Etchevers:Notebook/Genomics of hNCC/2009/04/17
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Endothelial cell cultures from mice
Nine adult mouse right eyes (after euthanasia for Stephane's other experiments) were made available for a trial in corneal endothelial cell culture.
One eye, Pierre showed me how he dissectes them out under the operating scope - not sterile, not retained.
One eye, I tried under the PSM using the in-hood loupe. But there is no field depth and I could not really see what I was doing. So I gave up and came back to lab. Dissected the seven remaining eyes. Cut carefully around limbal edge of cornea, remove to 2nd dish and scrape inner surface with flat edge of scissors to detach Descemet's membrane with adherent endothelium. (Apparently impossible to peel as one can with rabbit or human.) Resuspended mush in some DMEM had on hand, transferred to sterile 15 mL tube.
In culture room, spun down 4' 1200 rpm, removed medium, rinsed in 20% FCS/DMEM/ pen/strep + 0.1% fungizone, spun down same, resuspended in 2.4U/mL Dispase/PBS, spun down again and resuspended in 1 mL Dispase. Incubated for 1.5h at 37°C. Dissociated by trituration in flame-polished pipette for 20 strokes (see membranes floating).
Cell count after resuspension 0.2 mL - approximately 15,000 cells total, mostly in small groups. Trypan blue-excluding.