Original Article

Journal of Investigative Dermatology (1997) 109, 289–295; doi:10.1111/1523-1747.ep12335766

Regulation of Pigmentation in Cultured Skin Substitutes by Cytometric Sorting of Melanocytes and Keratinocytes

Viki B Swope1, Andrew P Supp2, James R Cornelius2, George F Babcock1,2 and Steven T Boyce1,2

  1. 1Department of Surgery, University of Cincinnati, U.S.A.
  2. 2Shriners Burns Institute, Cincinnati, Ohio, U.S.A.

Received 3 February 1997; Revised 25 April 1997; Accepted 12 May 1997.

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Abstract

Unpredictable pigmentation in cultured skin substitutes (CSS) is an anatomic deficiency after wound treatment and can require years to normalize. Variable numbers of human melanocytes (HM) survive in cultures of human keratinocytes (HK) as demonstrated by focal areas of pigmentation in CSS after healing. The purposes of this study were to deplete MM from HK cultures and to regulate the numbers of HM contained in CSS. A highly pigmented HM cell strain was chosen for these studies to emphasize the differences in light scattering between HK and HM by flow cytometry. Cytometric gates were set with selective cultures of HM and HK and were used to sort a mixed population of HK + 4% HM. After sorting, CSS were prepared from human fibroblasts attached to collagen-glycosaminoglycan sponges combined with cells from the FIX + 4% MM (Ire-treatment control), the sorted HR (experimental), or sorted HK + 3% HM (post-treatment positive control) subpopulations and grafted to athymic mice. Grafted wounds were assessed for 6 wk by planimetry for area of pigment and by a Minolta Chromameter for color density and hue in situ. Histology and staining of HLA-ABC were performed at 6 wk. Data from percent pigmented area and chromameter measurements identified quantitative and statistically significant decreases in color of healed skin after flow cytometric separation of HK and HM. Therefore, a purified HK subpopulation depleted of HM was isolated by flow cytometry that generated healed skin with reduced pigmentation. These results suggest that HM can be selectively depleted from HR cultures and then added to cultured skin substitutes at specific densities to generate predictable pigmentation for improved function and cosmesis in healed wounds.

Keywords:

athymic mice

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