Melanocytes in Cultured Epithelial Grafts Are Depleted With Serial Subcultivation and Cryopreservation

Compton, Carolyn C.; Warland, Gretchen; Kratz, Gunnar

doi:10.1097/00004630-199807000-00011

Cited by 19 publications

(14 citation statements)

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“…40 Indeed, when primary keratinocytes were serially cultivated under conditions usually adopted for the preparation of epidermal grafts destined for full-thickness burns (ie, plated at a density of 7.5ϫ 10 3 cells/cm 2 and passaged during the exponential phase of growth), 28 a significant number of melanocytes were preserved only in primary and secondary cultures, and melanocyte concentration rapidly decreased during serial cultivation (Figure 1A, yellow bars). However, if keratinocytes were subcultivated 1 to 2 days after they reached confluence and seeded at a cell density of at least 4 ϫ10 4 cells/cm 2 , the melanocyte/keratinocyte ratio was several-fold higher ( Figure 1A, blue bars) and was preserved also during repeated subcultivation.…”

Section: Human Epidermis Contains Approximately 4 ϫ10mentioning

confidence: 99%

Treatment of "Stable" Vitiligo by Timedsurgery and Transplantation of Cultured Epidermal Autografts

et al. 2000

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Section: Human Epidermis Contains Approximately 4 ϫ10mentioning

confidence: 99%

Treatment of "Stable" Vitiligo by Timedsurgery and Transplantation of Cultured Epidermal Autografts

et al. 2000

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“…Certainly, melanocytes can be identified within CEA sheets (9,13,14), but CEA culture has been optimised for the keratinocyte, not the melanocyte (19). There is literature available that contends that keratinocyte density will adversely affect melanocyte density (20).…”

Section: Influence Of Keratinocyte Density and Feeder Layers On Melanmentioning

confidence: 99%

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

Phillips¹,

Gawkrodger²,

Caddy³

et al. 2001

Pigment Cell Research

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A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well-integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co-cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down-regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase-related protein-1 (TRP-1) negative in confluent well-integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP-1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP-1 expression.

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“…Despite the success with CSS in treating burn wounds, CSS are deficient in many of the structures and functions of uninjured skin, such as sebaceous glands, melanocytes, hair follicles, endothelial cells, nerve cells and cell‐mediated immunity 3 . In postburn healing wounds, abnormal pigmentation patterns include both hyperpigmentation and hypopigmentation 4–6 . The skin dyschromia can take months to years to normalize.…”

mentioning

confidence: 99%

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

Swope

Supp²,

Boyce

2002

Wound Repair Regeneration

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Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.

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Melanocytes in Cultured Epithelial Grafts Are Depleted With Serial Subcultivation and Cryopreservation

Cited by 19 publications

References 0 publications

Treatment of "Stable" Vitiligo by Timedsurgery and Transplantation of Cultured Epidermal Autografts

Treatment of "Stable" Vitiligo by Timedsurgery and Transplantation of Cultured Epidermal Autografts

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

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