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

• Published in: PloS one Vol. 7; no. 5; p. e37333
• Main Authors: Farahat, Waleed A., Wood, Levi B., Zervantonakis, Ioannis K., Schor, Alisha, Ong, Sharon, Neal, Devin, Kamm, Roger D., Asada, H. Harry
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 25.05.2012; Public Library of Science (PLoS)
• Subjects: Angiogenesis; Angiogenesis Inducing Agents - pharmacology; Artificial intelligence; Aspect ratio; Assaying; Background levels; Baseline studies; Bioengineering; Biology; Biotechnology; Cell culture; Cell Culture Techniques - instrumentation; Cell Culture Techniques - methods; Design of experiments; Endothelial Cells - drug effects; Endothelial Cells - physiology; Endothelium; Engineering; Flow cytometry; Growth; Growth factors; Humans; Image acquisition; Interdisciplinary aspects; Lysophospholipids - pharmacology; Mathematical models; Mechanical engineering; Medical imaging; Medicine; Microfluidics; Microfluidics - instrumentation; Microfluidics - methods; Monomolecular films; Morphology; Neovascularization, Physiologic - drug effects; Neovascularization, Physiologic - physiology; Parallel processing; Phosphates; Quantitative analysis; Signaling; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; Statistical analysis; Stochasticity; Studies; Synergistic effect; Tissue engineering; Vascular endothelial growth factor; Vascular Endothelial Growth Factor A - pharmacology; United States > US; Massachusetts; Singapore
• DOI: 10.1371/journal.pone.0037333
• ISSN: 1932-6203

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.