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Presenter: Minoru, Fujita, Pittsburgh, United States
Authors: Minoru Fujita1, Hidetaka Hara1, Danny S. Roh2, Ruhina Mehra1, James L. Funderburgh2, David Ayares3, David KC. Cooper1
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Comparison of proliferative capacity of genetically-engineered pig and human corneal endothelial cells
Minoru Fujita1, Hidetaka Hara1, Danny S. Roh2, Ruhina Mehra1, James L. Funderburgh2, David Ayares3, David KC. Cooper1
1Department of Surgery, University of Pittsburgh, Pittsburgh, PA; 2Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA; 3Revivicor, Inc., Blacksburg, VA; United States
Background: The worldwide need for human donor corneas far exceeds supply. Although human (h) and pig (p) corneal endothelial cells (CECs) are considered to be non-proliferative in vivo, both can proliferate in vitro. This provides the possibility of providing corneal endothelial sheets for clinical transplantation. A single cornea could provide enough cultured CECs for several patients. Current evidence is that CECs are less likely to cause sensitization and require less immunosuppressive therapy than full-thickness corneal transplants.
Purpose: To compare the proliferative capacity of CECs from (i) wild-type (WT) pigs, (ii) genetically-modified pigs (which are largely protected from the human immune response), and (iii) humans.
Methods: The following CECs were cultured – hCECs from donors (i) <40 years (young), (ii) >40 years (old), and WT pCECs from (iii) neonatal (<10 days), (iv) young (2 months), and (v) old (>3 years) pigs, and pCECs from young (vi) GTKO/CD46 and (vii) GTKO/CD46/CD55 pigs. Direct cell counting over 15 days culture measured (a) cell numbers, and (b) speed of proliferation. The BrdU assay assessed (c) the phase of cell cycle (reflecting proliferation), and the MTT assay assessed (d) cell metabolic activity.
Results: pCECs proved easy to culture. There was significantly lower proliferative capacity of old CECs than of young CECs (p<0.01). There was no significant difference in proliferative capacity/metabolic activity between young h and p CECs. There was a significantly higher percentage of cell death in h compared to p CECs during culture (p<0.01). Young GTKO/CD46 and GTKO/CD46/CD55 pCECs showed similar proliferative capacity/metabolic activity to young WT p and h CECs.
Conclusions: Because of the greater availability of young pigs and the proliferative capacity of cultured GTKO/CD46/CD55 pCECs, genetically-engineered pigs could provide a source of corneal endothelial sheets for clinical Tx.
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