June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Engineering a photoreceptor cell-RPE microphysiological system
Author Affiliations & Notes
  • Laura R Bohrer
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Arwin Shrestha
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Erin R Burnight
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Luan M. Streb
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Robert Mullins
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Budd A. Tucker
    Institute for Vision Research, University of Iowa, Iowa City, Iowa, United States
    Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Laura Bohrer None; Arwin Shrestha None; Erin Burnight None; Luan Streb None; Robert Mullins None; Edwin Stone None; Budd Tucker None
  • Footnotes
    Support  NIH EY033331, Elmer and Sylvia Sramek Charitable Foundation, Howard Ruby Chair and Professor of Regenerative Ophthalmology
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3177. doi:
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    • Get Citation

      Laura R Bohrer, Arwin Shrestha, Erin R Burnight, Luan M. Streb, Robert Mullins, Edwin M Stone, Budd A. Tucker; Engineering a photoreceptor cell-RPE microphysiological system. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3177.

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

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Abstract

Purpose : Induced pluripotent stem cells (iPSCs) provide the opportunity to derive differentiated tissues from patients with inherited retinal diseases and use these models to study disease mechanisms and test therapeutics. However, current protocols often fail to accurately recapitulate the cellular organization present in the native tissue. The purpose of this study was to engineer a human model system that accurately recapitulates the outer retinal unit.

Methods : Polycaprolactone (PCL) scaffolds were printed on glass coverslips using ultra-high resolution two-photon lithography. Several photoreceptor cell scaffold designs with different pore size (10-80µm) and base height (0-15µm) were produced and tested. IPSC-derived retinal organoids were generated using a stepwise 3D differentiation protocol, dissociated at Day 100-150 and loaded onto scaffolds dropwise with or without a custom cell loading ring. IPSC-derived RPE were generated using a directed differentiation protocol, plated on culture inserts (0.4µm pores) and photoreceptor cell scaffolds were placed on top. A photopolymerized PEGDA gel was fabricated and placed on top to prevent movement during media exchange.

Results : Unlike scaffolds with small pores (10µm), which were designed to allow individual photoreceptor cells to stack one on top of the other, scaffolds with larger pores (80µm) were easier to load and promoted excellent cell-cell contact with uniform post-seeding maturation. To promote consistent loading of all pores, a loading device that surrounded the scaffold was fabricated. Photoreceptor cell scaffolds were successfully matured in the presence or absence of RPE for up to 7 weeks post-seeding. Photoreceptor cells expressing CHX10, recoverin and arrestin extend outersegment like structures toward the basal surface of the scaffold (Fig 1A) and axonal projections toward the apical surface of the scaffold (Fig 1B). Dissociation via papain digestion revealed an average of ~6.4x104 cells per mm2 (+/- 6400) with a viability of >94%. The PCL scaffold and the PEGDA positioning gel were well tolerated by both iPSC-derived photoreceptor and RPE cells (i.e., no evidence of cytotoxicity).

Conclusions : The development of this system will be invaluable for the study and development of drug, gene, and cell-based therapeutics for the treatment of retinal degenerative disorders.

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

 

Fig 1: IHC of photoreceptor cell scaffolds stained for CHX10, recoverin(A) and arrestin(B). *=PCL scaffold

Fig 1: IHC of photoreceptor cell scaffolds stained for CHX10, recoverin(A) and arrestin(B). *=PCL scaffold

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