The Viennese culture method: cultured human epithelium obtained on a dermal matrix based on fibroblast containing fibrin glue gels

Abstract

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin glue gel containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on to the gel. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained. After 10 days an irregular multilayer could be observed. The cells showed active interaction with the fibrin support, presenting as cell formations projecting into the matrix. After 15 days a regular epithelial sheet consisting of three to four layers of cells was formed. A limiting membrane demarcating the keratinocytes from the fibrin matrix was discernible. Squamous differentiation similar to Strata reticulare and corneum found in vivo could be observed. Nuclei of basal cells were regularly spaced from each other and the chromatin was of homogeneous appearance without prominent nucleoli. The last time point (20 days) showed signs of disintegration of the epithelial sheet. A basement membrane-like structure could not be seen any more. Detachment of the basal cells was associated with subepithelial vacuoles. Basal cells contained irregular nuclei. Therefore, we conclude that 15 days of culture were optimal for the generation of a keratinocyte layers with signs of differentiation; this new culture system could be an important step forward in covering severely burned patients due to a number of advantages, as for example a large expansion factor, the shortening of the optimal culture time to 15 days, the usage of commercially available fibrin glue gels and the versatile manipulation of composite cultures.