Cell Dev Biol 25, 1065–1072 (1989). https://doi.org/10.1007/BF02624143

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