Abstract
Purpose: :
Create a new method for isolation of human corneal endothelial cell primary. Compare the results achieved by this method with other current protocols.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: cornea: endothelium • cell adhesions/cell junctions • cornea: basic science