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A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

Cytotechnology volume 63, pages 655–662 (2011)Cite this article

Abstract

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

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References

  • Atiyeh BS, Costagliola M (2007) Cultured epithelial autograft (CEA) in burn treatment: three decades later. Burns 33:405–413

    Article  Google Scholar 

  • Billingham RE, Medawar PB (1955) Contracture and intussusceptive growth in the healing of extensive wounds in mammalian skin. J Anat 89:114–123

    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 81:33s–40s

    Article  CAS  Google Scholar 

  • Boyce ST, Kagan RJ, Yakuboff KP, Meyer NA, Rieman MT, Greenhalgh DG, Warden GD (2002) Cultured skin substitutes reduce donor skin harvesting for closure of excised, full-thickness burns. Ann Surg 235:269–279

    Article  Google Scholar 

  • Coolen NA, Verkerk M, Reijnen L, Vlig M, van den Bogaerdt AJ, Breetveld M, Gibbs S, Middelkoop E, Ulrich MM (2007) Culture of keratinocytes for transplantation without the need of feeder layer cells. Cell Transpl 16:649–661

    Google Scholar 

  • Cooper ML, Spielvogel RL (1994a) Artificial skin for wound healing. Clin Dermatol 12:183–191

    Article  CAS  Google Scholar 

  • Cooper ML, Spielvogel RL (1994b) Artificial skin for wound healing. Clin Dermatol 12:183–191

    Article  CAS  Google Scholar 

  • Currie LJ, Martin R, Sharpe JR, James SE (2003) A comparison of keratinocyte cell sprays with and without fibrin glue. Burns 29:677–685

    Article  Google Scholar 

  • De Corte P, Verween G, Verbeken G, Rose T, Jennes S, De Coninck A, Roseeuw D, Vanderkelen A, Kets E, Haddow D, Pirnay JP (2011) Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety. Cell Tissue Bank [Epub ahead of print]

  • Gustafson CJ, Kratz G (1999) Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds. Burns 25:331–335

    Article  CAS  Google Scholar 

  • Harris KL, Bainbridge NJ, Jordan NR, Sharpe JR (2009) The effect of topical analgesics on ex vivo skin growth and human keratinocyte and fibroblast behavior. Wound Repair Regen 17:340–346

    Article  Google Scholar 

  • Heiskanen A, Satomaa T, Tiitinen S, Laitinen A, Mannelin S, Impola U, Mikkola M, Olsson C, Miller-Podraza H, Blomqvist M, Olonen A, Salo H, Lehenkari P, Tuuri T, Otonkoski T, Natunen J, Saarinen J, Laine J (2007) N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells 25:197–202

    Article  CAS  Google Scholar 

  • Horch RE, Bannasch H, Kopp J, Andree C, Stark GB (1998) Single-cell suspensions of cultured human keratinocytes in fibrin-glue reconstitute the epidermis. Cell Transpl 7:309–317

    Article  CAS  Google Scholar 

  • James SE, Booth S, Dheansa B, Mann DJ, Reid MJ, Shevchenko RV, Gilbert PM (2010) Sprayed cultured autologous keratinocytes used alone or in combination with meshed autografts to accelerate wound closure in difficult-to-heal burns patients. Burns 36:e10–e20

    Article  Google Scholar 

  • LaFrance ML, Armstrong DW (1999) Novel living skin replacement biotherapy approach for wounded skin tissues. Tissue Eng 5:153–170

    Article  CAS  Google Scholar 

  • Lamme EN, Van Leeuwen RT, Brandsma K, Van Marle J, Middelkoop E (2000) Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation. J Pathol 190:595–603

    Article  CAS  Google Scholar 

  • Martin MJ, Muotri A, Gage F, Varki A (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 11:228–232

    Article  CAS  Google Scholar 

  • Martin Y, Eldardiri M, Lawrence-Watt DJ, Sharpe JR (2011) Microcarriers and their potential in tissue regeneration. Tissue Eng Part B Rev 17:71–80

    Article  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 on use of autologous keratinocytes on a transfer dressing to treat nonhealing diabetic ulcers. Regen Med 2:887–902

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Navarro FA, Stoner ML, Park CS, Huertas JC, Lee HB, Wood FM, Orgill DP (2000) Sprayed keratinocyte suspensions accelerate epidermal coverage in a porcine microwound model. J Burn Care Rehabil 21:513–518

    Article  CAS  Google Scholar 

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

    Google Scholar 

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

    Article  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 12:626–634

    Article  Google Scholar 

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Acknowledgments

This work was funded by the Blond McIndoe Research Foundation.

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Affiliations

  1. Blond McIndoe Research Foundation, Queen Victoria Hospital, Holtye Road, East Grinstead, West Sussex, RH19 3DZ, UK

    K. Jubin, Y. Martin, D. J. Lawrence-Watt & J. R. Sharpe

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  1. K. Jubin
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  2. Y. Martin
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  3. D. J. Lawrence-Watt
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  4. J. R. Sharpe
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Correspondence to J. R. Sharpe.

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Jubin, K., Martin, Y., Lawrence-Watt, D.J. et al. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use. Cytotechnology 63, 655–662 (2011). https://doi.org/10.1007/s10616-011-9382-5

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  • DOI: https://doi.org/10.1007/s10616-011-9382-5

Keywords

  • Cell culture
  • Tissue engineering
  • Skin
  • Keratinocyte