Abstract
Neurovascular unit (NVU) is a basic unit in the brain, including neurons, glial cells, blood vessels and extracellular matrix. This concept implies the importance of a three-dimensional (3D) culture model including these cell types for investigating brain functions. However, little is known about the construction of an in vitro 3D NVU model. In the present study, we aimed at constructing 3D neurovascular tissues by combining in vitro neurogenesis and angiogenesis models using a microfluidic platform, which is a critical step toward the NVU construction in vitro. Three gel conditions, which were fibrin gel, fibrin-Matrigel mixed gel and fibrin-hyaluronan mixed gel, were investigated to optimize the gel components in terms of neurogenesis and angiogenesis. First, fibrin-Matrigel mixed gel was found to promote neural stem cell (NSC) differentiation into neurons and neurite extension. In particular, 3D neural networks were constructed in 2–8 mg/ml fibrin-Matrigel mixed gel. Second, we found that capillary-like structures were also formed in the fibrin-Matrigel mixed gel by coculturing brain microvascular endothelial cells (BMECs) and human mesenchymal stem cells (MSCs). Finally, we combined both neural and vascular culture models and succeeded in constructing 3D neurovascular tissues with an optimized seeding condition of NSCs, BMECs and MSCs.
Highlights
The brain consists of neural tissues including neurons and glial cells, blood vessels wrapped by smooth muscle cells or pericytes, and extracellular matrix (ECM)
Previous studies reported in vitro Neurovascular unit (NVU) models using Transwell or microfluidic devices that were composed of endothelial cells (ECs) with/without astrocytes and pericytes to investigate blood brain barrier (BBB) functions of ECs2,8–11
Culturing brain cells in ECM hydrogel, which is closer to physiological conditions than a plastic substrate such as well plates, will lead to recapitulate more physiologically relevant morphogenesis
Summary
The brain consists of neural tissues including neurons and glial cells, blood vessels wrapped by smooth muscle cells or pericytes, and extracellular matrix (ECM). We present a 3D culture model in a microfluidic platform that allows us to construct neurovascular tissues by triculture of human NSCs, human brain microvascular ECs (BMECs) and human mesenchymal stem cells (MSCs). Our previous study demonstrated that MSCs promoted vascular formation of human umbilical vein endothelial cells (HUVECs) and differentiated into α-smooth muscle actin (α-SMA)-positive perivascular cells[6] These MSC-derived pericyte-like cells sparsely wrapped constructed vasculatures, which had continuous luminal structures. The aim of this study was to investigate 3D microenvironments for culturing NSCs and BMECs with induction of neurogenesis and angiogenesis in a microfluidic platform, which is the critical step toward the development of an in vitro NVU model
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