July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells
Author Affiliations & Notes
  • Jae Chan Kim
    Ophthalmology, Cheil eye hospital, Daegu, Korea (the Democratic People's Republic of)
    Chung-Ang University, Seoul, Korea (the Republic of)
  • Kyoung Woo Kim
    Ophthalmology, Chung-Ang University Hospital, Seoul, Korea (the Republic of)
  • Soo Jin Lee
    Ophthalmology, Cheil eye hospital, Daegu, Korea (the Democratic People's Republic of)
  • Footnotes
    Commercial Relationships   Jae Chan Kim, None; Kyoung Woo Kim, None; Soo Jin Lee, None
  • Footnotes
    Support  NRF-2017R1A2A2A05001128
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2182. doi:https://doi.org/
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      Jae Chan Kim, Kyoung Woo Kim, Soo Jin Lee; Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2182. doi: https://doi.org/.

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

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Abstract

Purpose : Human corneal endothelial cells (HCECs) are scarcely proliferative in vivo. In this study, we prepared cultured HCECs engineered to overexpress ribonuclease (RNase) 5 (R5-HCECs). Given that preparation of cell-seeded transplantable grafts must be quick and easily reproducible, we directly deposited cultured R5-HCECs on a carrier by extrusion-based printing and further evaluated the ex vivo functionality of 3D bioprinted corneal endothelial graft.

Methods : After HCECs were isolated from human donor corneas and were expanded in vitro, they were treated with RNase 5 plasmid to become R5-HCECs which overexpress RNase 5. Cell viability, proliferation, growth velocity and phenotypical marker expression were analyzed in accordance with RNase 5 expression. R5-HCECs were 3D bioprinted on lyophilized bovine amniotic membrane (AM) to prepare transplantable corneal endothelial graft, thereafter grafts were transplanted in rabbit corneas. Corneal clarity and central corneal thickness (CCT) were evaluated every 1 week over a month in R5-HCEC-bioprinted graft (R5-Graft) group and in control HCEC-bioprinted graft (Ct-Graft) group. At postsurgical 4 weeks, the grafts were observed histologically and their immune-staining of surface markers were examined.

Results : As candidate targets of RNase 5-elicited enhancement of viability and proliferation, PDCD4 was inhibited and cyclin D1 and cyclin E1 were activated in R5-HCECs. Cultured R5-HCECs showed more copious immune-staining of Na+-K+ ATPase with less fibroblastic morphology compared to control HCECs in vitro and this was supported by elevated gene expression of ATP1A1, TJP1 and ALCAM (i.e. CD166 gene). From 2 weeks after surgery, engrafted rabbit corneas in both R5-Graft and Ct-Graft groups started to recover corneal clarity and their CCTs were lower than Descemetorhexis-only group. At postsurgical 4 weeks, polygonal donor R5-HCECs were observed ex vivo on the posterior surface of the graft and they clearly expressed Na+-K+ ATPase, ZO-1 and CD166 along to hexagonal boundaries.

Conclusions : In corneal endothelial decompensated rabbit corneas, 3D bioprinted R5-HCECs-laden AM graft was well-engrafted and significantly improved corneal thickness and edema. Our results may provide proof-of-principle for future investigations of transplantation of genetically engineered 3D bioprinted corneal endothelial graft.

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

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