June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Incorporation of hPSC-derived photoreceptors and RPE cells into microfabricated, honeycomb-patterned microscaffolds for outer retinal reconstruction
Author Affiliations & Notes
  • Agustin Luz Madrigal
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • In–Kyu Lee
    Department of Electrical and Computer Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Ruosen Xie
    Departments of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Seunghwan Min
    Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Jingcheng Zhu
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Jiahe Jin
    Departments of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Allison L. Ludwig
    Comparative Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • M. Joseph Phillips
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Shaoqin Gong
    Departments of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Zhenqiang Ma
    Electrical and Computer Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • David M Gamm
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Agustin Luz Madrigal None; In–Kyu Lee WO2021076454, Code P (Patent); Ruosen Xie None; Seunghwan Min None; Jingcheng Zhu None; Jiahe Jin None; Allison L. Ludwig None; M. Joseph Phillips Opsis Therapeutics LLC , Code C (Consultant/Contractor), WO2021076454, Code P (Patent); Shaoqin Gong WO2021076454, Code P (Patent); Zhenqiang Ma WO2021076454, Code P (Patent); David Gamm US Patent No. US9752119B2 , Code P (Patent), Patent Application WO2021076454 , Code P (Patent), Opsis Therapeutics LLC , Code S (non-remunerative)
  • Footnotes
    Support  Department of Defense through the Vision Research Program under award no. W81XWH-20-1-0655; McPherson Eye Research Institute Sandra Lemke Trout Chair in Eye Research; Fighting Blindness Canada including philanthropic support from Donna Green and Goldie Feldman; NIH grant P51OD011106 to the Wisconsin National Primate Research Center; Slack Prevention of Blindness Fund-Muskingum County Community Foundation; Research to Prevent Blindness, Emmett A. Humble Distinguished Directorship of the Retina Research Foundation
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3173. doi:
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    • Get Citation

      Agustin Luz Madrigal, In–Kyu Lee, Ruosen Xie, Seunghwan Min, Jingcheng Zhu, Jiahe Jin, Allison L. Ludwig, M. Joseph Phillips, Shaoqin Gong, Zhenqiang Ma, David M Gamm; Incorporation of hPSC-derived photoreceptors and RPE cells into microfabricated, honeycomb-patterned microscaffolds for outer retinal reconstruction. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3173.

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

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Abstract

Purpose : Blinding retinal diseases such as late-stage of age-related macular degeneration require the replacement of both retinal pigment epithelium (RPE) and photoreceptors (PRs). However, co-delivery of these cell types as an organized bilayer is technically challenging. To address this issue, we developed a 3D honeycomb (HC)-patterned, poly(glycerol sebacate) (PGS) micro-molded scaffold capable of organized reconstruction of the RPE-PR bilayer.

Methods : HC-patterned PGS microscaffolds were generated using a novel process of photocuring that reduces the time required for scaffold production from days to minutes. Microscaffolds mounted in transwells were gas sterilized and laminin-coated before cell seeding. hPSC-RPE and retinal organoids (RO) were generated using an established protocol, followed by dissociation and seeding of each cell type alone or in combination to determine the optimum cell seeding density of RPE, PRs, or RPE + PRs. Live cell imaging was performed via widefield fluorescence microscopy. Scaffolds were then fixed and analyzed by immunocytochemistry (ICC) and high resolution 3-D reconstruction confocal microscopy.

Results : When seeded with hPSC-PRs alone, HC-patterned microscaffolds showed uniform cell incorporation and retention (~2x104 PRs cells/mm2). HC-patterned microscaffolds also supported uniform monolayer coverage of hPSC-RPE (9969 RPE cells/mm2) with proper cellular morphology. Importantly, following RPE seeding there remained a large space within each well conducive to secondary PR seeding. Dual RPE:PR seeding efficiency was tested by seeding PRs onto scaffolds pre-seeded with RPE the preceding day. Approximately 80% of all RPE-lined HC wells contained clusters of PRs. Overall, dual RPE:PR-seeded microscaffolds supported approximately 1.0 x104 RPE cells/mm2 and 1.3 x104 PRs cells/mm2.

Conclusions : We have optimized methods to generate a 3D outer retinal construct capable of organized co-delivery of RPE and PRs for the potential treatment of conditions such as late-stage AMD and other macular dystrophies resulting in the loss of both cell types.

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

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