Culture Media and Biopsy Location Affect the Proliferative Capacity and Phenotype of Ex Vivo Expanded Oral Mucosal Tissue

Abstract

Purpose: The oral mucosal epithelium shows substantial potential for use in regenerative medicine, including the treatment of ocular surface disease. The rationale for using oral mucosal cells is the possibility of treating bilateral ocular surface stem cell disease without the use of immunosuppression. The present project determines the ideal harvesting site for oral mucosal biopsies and the optimum culture media for these cells.

Methods: Pieces of oral epithelium were used from four locations: buccal mucosa (BM), hard palate (HP), lower lip (LL), and transition zone of the lower lip (TZ) of Sprague-Dawley rats. Explants were grown in four different culture media for six days. The media were 1) RPMI 1640 with 10% heat- inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 50 IU/mL penicillin-streptomycin; 2) EpiLife with epidermal growth supplement; 3) oral keratinocyte media (OKM) with oral keratinocyte growth factor; and 4) DMEM and Ham 12 (1:1 mixture) supplemented with 10% FBS, 5 μg/mL insulin, 0.1 nmol/L cholera toxin, 10 ng/mL human recombinant epidermal growth factor, and 50 IU/mL penicillin-streptomycin. Inverted light microscopy was used for outgrowth measurements with ImageJ. Immunofluorescence microscopy was used for phenotype characterization using antibodies against proliferating cell nuclear antigen (PCNA) and neural growth factor p75 (NGF P75). Stemness was assayed by colony-forming efficiency assay.

Results: DMEM and RPMI yielded higher explant outgrowth than OKM and Epilife. Using DMEM and RPMI, fold growth was superior from LL biopsies (36.43 ± 7.42 and 32.34 ± 12.22, respectively) compared to biopsies from HP (7.46 ± 0.69 and 10.14 ± 1.14, respectively), BM (11.15 ± 2.94 and 22.40 ± 8.45, respectively) and TZ (19 ± 3 and 15 ± 4.02, respectively) (p<0.05; N=4-11). PCNA (proliferating cells) and NGF P75 expression were detected in all conditions. NGF P75 expression appears to be lower in the cells cultured in RPMI compared to DMEM. In DMEM, TZ showed higher colony forming efficiency (0.057% ± 0.006%) than BM (0.004% ± 0.004%), HP (0.017% ± 0.004%) and LP (0.003% ± 0.002%) (p<0.05; N=2-6).

Conclusions: The transition zone of the lower lip, which showed the highest colony forming ability, may be most effective in treating limbal stem cell deficiency.