June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Feasibility of a Corneal Endothelial Cell Therapy using two in vitro corneal models.
Author Affiliations & Notes
  • Sergio Alonso-Alonso
    Regenerative medicine in ocular surface, Fundacion de Investigacion Oftalmologica, Oviedo, Asturias, Spain
  • Natalia Vázquez
    Regenerative medicine in ocular surface, Fundacion de Investigacion Oftalmologica, Oviedo, Asturias, Spain
  • Manuel Chacon
    Regenerative medicine in ocular surface, Fundacion de Investigacion Oftalmologica, Oviedo, Asturias, Spain
  • Alvaro Meana
    Regenerative medicine in ocular surface, Fundacion de Investigacion Oftalmologica, Oviedo, Asturias, Spain
  • Jesus Merayo-Lloves
    Regenerative medicine in ocular surface, Fundacion de Investigacion Oftalmologica, Oviedo, Asturias, Spain
    Universidad de Oviedo, Oviedo, Asturias, Spain
  • Mairobi Persinal-Medina
    Universidad de Oviedo, Oviedo, Asturias, Spain
  • Footnotes
    Commercial Relationships   Sergio Alonso-Alonso None; Natalia Vázquez None; Manuel Chacon None; Alvaro Meana None; Jesus Merayo-Lloves None; Mairobi Persinal-Medina None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 625. doi:
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      Sergio Alonso-Alonso, Natalia Vázquez, Manuel Chacon, Alvaro Meana, Jesus Merayo-Lloves, Mairobi Persinal-Medina; Feasibility of a Corneal Endothelial Cell Therapy using two in vitro corneal models.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):625.

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

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Abstract

Purpose : to study the feasibility of a corneal endothelial cell therapy to treat corneal endothelial dysfunction using two in vitro rabbit corneal models: one with Descemet's membrane preserved and one without.

Methods : Methods: Two models of endothelial dysfunction were established by removing corneal endothelium (Descemet's membrane + corneal endothelial cells) or scraping corneal endothelial cells. Corneal models were placed in a Transwell® insert and covered with culture medium until use. Corneal endothelial cells were obtained by processing twelve corneal endothelia obtained from rabbit corneas. Briefly, the corneal endothelium was dissected following the Schwalbe line and digested with TrypLE for 120 min at 37 °C. The detached cells were then centrifuged at 0.4 g for 10 min, the supernatant was removed, and the cells were seeded on the corneal model (n=6) or on a 2 cm2 culture plate (n=6) previously treated with FNC coating mix® for culture. The medium used used as a source of the growth factors rabbit Plasma rich in growth factors (Optimem I, 10 v/v% PRGF, 200 mg/L CaCl2 and 10 U/mL penicillin, 10 µg/mL streptomycin). Once confluent, corneal endothelial cell cultures were detached with TrypLE for 30 min, detached cells were centrifuged at 0.4 g for 10 min and seeded in an endothelial dysfunction model. After 24 h, the corneas were covered with culture medium and cultured at 37 °C, in a 5% CO2 atmosphere in an incubator for 7 seven days. The corneas were then fixed with ice-cold methanol overnight and embedded in OCT for histologic H-E analysis.

Results : The control corneas, both Descemetorhexis and scraping (n=4), do not present corneal endothelial cells. In corneas seeded with uncultured rabbit cells we can observe that if the model lacks Descemet's membrane the cells encounter problems to adhere and proliferate, however when Descemet's membrane is present these cells are able to form a monolayer. In corneas seeded with cultured rabbit cells, the cells were able to adhere and proliferate regardless of the presence or absence of Descemet membrane. In addition, after 7 days of culture, all corneas show edema, a normal condition when preserved in cell culture media.

Conclusions : Rabbit corneal endothelial cells are able to adhere to our ex vivo corneal model, moreover, after being cultured, their adhesion is improved in the absence of Descemet's membrane.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

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