All cell culture media and supplements were purchased from Cellgro (Mediatech, Inc., Tewksbury, MA, USA) unless otherwise indicated. Bovine retinal endothelial cells (BRECs) and pericytes were isolated according to a modified method, as described previously.
46 Bovine eyes were obtained from a local slaughterhouse (Country Home Meats, Oklahoma City, OK, USA). The retinas were removed, washed four times in Dulbecco's modified Eagle's medium (DMEM), dispersed, and centrifuged at 400
g for 10 minutes. The resultant pellet was resuspended in an isolation medium (DMEM with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin). Microvessels were trapped on an 85-μm nylon mesh and transferred to a petri dish (Falcon; Life Science, Corning, NY, USA) containing 10 ml of an enzyme cocktail, which contains 600 μg/ml DNase I, 165 μg/ml collagenase, and 700 μg/ml pronase E (Sigma-Aldrich Corp.) and were incubated at 37°C for 20 minutes. The resultant vessel fragments were trapped on a 53-μm nylon mesh, washed with the isolation medium, and centrifuged at 400
g for 5 minutes. For selective culture of pericytes, the resultant pellet was resuspended in 10 ml of the pericyte growth medium and transferred into 75-cm
2 plastic tissue culture flasks.
For selective culture of BRECs, the resultant cell pellet was resuspended in 10 ml of the BREC growth medium and transferred into 75-cm2 collagen-coated plastic tissue culture flasks. The BREC growth medium consisted of DMEM supplemented with 10% human serum, 1% glutamine, 1 mg/ml insulin, 550 μg/ml transferrin, 670 ng/ml selenium, 100 IU/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin, 90 μg/ml heparin (Sigma-Aldrich Corp.), and 15 μg/ml endothelial cell growth supplement. Cells were cultured at 37°C and 5% CO2. Confluent cultures were passaged by detaching the cells with 0.25% trypsin and plated at a split 1:3. The purity of BRECs and pericytes were confirmed by binding of Dil-Ac-LDL (Biomedical Technologies, Inc., Stoughton, MA, USA) to the LDL receptor on the surface of BRECs and immunolabeling with an anti-smooth muscle actin antibody (Sigma-Aldrich Corp.), respectively. At passage 2, BRECs and pericytes were stored in liquid nitrogen for future use.