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Gustavo T. Grottone, Priscila Cristovam, Renata R. Loureiro, Jose Alvaro P. Gomes; Optimized Isolation Of Human Corneal Endothelial Cells For Primary Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6459.
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© ARVO (1962-2015); The Authors (2016-present)
Create a new method for isolation of human corneal endothelial cell primary. Compare the results achieved by this method with other current protocols.
Nine human corneas received from BOS Eye Bank- Sao Paulo, were used for our experiment. Sheets of endothelial cells were placed in different medias for digestion and release of cells. Group A received a digestion treatment of SHEM media supplemented with collagenase A 1.5 mg /ml and incubated overnight. Group B received a mild digestion treatment of Opti-MEM media supplemented with collagenase D 0.5 mg/ml and incubated overnight. Group C received no digestion treatment and was incubated in Opti-MEM overnight. In group A, the media was replaced by SHEM and the pellet resuspended and plated. In group B, the media was replaced by EDTA 0,02% and left atin the incubator for 50 minutes. After that the cells were pipetted with a flame-polished pasteur pipette for 50 times and recentrifuged. The residual pellet was washed by Opti-MEM and plated on FNC coated 12-well dishes. Finally, group C, had media by EDTA 0,02% and left in the incubator for 30 minutes. After that the cells were pipetted with a 1000 ul micropipette for 30 times and recentrifuged. The residual pellet was washed by Opti-MEM and plated on FNC coated 12-well dishes.Cells were left undisturbed for 48 hours. Primary cultures were checked for morphology of the resultant material, adherence and time for confluency.
After digestion and plating group A, showed cell aggregates with hundreds of cell and residual ECM, in a round shape. A few cells were loose corresponding to dead cells using trypan blue dying. Group B, showed a large number of single cells in suspension after isolation but still moderate number of viable cells attached to the ECM. Group C, showed a large number of single cells in suspension after isolation without any cell attached to the ECM. After 48 hours, all cell aggregates from group A were attached to the culture plate. Group B, showed 50% of cells attached to the bottom of the well with some remaining cells still attached at ECM. Group C, showed 60% of cells attached to the well. Confluency time was 3 weeks for group A, 1 week for group B and 1 week for group C.
Isolation methods using EDTA and mechanical shearing resulted in a more efficient single cell suspension. Prior digestion with collagenase D facilitates cell isolation from ECM providing good results using regular micropipettes. Groups B and C reached confluency quickier.
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