Speaker
Description
"Introduction
Anatomically-correct models of human skin are highly desired by both biomedical sciences and industry. Organotypic skin cultures, termed full-thickness skin equivalents, including fibroblasts and keratinocytes, thus forming dermis and epidermis are in use. However, a more detailed model of the skin is difficult to set up due to its cellular heterogeneity, complex vascular anatomy, and intricate interaction between the substructures. Therefore, the next generation of in vitro skin models needs to provide an accurate depiction of homeostatic, aging, or injured skin that capture the skin's cellular heterogeneity and complex anatomy. Since current full-thickness skin equivalents lack functional vasculature, circulation, perfusion or inclusion of the immune system is not possible. As a solution, we here engineered a vascularized 3D full-thickness in vitro skin model (vascuSKINs) including epidermis, dermis, and vasculature by combining vascular self-assembly and full-thickness skin equivalent culture.
Methodology
Human dermal fibroblasts, endothelial cells, and adipose-derived stromal cells were embedded in fibrin-collagen co-gels before seeding of keratinocytes and epidermal differentiation at air-liquid interface culture for 14 days. Skin equivalents were fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained using immunofluorescence for histological analysis.
Results
In order to establish the vascuSKINs, we first investigated the suitability of fibrin-collagen co-gels as an extracellular matrix to support epidermal differentiation and microvascular self-assembly. In contrast to collagen-only hydrogels, the fibrin-collagen co-gels indeed supported vascularization and showed a limited contraction, thus enabling the formation and persistence of open lumina. In addition, histological analysis showed no differences in epidermal stratification of the collagen-fibrin co-gels compared to traditional collagen hydrogels: the epidermis stained positive for keratin 14, keratin 10, and loricrin, suggesting that it well supports keratinocyte differentiation and stratification. Importantly, endothelial cells embedded into the dermis formed a continuous, lumenized microvasculature as visualized by CD31 staining.
Conclusion
In summary, these results show the successful establishment of full-thickness vascularized skin equivalents using collagen-fibrin co-gels and human cells. Furthermore, our model resembles skin by including a dermis and an epidermis that consists of the essential cell types associated with wound healing. The next step of our project is to establish our vascuSKINs as models of skin injury and regeneration."
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