Etchevers:Notebook/Genomics of hNCC/2008/09/26
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hNCC injection in rabbit corneas for integration and repair
Today: redid the CMFDA (Molecular Probes) green membrane coloration of the cephalic and trunk hNCC in the flasks. This time, resuspended in F12/DMEM without serum at 25 μM, 10.5 ml per flask. Left in incubator for 50 minutes (maximum treatment).
Rinsed in warm DMEM then applied 8 ml trypsin for 3.5 minutes (it wasn't very warm when put it on) and stopped with 7 ml 15% serum. Spun down for 5 minutes. Cells are BRIGHT yellow. So the serum is a real problem and could have stayed at 5 μM probably.
Resuspended in 320 μL medium for the cephalic cells and 120 μL for the trunk-level cells. Removed 12 μL each for direct counting.
Cephalic: some debris. 208 x 104 cells for a single count over 1 mm2 = 2 x 106 cells / 320 μL or 665600 cells in flask, of which 624000 are remaining, in a wee bit more than 300 μL. So for a dose of 50 μL injected, should apply 100000 cells.
Trunk: cells seemed bigger, perhaps 30 μm diameter, and counted 150 x 104 cells for a single count over 1 mm2 = 1.5 x 106 cells / 120 μL = 180000 cells total (they did seem pretty sparse) and 150000 left in the 100 μL remaining. Added in 50 μL medium to bring down to 50000 cells per 50 μL injection.
Rabbits: protocol under the responsability of F. Malecaze.
Jérôme (animal care facility) premedicated (sedated) the rabbits that had been operated two weeks ago. L eyes had grade 2 corneal opacity after laser ablation of 1/2-2/3 stromal thickness two weeks ago; right eyes unoperated.
Jérôme injected them with ketamine 4 cc and reboosted with addition 1 cc (at what dose?) after 1/2 hour because they still seemed a bit reactive to light stimulus on eye.
Pierre applied drops of oxybuprocaine as local anaesthetic on the cornea, again I don't know the dose, but it's what is used in human corneal surgery.
Then on the right eye of the first (male) rabbit, he injured the corneal endothelium by passing through with 30.5G needle (yellow Microlance 3) and hooked up the tip a bit with tweezers, to scratch the endothelium pretty widely (sort of pine-needle cluster) centered. This should heal spontaneously anyhow and does not impair vision. He then injected 50 μL F12/DMEM medium.
On the left eye, with the corneal opacity, he simply injected 50 μL F12/DMEM medium in the stroma within/adjacent to the opacity. This makes a little liquid-filled vesicle, but stayed pretty flat.
Would have liked to backfill the Hamilton syringe but the dead volume of the hypodermic needle is 50 μL just like the syringe itself. So I filled it by aspiration. There was little resistance by the plunger but sometimes there were approx 5 μL bubbles. However, drawing the cells up went with no resistance at all. They had to be often resuspended; they pelleted quite quickly.
I handed the syringe with needle to Pierre and depressed the plunger for him as he was holding the needle in position with right hand and steadying the eye with the left. Cells went in with no problem either on right or on left. Second (male) rabbit had a spasmodic movement during the trunk-level cell injection into the stroma on left eye, so only received 1/2 dose (25 μL) instead of the foreseen 50 μL (25000 cells instead of 50000).
Otherwise, two rabbits treated in this way with the full doses of cephalic hNCC - four eyes - and one rabbit with the full dose of trunk hNCC in anterior chamber behind damaged endothelium on R and 1/2 dose of cells in opaque stroma on L.
In all stromal injections the medium + fluorescent cells was quite visible at injection and spread to make a 5 mm diameter disk. So cells may be at periphery of said disk; if beyond, they have migrated. (If there.)
Post-operative, applied a squeeze of antibiotic eye ointment in vaseline - probably neomycine but I didn't note this - and passed under eyelids, then let rabbits rest and supervised their breathing. They will wake up around now.
Discussing this afterward we think that inhaled isofluorane would be a better protocol (needs equipment but there is oxygen and compressed air piped into animal surgery buildin already) because it would avoid any possibility of movement and the animals wake up more quickly.
I had brought the cells to the animal care facility at 11AM, we made the injections, then I threw away the few microliters left in the cephalic and trunk microcentrifuge tubes (I had autoclave sterilized the low-adherence tubes: G017 Quali-"Low Retention" Mikrozentrifugengefasse zertifiziert RNase-, DNase + Pyrogenfrei, 1,7ml from kisker-biotech.com). So at 2PM I went back and fished them out of the incineration bin - luckily they were closed so had not evaporated - and resuspended each residue in 1 ml Rich medium, plating on 35 mm dishes. There are actually lots of whole cells. Will look on Monday at the confocal to see membrane retention of fluorescent dye.