June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Oral mucosa culture system for ocular surface treatment avoiding the use of xenogeneic components
Author Affiliations & Notes
  • Alvaro Meana
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Mairobi Persinal-Medina
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Natalia Vázquez
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Manuel Chacon
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Sergio Alonso-Alonso
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Carlos Fernández-Vega-González
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Jose I Blazquez
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Sara Llames
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
    U714, Centro de Investigacion Biomedica en Red de Enfermedades Raras, Valencia, Valenciana, Spain
  • Jesus Merayo-Lloves
    Instituto Universitario Fernández-Vega - Fundación de Investigación Oftalmológica - Universidad de Oviedo, Oviedo, Asturias, Spain
  • Footnotes
    Commercial Relationships   Alvaro Meana, None; Mairobi Persinal-Medina, None; Natalia Vázquez, None; Manuel Chacon, None; Sergio Alonso-Alonso, None; Carlos Fernández-Vega-González, None; Jose Blazquez, None; Sara Llames, None; Jesus Merayo-Lloves, None
  • Footnotes
    Support  RTC-2017-6760-1 (AUTOCELL)
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 758. doi:
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    • Get Citation

      Alvaro Meana, Mairobi Persinal-Medina, Natalia Vázquez, Manuel Chacon, Sergio Alonso-Alonso, Carlos Fernández-Vega-González, Jose I Blazquez, Sara Llames, Jesus Merayo-Lloves; Oral mucosa culture system for ocular surface treatment avoiding the use of xenogeneic components. Invest. Ophthalmol. Vis. Sci. 2021;62(8):758.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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