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Hung-Chi Chen, David Hui-Kang Ma, Hsiang-Fu Huang, Jesseica Ma, Yi-Jen Hsueh; Stem/progenitor properties are preserved during ex vivo expansion of human oral mucosal epithelial cell sheet by collagenase but not dispase. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1444. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, in the Phase I trial of cultivated oral mucosal epithelial transplantation (COMET), we have substantiated efficacy of such cell therapy in promoting wound healing in patients with severe ocular surface burns, identified long-term persistence of transplanted oral mucosal epithelial cells (OMEC) in the cornea, and concluded that COMET in suitable in promoting corneal wound healing due to its inferior inhibitory effect on corneal neovascularization. Recently, collagenase, but not dispase, has been reported to isolate cell spheres mimicking stem/progenitor cells from the corneal endothelium or epithelium. In this Phase II trial, we aimed to investigate the safety and efficacy of isolating OMEC with collagenase.
Human oral mucosal remnants removed during oral surgeries were digested either by 1% collagenase A in PBS overnight or 1.2 U dispase II/0.25% trypsin EDTA at room temperature for 1.5 hours followed by scrapping. The yielded cell pellets were seeded on denuded amniotic membrane (DAM) with or without coculture with 3T3 cells. BrdU incorporation assay, colony formation assay (CFA), immunostaining and Western blot for stem/progenitor cell markers (p63 and p75) and differentiation markers (keratins 3 and 13) were performed. Scanning electron microscopy (SEM) was used to compare surface structures. Finally, Western blot for nuclear β-catenin was performed.
Collagenase treatment group generated cell sheets that retained regular cell morphology, and could be maintained in serum-free media, and even without 3T3 coculture. In contrast, 3T3 coculture was indispensible in the initial culture stage using dispase II/trypsin. BrdU label index and CFA were both higher in collagenase group, and so was a prominent p63 expression by Western blot in that group. Finally, more prominent nuclear β-catenin expression by Western blot was also noted in the collagenase group.
Collagenase treatment is superior to dispase II/trypsin treatment in generating human OMEC sheet, and is free of animal serum and 3T3 cells, which better conforms with contemporary regulations of cell therapy. Therefore, we suggest adopting such a cell isolation method in the Phase II clinical trial of COMET. Future goals are to investigate the molecular mechanisms through which collagenase treatment can better preserve the stem/progenitor cells of OMEC.
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