Construction of a Bilayered Dermal Equivalent Containing Human Papillary and Reticular Dermal Fibroblasts: Use of Fluorescent Vital Dyes

Bilayered dermal equivalents were constructed by seeding human papillary and reticular dermal fibroblasts into separate layers of type I collagen and allowing these layers to gel into a single entity. That these bilayered gels had fused was established through histologic examination and from the fact that these gels, when detached, contracted as a single unit. Papillary and reticular dermal fibroblasts remained in their respective layers as established by differentially labeling these dermal cells with fluorescent vital dyes l,l'-dioctacecyl-3,3,3',3'-tetramethylindocabocyanine perchlorate, DiIC₁₈(3) (Dil), and 3,3'-dioctadecyloxacarbocyanine perchlorate, DiOC₁₈(3) (DiO). The labeling with these vital dyes did not interfere with the ability of the cells to proliferate or to contract floating type I collagen gels. Thus, these bilayered gels can provide the means of creating dermal equivalents that contain a variety of different dermal cell types to assess their relative abilities, either alone or in various combinations, to support keratinocyte proliferation and differentiation and to contribute, eventually, to the formation of a multilayered skin equivalent.