July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Characterization of a collagen-based engineered corneal endothelium
Author Affiliations & Notes
  • Maria Dolores Montalvo Parra
    Tecnologico de Monterrey (ITESM), Monterrey, NUEVO LEON, Mexico
  • Judith Zavala
    Tecnologico de Monterrey (ITESM), Monterrey, NUEVO LEON, Mexico
  • Wendy Ortega-Lara
    Tecnologico de Monterrey (ITESM), Monterrey, NUEVO LEON, Mexico
  • Jorge E. Valdez-Garcia
    Tecnologico de Monterrey (ITESM), Monterrey, NUEVO LEON, Mexico
  • Footnotes
    Commercial Relationships   Maria Dolores Montalvo Parra, None; Judith Zavala, None; Wendy Ortega-Lara, None; Jorge E. Valdez-Garcia, None
  • Footnotes
    Support  CONACyT Ciencia Basica PN-207 6558
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4119. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Maria Dolores Montalvo Parra, Judith Zavala, Wendy Ortega-Lara, Jorge E. Valdez-Garcia; Characterization of a collagen-based engineered corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4119.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Corneal endothelium replacement must assure cell morphology and physiology. In order to do so, a biocompatible, transparent, scaffold is the best option. The purpose of our project was to characterize a construct produced by seeding corneal endothelial cells (CEC) on collagen vitrigel (CV) membranes.

Methods : CV membranes were produced modifying a previous methodology in two steps: 1) Gelification; ~2 µl/mm2 of collagen type I, HEPES solutions, and fetal bovine serum mixture per well were placed on a 12 well plate, 37°C, 5% CO2 for 2 hrs. 2) Vitrification: a sealed desiccation chamber with a saturated solution of K2CO3 was placed in an oven set to 40°C; collagen gels were left inside for 37 days to decrease relative humidity up to 40%. CV membranes were characterized with confocal microscopy, SEM and spectrophotometry to analyze surface, thickness and transparency respectively. CEC were isolated from young New Zealand rabbits. Descement’s membrane was peeled from rabbit cornea, digested and, CEC obtained were cultured until confluence in mitotic media (OptiMEM I, FBS 8%, nerve growth factor 20 ng/ml, endothelial growth factor 5ng/ml, CaCl2 200 µg/ml, ascorbic acid 20 µg/ml, chondroitin sulfate 0.08%, antibiotic 1%). Passages 1 and 2 were carried out in resting media (OptiMEM I 8%FBS). At passage 3, ~24,000 CEC were planted onto 8 mm Ø CV membranes.

Results : SEM and confocal microscopy tests showed CV membranes yielded a ~4 µm thickness and smooth surface upon 20 min hydration. SEM also showed collagen fibers merge to form a mesh-like laminar structure. Spectrophotometric scan from 450-700 nm showed a 94-95.5% transmittance. CEC seeded on CV membranes showed adhesion and proliferation at 24 hours; 72 hours served to reach confluence in a ~5 mm Ø. Culture on CV membranes reached canonical corneal endothelium shape. We produced 12 constructs made of collagen vitrigel and culture-expanded CEC from one cornea.

Conclusions : In conclusion, CV synthesis modified methodology offers a robust option to produce an scaffold with optical properties that can be easily stablished in every laboratory and can be scaled up. CEC cultured in a two-phase system adhere and proliferate over the CV membrane maintaining their canonical morphology.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Corneal endothelium cells (CEC) on collagen-vitrigel membrane, 48 hours after seeding. The CEC were isolated from a rabbit, second passage expansion under a two phase culture system.

Corneal endothelium cells (CEC) on collagen-vitrigel membrane, 48 hours after seeding. The CEC were isolated from a rabbit, second passage expansion under a two phase culture system.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×