High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels.

A simple and non-mechanical enzymatic method for the isolation and large-scale in vitro culture of bovine retinal endothelial cells (BRECs) is described. A sufficiently high yield of retinal microvessels was obtained from bovine eyes by brisk stirring of the retina with Minimum Essential Medium for 3 min, washing the pellet five times by repeated centrifugation at 300 x g and sieving in a single step. Pericyte contamination was eliminated by increasing the incubation period in an enzyme mixture and passaging the cells at a low concentration of trypsin for 1-2 min. The cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum. The cells were cobble-stone shaped and stained positive for von Willebrand Factor (vWF) and cluster differentiation factors, CD31 and CD146. These cells stained negative for alpha-smooth muscle actin (ASMA) and glial fibilary acidic protein (GFAP), forming characteristic capillary tube-like structures on matrigel. Here we optimized the method for the isolation of pure retinal endothelial cells without using complex procedures and sophisticated instruments. This method is simpler, more rapid and cost-effective, and also results in a higher yield of endothelial cells than other methods. Retinal endothelial cells cultured in this way will be used to study the pathogenesis of vascular eye diseases such as diabetic retinopathy and other neovascularization diseases. They can also serve as an excellent in vitro model system for drug analysis.

Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology.

Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of Nattokinase by Bacillus subtilis at pH 7.5. The four variables involved in this study were Glucose, Peptone, CaCl2, and MgSO4. The statistical analysis of the results showed that, in the range studied; only peptone had a significant effect on Nattokinase production. The optimized medium containing (%) Glucose: 1, Peptone: 5.5, MgSO4: 0.2 and CaCl2: 0.5 resulted in 2-fold increased level of Nattokinase (3194.25U/ml) production compared to initial level (1599.09U/ml) after 10h of fermentation. Nattokinase production was checked with fibrinolytic activity.