Development of ex vivo adult retinal explant organotypic tissue culture system for anti-angiogenic drug evaluation
,1 Zahra-soheila soheili
,2,* Mehdi sadeghi
,3 Shahram samiei
,4 Ehsan ranaei pirmardan
,5 Hoda shams najafabadi
1. National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
2. National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
3. National Institute of Genetic Engineering and Biotechnology, Tehran, Iran; School of Biological Sciences, Institute for Research in Fundamental Sciences, Tehran, Iran.
4. Blood Transfusion Research Centre High Institute for Research and Education in Transfusion, Medicine, Tehran, Iran
5. Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
6. National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Reliable disease models are essential for investigations on mechanisms and therapies. retinal explant culture systems can maintain the architecture and cellular connections within the tissue in vitro. it makes an intermediate model system between in vitro cell cultures and in vivo animal models. furthermore it is flexible enough for complicated experimental procedures and can be used efficiently to evaluate the effect of different anti-angiogenic factors. we developed a method for establishment of retinal tissue from adult rats to study the effect of anti-angiogenic drugs sufficiency.
Eyes from adult rats were enucleated and the neural retina was isolated. tissue was cut as yielding four retinal explants per animal. the resulted explants were cultured at a fluid/air interface on membranes. formation of endothelial sprouts was induced with 75 ng/ml of vegf. after 7 days of incubation with vegf, retina fragments were fixed within the culture insert by replacing the culture medium with 0.5 ml cold methanol followed by 20 min incubation at 4◦ c. then, samples were washed 2 times with pbs (5 min each) and incubated for overnight in blocking solution (10 mg bsa+ 3µl triton- up to 1 ml pbs). next, samples were washed 3 times with pbs/triton (20 min each) and then were incubated overnight at 4◦ c with the fitc-conjugated endothelial marker bandeiraea simplicifolia bs-i isolectin b4 (40 µg ml−1) followed by 2h incubation in dark. manipulated samples were washed 2 times with pbs (5 min each).
The results of staining revealed sprouting from the retinal explant.
In conclusion, this method represented a new ex vivo model of retinal neovascularization efficient for the rapid screening of novel anti-angiogenic therapeutics.