Abstract
Microfluidic devices have been successfully used to recreate in vitro biological microenvironments, including disease states. However, one constant issue for replicating microenvironments is that atmospheric oxygen concentration (21% O2) does not mimic physiological values (often around 5% O2). We have created a microfluidic device that can control both the spatial and temporal variations in oxygen tensions that are characteristic of in vivo biology. Additionally, since the microcirculation is responsive to hypoxia, we used a 3D sprouting angiogenesis assay to confirm the biological relevance of the microfluidic platform. Our device consists of three parallel connected tissue chambers and an oxygen scavenger channel placed adjacent to these tissue chambers. Experimentally measured oxygen maps were constructed using phosphorescent lifetime imaging microscopy and compared with values from a computational model. The central chamber was loaded with endothelial and fibroblast cells to form a 3D vascular network. Four to six days later, fibroblasts were loaded into the side chambers, and a day later the oxygen scavenger (sodium sulfite) was flowed through the adjacent channel to induce a spatial and temporal oxygen gradient. Our results demonstrate that both constant chronic and intermittent hypoxia can bias vessel growth, with constant chronic hypoxia showing higher degrees of biased angiogenesis. Our simple design provides consistent control of spatial and temporal oxygen gradients in the tissue microenvironment and can be used to investigate important oxygen-dependent biological processes in conditions such as cancer and ischemic heart disease.
Highlights
Hypoxia, or low oxygen tension, is a central feature of many different diseases, it is a part of normal physiological states [1,2,3]
Our approach utilizes a simple and inexpensive aqueous solution of sodium sulfite as an oxygen scavenger. This has several advantages over previous methods of controlling oxygen: 1) our method reduces pervaporation and avoids introduction of gas bubbles in long term cell cultures; 2) our method avoids exposure of cells to non-physiologic oxygen scavenging chemicals; 3) our method is cost effective as expensive gas tanks, gas mixers, or oxygen scavengers are not required; 4) our method offers more leverage to precisely control oxygen gradients as kinetic properties (e. g. concentration of sodium sulfite) in addition to mass transport (e. g. flow rate) can be finely regulated to control the gradients
In this study we show the effects of physioxia (P), constant chronic hypoxia (CH), and intermittent hypoxia (IH) on true 3D angiogenesis
Summary
Low oxygen tension, is a central feature of many different diseases, it is a part of normal physiological states [1,2,3]. Hypoxia can be chronic (sustained oxygen tension < 5% O2 for > 24 hours), as in the case of ischemic heart disease and wound healing, or intermittent (repeated oxygen tension < 5% O2 for < 24 hours) as in the case of some tumors, sleep apnea, and exercise [4,5,6,7]. Previous literature often compares hypoxic conditions to normoxic conditions, we chose to compare hypoxic conditions to physioxia (5% O2) conditions because it is more physiologically relevant To perform these studies, even though the incubator oxygen tension may be set to 5% O2, controlling the spatial distribution of oxygen within the cell culture is not possible. It has been shown that gradients are important for chemical and mechanical factors for recapitulating the physiological microenvironment [9,10]
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