Oral mucosa culture system for ocular surface treatment avoiding the use of xenogeneic components

Abstract

Purpose : To develop an easy and xenogeneic free culture system of oral mucosa stem cells

Methods : Tissue and blood collection. Oral mucosa biopsies were obtained from deceased organ donors following Spanish laws. Platelet Rich in Growth Factors (PRFG) used for media supplementation and PRGF fibrin membranes were obtained from voluntary donors and processed using a commercial kit (Endoret^®)
Cell culture. 3-4 mm² biopsies were cut in fragments and cultured as explants in a 12 well culture plate
Culture media. Cells were expanded in two different media: DMEM/F12 (2:1), 5 µg/ml insulin, 8.33 ng/ml choleric toxin, 24 μg/ml adenine, 1.3 ng/ml triiodothyronine, 0.4 µg/ml hydrocortisone, 40 µg/ml vancomycin-amikacin, 0.5 µg/ml amphotericin B and either 10% SBF or 10% PRGF
Data analysis. Expansion efficiency of SBF and PRGF cultures were compared quantitatively and qualitatively
PRGF membrane in vivo assay. Confluent cultures of epithelial cells expanded in PRGF were subcultured on 4 cm² PRGF fibrin membranes (2.5 x 10⁵ cells/cm²), cultured for 72h and grafted in the subcutaneous space of nude mice. After 21 days, animals were euthanized and tissue samples were subjected to immunohistochemical analysis
Immunofluorescence. Cultures and tissues were fixed with 4% paraformaldehyde and immunofluorescence studies were performed using anti-p63, anti-CK5 and anti-CK14 monoclonal antibodies

Results : Cell culture. 93±5% and 91±9% of cultures were successfully expanded from initial biopsies cultured in SBF and PRGF, respectively. The expanded cells showed epithelial cells, fibroblast or a mix of both. Pure epithelial cell cultures without contaminant fibroblasts were observed in 61±6% of SBF cultures and in 69±13% of PRGF cultures. Data analysis showed that there were no statistically significant differences between the media
Immunofluorescence. Oral mucosal epithelial cells were positive against CK5, CK14, and p63 either cultured with SBF or PRGF. Cells cultured on the PRGF membrane still retained their morphological and phenotypic markers
In vivo assay. Oral mucosal epithelial cells remained viable and retained its characteristic morphology and phenotypic markers without any apparent tissue defect in the in vivo model

Conclusions : It is possible to isolate oral mucosal epithelial stem cells without the need of xenogenic factors. These cells could be grafted and represent a potential alternative for ocular tissue engineering

This is a 2021 ARVO Annual Meeting abstract.