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Olga Kuzmenko, Alena Bartakova, Noelia J Kunzevitzky, Jeffrey L Goldberg; Clinical Grade Magnetic Human Corneal Endothelial Cells for Cell Therapy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2255. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cell-based therapies to replace corneal endothelium rely on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT), consistent magnetic nanoparticle incorporation for maximum cell delivery, and stability after shipping.
HCECs were isolated from cadaveric donor corneas and expanded in vitro in the presence of mitogenic growth factors and in low-mitogen media, as well as . superparamagnetic nanoparticles (MNPs) prior to harvesting. To quantify the effectiveness of MNP incorporation, Prussian Blue staining and colorimetric methods were used. Resilience to shipping was tested using commercial overnight services, and HCEC identity, viability and function were assessed over time using marker expression, flow cytometry, morphology, and trans-endothelial electrical resistance (TEER) assays.
The use of low-mitogenic medium at confluence before each passage increased the number of passages HCECs could undergo while maintaining canonical morphology, identity and ability to form tight junctions. Prussian Blue staining and colorimetry revealed consistency in MNP binding across biological replicates. In shipping stability experiments, HCECs transported in 1 ml syringes were viable (>70%) for up to 120 hours when shipped and stored at 4°C, and these cells expressed identity and functional markers similar to cryopreserved cells after thawing.
HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and avoid EnMT, increasing the number of passages and total canonical cell yield. These data may facilitate production of a reliable HCEC therapy, and a path towards cell transplant for human studies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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