Compositions and methods for treating retinal diseases
A retinal and retinal vascular technology, applied in the fields of stem cell biology and regenerative medicine, can solve the problems of pluripotent cell contamination, low graft yield and efficiency
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Embodiment 1
[0242] Example 1: Isolation and culture of retinal progenitor cells
[0243] Enzymatic digestion was performed by incubating retinal tissue in 0.8 ml undiluted TrypLE Express (Invitrogen) for 40 seconds at room temperature. Then pipette the trypsinized tissue up and down with a 1 ml pipette tip. The trypsin was then neutralized by adding 10 ml of cold fresh serum-free cell culture medium and the mixture was collected by centrifugation at 1000 rpm (179 xg) for 4 minutes. The supernatant was removed and the pellet was resuspended in cold fresh cell culture medium before cell viability and cell number were determined by trypan blue (Invitrogen) exclusion using Countess (Invitrogen) or manual counting. get about 10 x 10 6 of which about 80% are small / medium cell clumps, about 9 to 18% are single cells, and about 1 to 2% are large cell clumps. This technique resulted in cell viability around 92%.
[0244] Cells were then seeded into two T75 culture flasks previously coated with...
Embodiment 2
[0257] Example 2: Characterization of RPC
[0258] Immunocytochemistry
[0259] Cells were dissociated and grown on 4- or 8-well chamber slides for 1-3 days, then fixed in 4% paraformaldehyde for 15 minutes and washed 3 times in PBS. Slides were blocked for 1 hour in a solution containing 5% donkey serum and / or 0.3% Triton X-100, then washed again with PBS. It was then incubated overnight at 4°C with a panel of antibodies to detect antigens expressed by the progenitor cells. This includes Anti-Nestin (Chemicon 1:200), Anti-Vimentin (Sigma 1:200), Anti-Sox2 (Santa Cruz 1:400), Anti-SSEA-1 (BD 1:200), Anti-GD2 (Chemicon 1:100), anti-Ki-67 antibody (BD 1:200), anti-β3-tubulin antibody (Chemicon 1:400), anti-GFAP antibody (Chemicon 1:400), and anti-GDNF antibody (Santa Cruz 1:200). This was followed by incubation with anti-mouse Alexa 546 (Invitrogen 1:400), anti-goat Alexa 488 (Invitrogen 1:400), or anti-rabbit FITC (Chemicon 1:800) secondary antibodies. Fluorescence was det...
Embodiment 3
[0315] Example 3: In vivo effectiveness of RPCs
[0316] RPCs for transplantation were first prepared by harvesting RPCs with TrypLE Express and centrifuging at 1000 rpm for 5 minutes to collect the RPCs. Cells were washed once in HBSS and then resuspended in cold HBSS to determine cell viability and cell number. For human transplants, use 0.5 × 10 in 100 μl HBSS 6 cells. For transplantation into rats, different doses ranging from 4000 to 75,000 cells in 2 μl of HBSS were used.
[0317] Human RPCs were transplanted as a suspension into the vitreous or subretinal space of dystrophic hooded RCS rats. Rats were given cyclosporin A and steroids to avoid xenograft rejection. Control eyes received sham injections (subretinal, intravitreal) consisting of vehicle only. Transplanted animals were functionally tested in an unrestrained wakefulness state using a commercial instrument designed to quantify optokinetic response (OR). Test the brightness threshold of a subgroup of anima...
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