Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions

POLYESTER FILM STRIPS COATED WITH PHOTOGRAPHIC GELATIN CONTAINING IMMOBILIZED GLUCOSE-OXIDASE HARDENED BY CHROMIUM(III) SULFATE

Glucose oxidase was immobilized into photographic gelatin hardened by chromium(III) sulphate. The enzyme-gelatin mixture was coated on polyester film strips which allowed easy and simple handling during assays. The effect of gelatin and cross-linker concentrations on water content and enzymatic activity was studied. The effect of pH during immobilization and that of incubation temperature on maximum activity were examined- Enzyme leakage tests were carried out during reuse number studies. Consecutive use of strips followed by washing and resting between uses were found to affect the reuse number. A maximum immobilization of 68% was reached under optimal conditions. Mechanical stability and leakage were found to be functions of gelatin and cross-linker concentrations. Photographic gelatin was found to have many capabilities with extraordinary characteristics as a carrier on immobilization

IMMOBILIZATION OF UREASE INTO CARBOXYMETHYLCELLULOSE - GELATIN SYSTEM

In the present work carboxymethylcellulose (CMC) and CMC-gelatin were used as carrier systems for urease immobilization. Immobilization was based on the formation of insoluble salts of CMC and gelatin with chromium(111) ions. Chromium(111) acetate (CA) and chromium(111) sulfate (CS) were used for this purpose and their effect on urease activity was investigated. The activities of immobilized urease using pure and CMC-gelatin carrier systems were compared. Urease activity was determined by using Berthelot method. Reuse number, pH, enzyme and cross linker concentrations and the incubation period were the factors taken into account in this investigation. Activity of immobilized enzymes were found to be stable for at least 2 months and 16-24 usage. Immobilization percentage obtained under optimum conditions was 40%

GLUCOSE-OXIDASE IMMOBILIZED ON GELATIN BY VARIOUS CROSS-LINKERS

Immobilization of glucose oxidase have been realized with photographic gelatin as support for chemical coupling. Chromium acetate, potassium chromium sulfate and formaldehyde were used as crosslinking agents to immobilize glucose oxidase on film strips. Enzyme, crosslinker concentrations, temperature, pH and reuse number on native and immobilized glucose oxidase activity and water contents of unused and used films were investigated. The best results were achieved with the crosslinker chromium acetate as 50% maximum activity after 40 assays

STUDIES ON IMMOBILIZATION OF UREASE IN GELATIN BY CROSS-LINKING

Urease enzyme was immobilized in photographic gelatin by chemical cross-linking using formaldehyde, glutaraldehyde and chromium (III) acetate. The effects of enzyme and cross-linker concentrations, temperature, incubation time and pH on urea hydrolysis were investigated. Effect of reuse on the activity of immobilized enzyme was also studied. Glutaraldehyde (0.004 M) was the most suitable cross-linker; relative activities within 2.5 months after 24 reuses were stable (about 78%)