A Facile Method for Generating a Smooth and Tubular Vessel Lumen Using a Viscous Fingering Pattern in a Microfluidic Device.

Blood vessels are ubiquitous in the human body and play essential roles not only in the delivery of vital oxygen and nutrients but also in many disease implications and drug transportation. Although fabricating in vitro blood vessels has been greatly facilitated through various microfluidic organ-on-chip systems, most platforms that are used in the laboratories suffer from a series of laborious processes ranging from chip fabrication, optimization, and control of physiologic flows in micro-channels. These issues have thus limited the implementation of the technique to broader scientific communities that are not ready to fabricate microfluidic systems in-house. Therefore, we aimed to identify a commercially available microfluidic solution that supports user custom protocol developed for microvasculature-on-a-chip (MVOC). The custom protocol was validated to reliably form a smooth and functional blood vessel using a viscous fingering (VF) technique. Using VF technique, the unpolymerized collagen gel in the media channels was extruded by less viscous fluid through VF passive flow pumping, whereby the fluid volume at the inlet and outlet ports are different. The different diameters of hollow tubes produced by VF technique were carefully investigated by varying the ambient temperature, the pressure of the passive pump, the pre-polymerization time, and the concentration of collagen type I. Subsequently, culturing human umbilical vein endothelial cells inside the hollow structure to form blood vessels validated that the VF-created structure revealed a much greater permeability reduction than the vessel formed without VF patterns, highlighting that a more functional vessel tube can be formed in the proposed methodology. We believe the current protocol is timely and will offer new opportunities in the field of in vitro MVOC.

3D Self-Organized Human Blood-Brain Barrier in a Microfluidic Chip.

A preclinical blood-brain barrier (BBB) model is important for the study of fundamental transport mechanisms and in accessing the delivery of small molecules and antibodies that target brain. Transwell assays for BBB models are easy to create and use but lack the true 3D anatomy of the brain microvasculature and also often the cell-cell and cell-matrix interactions that are important in ensuring a tight BBB. Here we describe the formation of a BBB that expresses neurovascular membrane transporters, tight junction, and extracellular matrix proteins using the coculture of human-induced pluripotent stem cell-derived endothelial cells (iPSC-EC), brain pericytes (PC), and astrocytes (AC) in a microfluidic device. The BBB model recapitulates human brain vascular permeability with values that are lower than conventional in vitro models and are comparable to in vivo measurements in rat brain. This in vitro BBB model can therefore be used to screen for brain-targeting drugs or to study neurovascular functions.

In Vitro Microvessel Growth and Remodeling within a Three-dimensional Microfluidic Environment.

This paper presents in vitro microvascular network formation within 3D gel scaffolds made from different concentrations of type-I collagen, fibrin, or a mixture of collagen and fibrin, using a simple microfluidic platform. Initially, microvascular network formation of human umbilical vein endothelial cells was examined using live time-lapse confocal microscopy every 90 min from 3 h to 12 h after seeding within three different concentrations of collagen gel scaffolds. Among the three conditions of collagen gel scaffolds (2.0 mg/ml, 2.5 mg/ml, and 3.0 mg/ml), the number of skeleton within collagen gel scaffolds was consistently the highest (3.0 mg/ml), followed by those of collagen gel scaffolds (2.5 mg/ml and 2.0 mg/ml). Results demonstrated that concentration of collagen gel scaffolds, which influences matrix stiffness and ligand density, may affect microvascular network formation during the early stages of vasculogenesis. In addition, the maturation of microvascular networks in monoculture under different gel compositions within gel scaffolds (2.5 mg/ml) was examined for 7 d using live confocal microscopy. It was confirmed that pure fibrin gel scaffolds are preferable to collagen gel or collagen/fibrin combinations, significantly reducing matrix retractions during maturation of microvascular networks for 7 d. Finally, early steps in the maturation process of microvascular networks for 14 d were characterized by demonstrating sequential steps of branching, expanding, remodeling, pruning, and clear delineation of lumens within fibrin gel scaffolds. Our findings demonstrate an in vitro model for generating mature microvascular networks within 3D microfluidic fibrin gel scaffolds (2.5 mg/ml), and furthermore suggest the importance of gel concentration and composition in promoting the maturation of microvascular networks.

Complementary effects of ciclopirox olamine, a prolyl hydroxylase inhibitor and sphingosine 1-phosphate on fibroblasts and endothelial cells in driving capillary sprouting.

Capillary sprouting, a key step of neoangiogenesis in wound healing and tumor growth, also represents a therapeutic target for tissue repair. It requires crosstalk between endothelial cells (EC) and other cell types. We studied this process in a microfluidic platform that allows EC to migrate out of a channel across a collagen gel up a gradient of factors produced by a collection of encapsulated fibroblasts. Introduction of a prolyl hydroxylase inhibitor (PHi), ciclopirox olamine (CPX) to stabilize hypoxia inducible factor 1α (HIF-1α) predominantly in fibroblasts induced capillary sprouting in EC, but the most complex tubular networks with true lumina formed after combining CPX with the lysophospholipid sphingosine 1-phosphate (S1P). The enhanced angiogenesis is a possible consequence of the generation of mutually stimulating factors as each cell type responded differently to the compounds. The combination of CPX and S1P induced secretion of vascular endothelial growth factor (VEGF) in fibroblast culture whereas the angiogenic monocyte chemoattractant protein (MCP)-1 was exclusively secreted by fibroblasts, but only in the presence of EC-conditioned medium. Antibody interference with fibroblast-produced VEGF and MCP-1 inhibited the sprouting response. These observations not only demonstrate the collaboration of EC and fibroblasts in inducing capillary sprouting but also suggest that the combination of CPX and S1P enhances angiogenesis and thus might be of therapeutic value for the pharmacological induction of tissue repair and regeneration.