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Alfonso Luis Sabater, Adriano Guarnieri, María García Guzmán, Enrique Andreu, Felipe Prósper, Javier Moreno-Montanes; A Novel and Highly Efficient Method for the Expansion of Corneal Endothelial Cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2027.
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© ARVO (1962-2015); The Authors (2016-present)
To define a new culture medium that promotes the expansion and migration of primate corneal endothelial cells (PCEC), maintaining their characteristic hexagonal morphology.
Corneal Descemet’s membrane and corneal endothelial cells obtained from 5 cynomolgus macaque monkeys were digested with collagenase A in basal growth medium A for 14 hours. After digestion, PCEC aggregates were resuspended in basal growth medium (A or B) and plated in 1 well of a 48-well plate coated with FNC Coating Mix®. Basal growth medium A was composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 ng/ml bFGF and antibiotics. Basal growth medium B was composed of Knockout DMEM supplemented with 10% FBS, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 2 ng/ml bFGF and antibiotics. Additionally, IGF-1, Heregulin and Activin were added to the basal medium B in different proportions. A growth curve was performed to monitor cell proliferation. A wound-healing assay was performed by scratching the cell layer with a pipette tip, and phase-contrast images were taken at 0, 6, 12 and 24 hours later to assess cell migration into the open space.
Cell proliferation was significantly improved with basal medium B, and was even higher when IGF-1, Heregulin and Activin were added to this medium. Cell size was considerably smaller when basal medium B was used. Addition of IGF-1, Heregulin and Activin to the basal medium B significantly enhanced wound healing. PCEC were able to maintain their characteristic hexagonal morphology up to passage 2 with basal medium A, up to passage 4 with basal medium B and up to passages 6 or more when IGF-1, Heregulin and Activin were added to the basal medium B.
The findings of this study indicate that addition of IGF-1, Heregulin and Activin to the basal medium significantly increases the proliferative capacity of primate corneal endothelial cells. Additionally, these components enhance wound healing and maintain the characteristic hexagonal morphology of corneal endothelial cells.
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