June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Functionalising a PEG-based Hydrogel Scaffold Improves Human Corneal Endothelial Cell Culture for Tissue-engineered Endothelial Keratoplasty.
Author Affiliations & Notes
  • Karl David Brown
    Surgical Research Unit, Centre for Eye Research Australia Ltd, East Melbourne, Victoria, Australia
  • Jason Palmer
    Surgical Research Unit, Centre for Eye Research Australia Ltd, East Melbourne, Victoria, Australia
  • John Finnigan
    Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia
  • Paul Gurr
    Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia
  • Jaime Montenegro
    Surgical Research Unit, Centre for Eye Research Australia Ltd, East Melbourne, Victoria, Australia
  • Greg Dusting
    Surgical Research Unit, Centre for Eye Research Australia Ltd, East Melbourne, Victoria, Australia
  • Greg Qiao
    Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia
  • Mark Daniell
    Surgical Research Unit, Centre for Eye Research Australia Ltd, East Melbourne, Victoria, Australia
  • Footnotes
    Commercial Relationships   Karl Brown None; Jason Palmer None; John Finnigan None; Paul Gurr None; Jaime Montenegro None; Greg Dusting None; Greg Qiao None; Mark Daniell None
  • Footnotes
    Support  Portions of this work were funded by Australian Federal Government Medical Research Futures Fund Grant and an EBAA High Impact Grant. Dr Brown was supported by a DHB Foundation Fellowship
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3812. doi:
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      Karl David Brown, Jason Palmer, John Finnigan, Paul Gurr, Jaime Montenegro, Greg Dusting, Greg Qiao, Mark Daniell; Functionalising a PEG-based Hydrogel Scaffold Improves Human Corneal Endothelial Cell Culture for Tissue-engineered Endothelial Keratoplasty.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3812.

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

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Abstract

Purpose : We previously developed a PEG-based hydrogel film for transplantation of cultured corneal endothelial cells. We hypothesised that functionalisation of our film with either carboxylic acid or a peptide with the arginine-glycine-aspartate (RGD) signalling sequence would improve the quality of cultured human corneal endothelial cells (hCEC) compared to non-functionalised films. Key indicators of quality are cells per mm2 and cell shape.

Methods : B4G12 hCEC line seeded at 200 cell/mm2 was cultured on PEG-based hydrogel (non-functionalised control), RGD functionalised PEG-based hydrogel, carboxylic acid functionalised hydrogel, and tissue culture plastic (positive control) for 14 days, fixed in 4% PFA and immunofluorescence for zonula occuludens-1 (ZO-1) was performed. Morphometrics including confluence, cell area, and cell circularity was performed on immunofluorescent images using ImageJ. Cell/mm2 was calculated from cell area and confluence.

Results : On all surfaces B4G12 cells immunofluorescence demonstrated cells expressed ZO-1 and had polygonal morphology indicating the cells maintained a corneal endothelial phenotype. As hypothesised, functionalised hydrogels produced superior cultures with higher confluence, smaller cells, and therefore higher cells/mm2 than non-functionalised hydrogels. Mean results (confluence, area, calculated cell/mm2, circularity): non-functionalised hydrogel (50 %, 317 µm2, 1577 cell/mm2, 0.74), RGD functionalised hydrogel (93%, 249 µm2, 3735 cell/mm2, 0.71), carboxylic acid functionalised hydrogel (86 %, 263 µm2, 3270 cell/mm2, 0.73), tissue culture plastic (100 %, 291 µm2, 3436 cell/mm2, 0.69). A donor corneal endothelium had a circularity of 0.76. Eyebanks typically require donor to have greater than 2500 cell/mm2.

Conclusions : B4G12 cells maintain their phenotype when cultured on our PEG-based hydrogel. On functionalised hydrogels high quality cell cultures were produced that exceeded the quality measures required by eyebanks. Peptide functionalised hydrogels were superior to those functionalised with carboxylic acid groups. Ongoing work is validating these results with cadaveric donor primary corneal endothelial cells.
There is a global shortage of donor corneas for transplantation. A tissue engineered corneal endothelium could remove corneal endothelial disease cases from the waiting lists for donor corneas.

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

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