Abstract
Microfluidic technology is an important research tool for investigating angiogenesis in vitro. Here, we fabricated a polydimethylsiloxane (PDMS) microfluidic device with five cross-shaped chambers using a coverslip molding method. Then, the perforated PDMS microhole arrays prepared by soft lithography were assembled in the device as barriers; a single microhole had a diameter of 100 μm. After injecting type I collagen into the middle gel chamber, we added a culture medium containing a vascular endothelial growth factor (VEGF) into the middle chamber. It would generate a linear concentration gradient of VEGF across the gel region from the middle chamber to the four peripheral chambers. Human umbilical vein endothelial cells (HUVECs) were then seeded on the microhole barrier. With VEGF stimulation, cells migrated along the inner walls of the microholes, formed annularly distributed cell clusters at the gel-barrier interface, and then three-dimensionally (3D) sprouted into the collagen scaffold. After 4 days of culture, we quantitatively analyzed the sprouting morphogenesis. HUVECs cultured on the microhole barrier had longer sprouts than HUVECs cultured without the barrier (controls). Furthermore, the initial distribution of sprouts was more regular and more connections of tube-like structures were generated when the microhole barrier was used. This study introduces a novel microfluidic device containing both microtopographic structures and 3D collagen. HUVECs cultured with the microhole barrier could form well-interconnected tube-like structures and are thus an ideal in vitro angiogenesis model.
Highlights
IntroductionAngiogenesis is the physiological process of growing new microvessels from pre-existing vessels, and it plays an important role in regenerative repair, tumor growth, and numerous other angiogenesis-dependent diseases. The critical steps during new blood vessel formation are vascular basement membrane degradation, directional migration of endothelial cells (ECs), sprouting morphogenesis, and the formation of tube-like structures. Since the first in vitro angiogenesis model and the hypothesis that solid tumors are angiogenesis dependent were presented by Folkman, the study of angiogenesis-dependent diseases has become a hotspot. The development of a suitable in vitro angiogenesis model has become a key step to understanding the cellular and molecular mechanisms of angiogenesis.1932-1058/2017/11(5)/054111/12VC Author(s) 2017.054111-2 Chen et al.Biomicrofluidics 11, 054111 (2017)Recently developed microfluidic technology, which is based on microelectromechanical systems (MEMS) technology, has gradually become an important method for establishing in vitro angiogenesis models. The microfluidic technologies make it possible to better control studies of the effects of physical and chemical factors on angiogenesis in three dimensions (3D)
The various existing microfluidic devices have allowed for the construction of microvascular networks,13,15 3D co-culture of endothelial cells (ECs) and angiogenesis-related cells,14,16 the establishment of controllable concentration gradients of angiogenesis-related factors,12,13 investigations of the effect of extracellular matrix (ECM) biophysical and biochemical properties on angiogenesis,17 investigations of the effect of mechanical stimulation on angiogenesis,18,19 and other experiments
It was conducive to the adhesion of the PDMS microhole barrier to the device and could prevent the gel in the middle chamber from spilling into the cell chambers
Summary
Angiogenesis is the physiological process of growing new microvessels from pre-existing vessels, and it plays an important role in regenerative repair, tumor growth, and numerous other angiogenesis-dependent diseases. The critical steps during new blood vessel formation are vascular basement membrane degradation, directional migration of endothelial cells (ECs), sprouting morphogenesis, and the formation of tube-like structures. Since the first in vitro angiogenesis model and the hypothesis that solid tumors are angiogenesis dependent were presented by Folkman, the study of angiogenesis-dependent diseases has become a hotspot. The development of a suitable in vitro angiogenesis model has become a key step to understanding the cellular and molecular mechanisms of angiogenesis.1932-1058/2017/11(5)/054111/12VC Author(s) 2017.054111-2 Chen et al.Biomicrofluidics 11, 054111 (2017)Recently developed microfluidic technology, which is based on microelectromechanical systems (MEMS) technology, has gradually become an important method for establishing in vitro angiogenesis models. The microfluidic technologies make it possible to better control studies of the effects of physical and chemical factors on angiogenesis in three dimensions (3D). The various existing microfluidic devices have allowed for the construction of microvascular networks,13,15 3D co-culture of ECs and angiogenesis-related cells, the establishment of controllable concentration gradients of angiogenesis-related factors (such as vascular endothelial growth factor, VEGF), investigations of the effect of extracellular matrix (ECM) biophysical and biochemical properties on angiogenesis, investigations of the effect of mechanical stimulation on angiogenesis, and other experiments. Considering that the basic structure of blood vessels is a circular tube-like structure with a lumen, it may be more appropriate to construct circular structures on the cell-gel chamber (or channels) interfaces to replace the common rectangular structures. This will be closer to the real situation of vascular sprouting.
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