Standardization of Isolation and Expansion of Oral Mucosa Connective Tissue Cells

Objective: The aim of this study was to establish a protocol for the isolation and expansion of a fibromesenchymal cell population from the connective tissue of the oral mucosa for future bioengineering protocols. Methods: For the isolation of fibroblasts and progenitor cells, we used pieces of surgical samples from patients with an indication for oral surgery. The protocol for isolation was as follows: the tissue was washed with Phosphate-Buffered Saline (PBS) supplemented with antibiotic-antimycotic (PSA). The tissue was placed in a test tube containing collagenase type II and was incubated overnight in the oven. After incubation, the collagenase was collected and the tissue was again washed once with PBS +PSA. Subsequently, the Colony-Forming Unit (CFU) test was performed. The cells (1.0 × 105 ) were plated on a 10 cm² dish containing Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, supplemented with 10% Fetal Bovine Serum (FBS). The cells were fixed with 10% formalin and stained with crystal violet before counting the colonies. The assay was performed in triplicates. Results: The cells from all samples showed a homogeneous morphology with a characteristic stellate appearance. The only difference was in the number of colonies formed. There was a significant increase in the number of colonies formed on day 1 when compared with those formed at day 0, and a significant decrease when compared with those formed at day 2. Conclusion: It was possible to establish a protocol for the primary culture of fibroblasts derived from human oral mucosa.