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Himi Singh, Radhika Tandon, Sujata Mohanty, Anil Kumar; Study of Effect of Donor Age and Death Enucleation Time on in-vitro Culture of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6018.
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The purpose of this study was to review the experience of our laboratory considering the role of donor age and death enucleation time (DET) for optimization of in-vitro Human Corneal Endothelial Cell (HCEC) culture.
Human corneal tissues not suitable for transplantation, based upon their endothelial cell count (<1500cells/mm2), were used to establish the primary culture. The explants of the endothelial cell layer with the Descemet’s membrane were propagated in culture dishes coated with fibronectin. The basal media used for establishment of culture was OptiMEM-I supplemented with 15% FBS, 10ng/ml EGF, pituitary extract 100µg/ml, 20 µg/ml ascorbic acid, 0.08% chondroitin sulphate, 20ng/ml nerve growth factor and 100 U/ml penicillin and 100μg/ml streptomycin. Established endothelial cells were subsequently expanded for two passages and assessed morphologically. The corneal endothelial cultures were also analyzed for their propensity to proliferate using Ki67and for characteristic markers ZO-1, and Na+K+ATPase through RT-PCR and indirect immunofluorescence in both the age groups.
The endothelial culture success rate was calculated based on two criteria i.e. age group and DET .For the age group of 20-60 years and >60 years the successful culture rate was 11% (4/36) and 18% (20/110)respectively and successful culture rate for DET ≤6hrs and>6 hrs was 19% and 12%. Further culture success rate in age groups of 20-60 yearsDET≤6hrs (10%), DET>6hrs (12%), and for age group >60 years it was DET≤6 hrs (19%), DET> 6hrs (10%) respectively. Corneal Endothelial Cell cultures were established in both the age groups and they retained polygonal cellular morphology during their initial passages. However, the phenotype of HCEC’s from old donors could make less compact monolayer in comparison to young donors. The cultured HCECs expressed markers characteristics of the human corneal endothelium- ZO-1 and Na+K+ATPase which were confirmed by RT-PCR and indirect immunoflouresence in both the age groups. Immunostaining for Ki67, a proliferation marker were positive for both the age groups indicating proliferation capacity of old donor tissue.
These results indicate that shorter DET helps in successfully establishing the HCEC’s despiteolder age. The ability to obtain cultured endothelial cells from old age can be useful in field of tissue engineering and for extended utilization of donated corneas.
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