A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin
Received: 12 September 1994 Accepted: 10 February 1995 DOI:
Cite this article as: Normand, J. & Karasek, M.A. In Vitro Cell Dev Biol - Animal (1995) 31: 447. doi:10.1007/BF02634257 Summary
When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin.
To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions.
To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment to
Ulex europeus agglutinin I-coated paramagnetic beads.
To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS).
Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.
Key words isolation propagation keratinocytes microvascular endothelial cells fibroblasts human skin biopsy References
Auerbach, R.; Plendl, J.; Kusha, B. Endothelial cell heterogeneity and differentiation. In: Maragoudakis, M., ed. Angiogenesis in health and disease. New York: Plenum Press; 1992:55–62.
Bensch, K. G.; Davison, P. M.; Karasek, M. A. Factors controlling the in vitro growth pattern of human microvascular endothelial cells. J. Ultrastruct. Res. 82:76–89; 1983.
Birdwell, C. R.; Gospodarowicz, D. Factors from 3T3 cells stimulate proliferation of cultured vascular endothelial cells. Nature 268:528–531; 1977.
Breidahl, A. F.; Judson, R. T.; Clunie, G. J. A. Review of keratinocyte culture techniques: problems of growing skin. Aust. N. Z. J. Surg. 59:485–497; 1989.
Cox, R.; Masson, W. K. Quantitative clonogenic cell techniques in studies with human diploid fibroblasts. In: Potten, C. S.; Hendry, J. H., eds. Cell clones: manual of mammalian cell techniques. New York: Churchill-Livingstone; 1985:170–174.
Daley, J. P.; Epstein, D. A.; Hawley-Nelson, P. Growth of human epidermal keratinocytes in keratinocyte serum-free medium. Focus 12:68–71; 1990.
Davison, P. M.; Bensch, K.; Karasek, M. A. Isolation and growth of endothelial cells from the microvessels of the newborn human foreskin in cell culture. J. Invest. Dermatol. 75:316–321; 1980.
Davison, P. M.; Bensch, K.; Karasek, M. A. Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis. In Vitro 19:937–945; 1983.
Davison, P.; Karasek, M. A. Human dermal microvascular endothelial cells in vitro: effect of cyclic AMP on cellular morphology and proliferation rate. J. Cell Physiol. 106:253–258; 1981.
Hawley-Nelson, P.; Sullivan, J. E.; Kung, M., et al. Optimized conditions for the growth of human epidermal cells in culture. J. Invest. Dermatol. 75:176–182; 1980.
Hewett, P. W.; Murray, J. C. Human microvessel endothelial cells: isolation, culture and characterization. In Vitro Cell. Dev. Biol.:823–830; 1993.
Hewett, P. W.; Murray, J. C. Immunomagnetic purification of human microvessel endothelial cells using dynabeads coated with monoclonal antibodies to PECAM-1. Eur. J. Cell Biol. 62:451–454; 1993.
Hewett, P. W.; Murray, J. C.; Price, E. A., et al. Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. In Vitro Cell. Dev. Biol. 29A:325–331; 1993.
Holthöfer, H.; Virtanen, I.; Kariniemi, A. L., et al.
I lectin as a marker for vascular endothelium in human tissues. Lab. Invest. 47:60–66; 1982.
Hormia, M.; Lehto, V. P.; Virtanen, I. Identification of UEA-1 binding surface glycoproteins on cultured human endothelial cells. Cell Biol. Int. Rep. 7:467–475; 1983.
Jackson, C. J.; Garbett, P. K.; Nissen, B., et al. Binding of human endothelium to
I-coated Dynabeads: application to the isolation of microvascular endothelium. J. Cell Sci. 96:257–262; 1990.
Johnson, E. W.; Meunier, S. F.; Roy, C. J., et al. Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation. In Vitro Cell. Dev. Biol. 28A:429–435; 1992.
Jones, G. E. Establishment, maintenance and cloning of human primary cell strains. In: Walker, J. M., ed. Methods in molecular biology: animal cell culture. Vol. 5. Totowa, NJ: Humana Press; 1990:13–23.
Karasek, M. A. Cell culture of human skin epidermal cells. In: Goldsmith, L. A., ed. Biochemistry and physiology of the skin. New York: Oxford University Press; 1983:230–240.
Karasek, M. A. Microvascular endothelial cell culture. J. Invest. Dermatol. 93:33–38; 1989.
Kramer, R. H.; Karasek, M. A.; Bensch, K. G. Isolation and characterization of human vascular endothelial cells with application to studies of the subendothelial matrix. In: Pretlow, T. P.; Pretlow, T. G., eds. Cell separation: methods and selected applications. Vol. 5. San Diego, CA: Academic Press; 1987:1–27.
Kumar, S.; West, D. C.; Ager, M. Heterogeneity in endothelial cells from large vessels and microvessels. Differentiation 36:57–70; 1987.
Lipton, B. H.; Bensch, K. G.; Karasek, M. A. Microvessel endothelial cell transdifferentiation: phenotypic characterization. Differentiation 46:117–133; 1991.
Liu, S. C.; Karasek, M. A. Isolation and growth of adult human epidermal keratinocytes in cell culture. J. Invest. Dermatol. 71:157–162; 1978.
Maciag, T.; Nemore, R. E.; Weinstein, R., et al. An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes. Science 211:1452–1453; 1981.
Marks, R. M.; Czerniecki, M.; Penny, R. Human dermal microvascular endothelial cells: an improved method for tissue culture and a description of some singular properties in culture. In Vitro Cell. Dev. Biol. 21:627–635; 1985.
McAuslan, B. R.; Hannan, G. N.; Reilly, W. Characterization of an endothelial cell proliferation factor from cultured 3T3 cells. Exp. Cell Res. 128:95–101; 1980.
Moerman, E. J.; Goldstein, S. Culture of human fibroblasts. In: Clarke, W. L.; Larner, J.; Pohl, S., eds. Methods in diabetes research. Vol. II. New York: John Wiley & Sons; 1986:283–312.
Normand, J.; Karasek, M. A. Micromethod for the isolation of keratinocytes, endothelial cells and fibroblasts from a single punch biopsy of human skin. Clin. Research. 42:11A; 1994 (abstract).
Page, C.; Rose, M.; Yacoub, M., et al. Antigenic heterogeneity of vascular endothelium. Am. J. Pathol. 141:673–683; 1992.
Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–344; 1975.
Sherer, G. K.; Fitzharris, T. P.; Faulk, W. P., et al. Cultivation of microvascular endothelial cells from human preputial skin. In Vitro 16:675–684; 1980.
Thilo, D. G. S.; Müller-Küsel, S.; Heinrich, D., et al. Isolation of human venous endothelial cells by different proteases. Artery 8:259–266; 1980.
Tsao, M. C.; Walthall, B. J.; Ham, R. G. Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell. Physiol. 110:219–229; 1982.
Tuder, R. M.; Karasek, M. A.; Bensch, K. G. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells. J. Cell. Physiol. 142:272–283; 1990.
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