The use of in vitro 2d co-culture models to determine the optimal keratinocyte: melanocyte ratio to be used in the development of pigmented 3d skin model

Doctoral Thesis


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University of Cape Town

Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.

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