Tumorigenicity Testing in Athymic Mice of Cultured Human Melanocytes for Transplantation in Engineered Skin Substitutes

Abstract

Autologous engineered skin substitutes (ESS) have been shown to close excised, full-thickness burns, but are consistently hypopigmented due to depletion of human melanocytes (hM) during culture of keratinocytes. Hypothetically, addition of hM to ESS may restore uniform pigmentation, but may also promote neoplasia and tumor formation. To evaluate this risk, 16 strains of hM were isolated and propagated in selective culture medium, then injected subcutaneously into athymic mice (1 × 10 7 hM/animal; n = 6/strain) and followed for 24 weeks. Human melanoma cells (SK-Mel-2, SK-Mel-5) served as positive controls. No detectable tumors formed from hM strains derived from normal skin. In contrast, SK-Mel-2 formed tumors in 50% of mice, and SK-Mel-5 formed tumors in 83% of mice. Histopathology confirmed the tumorigenic anatomy of the controls and the presence of hM that were not tumorigenic in the test groups. These results support the safety of cultured hM for transplantation to restore uniform skin pigmentation in wounds closed with ESS.

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

“…Transplantation studies of two types have been performed to evaluate tumorigenicity as an index of safety (Boyce, Lloyd, Kleiner, & Supp, ; Boyce et al., ) and pigment uniformity and density as reported here. In tumorigenicity studies, no tumors were detected during 24 weeks of observation (Boyce et al., ), and earlier restoration of pigmented area and pigment density was found after incubation of ESS‐P in UCDM1 compared with UCMC160.…”

“…To prevent this risk, phorbol esters have been eliminated from media for hM (Swope, Medrano, Smalara, & Abdel‐Malek, ; Swope, Supp, Cornelius, Babcock, & Boyce, ). In previous studies from these investigators, culture of hM in media with only physiologic mitogens (i.e., basic fibroblast growth factor, endothelin‐1, α‐melanocyte‐stimulating hormone [α‐MSH], triiodothyronine) resulted in no detectable tumor formation after subcutaneous injection in immunodeficient mice (Boyce, Zimmerman, & Supp, ).…”

Evaluation of Cultured Melanocytes in Terms of Pigmentation, Genetic Stability and Tumorigenicity in order to Vitiligo patients Transplantation

“…The NA8 melanoma cell line displayed a modal distribution of chromosome numbers 59-61 (hypotriploid), A375 cells displayed a modal distribution of chromosome number 62 (hypotriploid), and D10 cells displayed a bimodal distribution of chromosomes 46-48 (near triploid, Figure 3A and Table 4). [4], +4 [4],del(4)(q33) [2],del(4)(q33) [1],dup(4)(q21q24) [4],+5 [4],del(5)(p15) [4],rev( 5)(p?) [4],-6 [4],del(7)(q31) [4],der(7)t(7;10)(q32;q23) [4],8 [4],+10 [4],del(10)(p13) [4],del(10)(p13) [4],add(11)(p11.2) [4],der( 12)t(1;17)(q21;p11.1) [4],der(12)t(9;12)(q21;p13) [2],add(13)(p13) [3],der(13)t(5;13)(q11.2;p13) [2],-14 [3], add( 14)(q32) [2],t(14;14)(q10;q10) [1],…”

Advances in Skin Tissue Bioengineering and the Challenges of Clinical Translation

“…However, this model also has limitations (lack of other cell types and adnexal structures, contraction of the collagen scaffold during ESS fabrication, relatively high cost and regulatory complexity); and is not commercially available. Pre-clinical studies have recently demonstrated the successful incorporation of melanocytes ( 108 , 109 ), microvascular endothelial cells ( 110 ), and hair follicles ( 111 ) into the ESS model.…”