Skip to main content

Advertisement

Log in

Skin explant cultures as a source of keratinocytes for cultivation

  • Original Paper
  • Published:
Cell and Tissue Banking Aims and scope Submit manuscript

Abstract

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this “passaging” yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84(8):2302–2306

    Article  PubMed  CAS  Google Scholar 

  • Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81(1 Suppl):33–40

    Article  Google Scholar 

  • Cruickshank CN, Cooper JH, Hooper C (1960) The cultivation of cells from adult epidermis. J Invest Dermatol 34:339–342

    PubMed  CAS  Google Scholar 

  • Dragúňová J, Kabát P, Koller J, Jarabinská V (2011) Experience gained during the long term cultivation of keratinocytes for treatment of burns patient. Cell Tissue Bank. doi:10.1007/s10561-011-9275-z

  • Green H, Kehinde O, Thomas J (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 76(11):5665–5668

    Article  PubMed  CAS  Google Scholar 

  • Guo A, Jahoda CA (2009) An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells. Exp Dermatol 18(8):720–726

    Google Scholar 

  • Halprin KM, Lueder M, Fusenig NE (1979) Growth and differentiation of postembyonic mouse epidermal cells in explant culture. J Invest Dermatol 72(2):88–98

    Article  PubMed  CAS  Google Scholar 

  • Hammr H (1981) Stimulated mouse ear epidermis in explant culture—the effect of retinoic acid and hexadexane. Arch Dermatol Res 270(4):469–481

    Article  Google Scholar 

  • James SE, Booth S, Dheansa B, Mann DJ, Reid MJ, Shevchenko RV, Gilbert PM (2010) Strayed cultured autologous keratinocytes used alone or in combination with meshed autografts to accelerate wound closure in difficult-to-heal burn patients. Burns 36(3):10–20

    Article  Google Scholar 

  • Leigh I, Watt FM (1994) Keratinocyte methods. Cambridge University Press, Cambridge

    Google Scholar 

  • Ljunggren CA (1897) Von Fahigkeit des Hautepithels ausserhalb des Organismus sein Leben zu behalten mit Beruchsichtigung der Transplantation. Deutsche Zietschrift fur Chirurgie 47:608–615

    Article  Google Scholar 

  • Medawar PB (1941) Sheets of pure epidermal epithelium from human skin. Nature 148:783

    Article  Google Scholar 

  • Morris RJ, Fischer SM, Slaga TJ (1985) Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations. J Invest Dermatol 84(4):277–281

    Article  PubMed  CAS  Google Scholar 

  • Moustafa M, Bullock AJ, Creagh FM, Heller S, Jeffcoate W, Game F, Amery C, Tesfaye S, Ince Z, Haddow DB, MacNeil S (2007) Randomized controlled, single blind study of use of autologous keratinocytes on a transfer dressing to treat nonhealing diabetic ulcers. Regen Med 2(6):887–902

    Google Scholar 

  • Mujaj S, Manton K, Upton Z, Richards S (2010) Serum-free primary human fibroblasts and keratinocyte coculture. Tissue Eng Part A 16(4):1407–1420

    Article  PubMed  CAS  Google Scholar 

  • Myers S, Navsaria H, Sanders R, Green C, Leigh I (1995) Transplantation of keratinocytes in the treatment of wounds. Am J Surgery 170(1):75–83

    Article  CAS  Google Scholar 

  • Nakamura M, Rikimaru T, Yano T, Moore G, Pula PJ, Schofield BH, Dannenberg AM Jr (1990) Full-thickness human skin explants for testing the toxicity of topically applied chemicals. J Invest Dermatol 95(3):325–332

    Google Scholar 

  • Navsaria HA, Sexton C, Bouvard V, Leigh IM, Watt FM (1994) Human epidermal keratinocytes. In: Leigh IM, Watt FM (eds) Keratinocyte methods. Cambridge University Press, Cambridge, pp 5–12

    Google Scholar 

  • Normand J, Karasek MA (1995) A method for the isolation and serial propagation of keratinocytes, endothelial cells and fibroblasts from a single punch biopsy of human skin. In Vitro Cell Dev Biol Anim 31(6):447–455

    Article  PubMed  CAS  Google Scholar 

  • Peramo A, Marcelo CL, Goldstein SA, Matrin DC (2009) Novel organotypic cultures of human skin explants with an implant-tissue biomaterial interface. Ann Biochem Eng 37(2):401–409

    Google Scholar 

  • Rheinwald JG, Green H (1975a) Formation of a keratinizing epithelium in culture by a cloned cells line derived from a teratoma. Cell 6(3):317–330

    Article  PubMed  CAS  Google Scholar 

  • Rheinwald JC, Green H (1975b) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6(3):331–343

    Article  PubMed  CAS  Google Scholar 

  • Smirnov SV, Kiselev IV, Rogovaya OS, Vasilev AV, Terskikh VV (2003) Skin repair by transplantation of cultured keratinocytes. Bulleten Experimentalnoj Biologii i Meditsiny 135(6):608–609

    CAS  Google Scholar 

  • Sun T, Higham M, Layton C, Haycock J, Short R, MacNeil S (2004) Developments in xenobiotic-free culture of human keratinocytes for clinical use. Wound Repair Regen 12(6):626–634

    Article  PubMed  Google Scholar 

  • Taylor JR, Halprin KM, Levine V, Woodyard C (1983) Effects of methotrexate in vitro on epidermal proliferation. Br J Dermatol 108(1):45–61

    Article  PubMed  CAS  Google Scholar 

  • Varani J, Perone P, Spahliger DM, Singer L, Diegel KL, Bobrowski WF, Dunstan R (2007) Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage. Toxicol Pathol 35:693–701

    Google Scholar 

  • Wood FM, Kolybaba ML, Allen P (2006) The use of cultured epithelial autograft in the treatment of major burn wounds: eleven years of clinical experience. Burns 32(5):538–544

    Google Scholar 

Download references

Acknowledgments

The authors thank Joanne Martin for critical comments on an early version of the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to P. Kabát.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dragúňová, J., Kabát, P. & Koller, J. Skin explant cultures as a source of keratinocytes for cultivation. Cell Tissue Bank 14, 317–324 (2013). https://doi.org/10.1007/s10561-012-9330-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10561-012-9330-4

Keywords

Navigation