Feeder-Free Cell Culture of Labial Oral Mucosal Epithelium for Tissue-Engineering and Regenerative Medicine

Background. The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions. Aims — to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering. Methods. Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patient’s lip mucosa were used to set the explant (n = 18) and enzymatic (n = 17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06 mM Calcium contained 5% fetal bovine serum, 5 μg/ml human insulin, 5 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression. Results. The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p = 0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.1 ± 3.6 μm thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p = 0.001) in comparison with the enzymatic method. Stable cultures had a “cobble-stone” morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mM Ca⁺⁺ medium (p = 0.0006). Conclusions. Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.