Primary Mucosal Epithelial Cell Cultivation: A Reliable and Accelerated Isolation

Background: Our aim was to establish a reliable protocol for the isolation of primary human oral mucosal epithelial cells that ensured a successful, efficient, and reproducible cell culture for the utilization in tissue engineering purposes. We evaluated various isolation routines and cultivation protocols. Furthermore, we investigated the functionality of isolated epithelial cells for tissue engineering approaches in an in vitro mucosa equivalent cell culture model. Methods: Oral mucosal biopsies were obtained from patients undergoing oral surgery. Different enzymatic and nonenzymatic isolation protocols were carried out. Media supplements and the size of cut biopsy material were analyzed to determine their effect on epithelial cell outgrowth. The epithelial phenotype of the isolated cells was demonstrated by immunohistochemical staining of various cell markers. The functionality of the isolated primary oral epithelial cells was evaluated in an established in vitro mucosa model in coculture with fibroblasts and in triculture with the addition of endothelial cells. Results: We found that enzymatic digestion has no advantage to nonenzymatic preparation of oral mucosal tissues. A reliable outgrowth of epithelial cells could be obtained by increasing the insulin concentration in culture media. Other media supplements analyzed such as epidermal growth factor did not show any benefits. Tissue sizes between 1 and 3 mm(2) seemed to be the most effective for the isolation of cells. Stable epithelial cell cultures with confluent monolayers could be achieved. The cells stained positive for typical epithelial markers such as cytokeratin 13, cytokeratin 8/18, and cytokeratin 19. In tissue engineered tricultures on collagen scaffolds, epithelial cells formed a simple epithelium including a cell multilayer and reached terminal differentiation. Conclusion: A comparatively high insulin concentration results in a reliable outgrowth of epithelial cells in explant culture, presumably by stimulating cell proliferation. Enzymatic digestion does not seem to be necessary for epithelial cell isolation, but rather is disadvantageous compared with mechanical tissue processing. The method described is an easy, fast, and reliable way to rapidly obtain functional primary human epithelial cell cultures. The cells are suitable for tissue engineering applications as confirmed by the organization of a terminally differentiated epithelium in an in vitro mucosa equivalent cell culture model. Impact Statement We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevertheless reliable method.