Integration of neurogenesis and angiogenesis models for constructing a neurovascular tissue

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.

Figure 1

Schematic illustrations of a microfluidic device. (A) A PDMS device copied by an SU-8 mold was plasma-bonded with a coverglass to form microfluidic channels. Hydrogel pre-polymer was then injected into the central channel from two gel inlets (arrows). (B) There are two parallel microchannels separated by the gel region. BMECs and NSCs were injected into one of the microchannels (arrows).

Figure 2

Neurite formation of NSC-derived neurons in different gel scaffolds. (A) Schematic illustrations of NSC culture for constructing a neural network. (B) Representative phase-contrast images of NSCs cultured in three different gel scaffolds; fibrin (left), fibrin-Matrigel mixed gel (middle) and fibrin-hyaluronan mixed gel (right). Arrowheads in insets indicate a round shape of NSCs (Fibrin, Fibrin-Hyaluronan), while arrows in an inset indicate an extended NSCs (Fibrin-Matrigel). (C) Representative immunofluorescence images of neurons (MAP2, green) and nuclei (DAPI, blue) on day 9. (D) Quantitative analysis of neurite length. Data are shown as the mean ± s.e.m. (N = 3, n ≥ 16). *p < 0.05 vs. Fibrin. Scale bars, 100 µm.

Figure 3

Formation of capillary-like structures in BMEC-MSC coculture in fibrin, fibrin-Matrigel mixed gel, and fibrin-hyaluronan mixed gel. (A) Schematic illustrations of BMEC-MSC coculture. (B,C) Representative phase-contrast images on days 1, 5 and 9, and corresponding immunofluorescence images of BMECs (PECAM-1, red), pericytes (α-SMA, green), and nuclei (DAPI, blue) on day 9. Arrowheads in an inset indicates vascular sprouts (Fibrin-Matrigel, day 1). Scale bar, 200 µm. (D) An enlarged image of cells in the fibrin-hyaluronan mixed gel. Arrowheads indicate a sheet-like structure of BMECs. Scale bar, 200 µm. (E) Quantitative analysis of network length. Data are shown as the mean ± s.e.m. (N = 3, n ≥ 9). *p < 0.05 vs. Fibrin-Hyaluronan.

Figure 4

Optimization of the concentration of fibrin-Matrigel mixed gel for 3D migration of neurons. (A–D) Immunofluorescence projection images and corresponding 3D views of neurons in 2–2 mg/ml and 2–8 mg/ml fibrin-Matrigel mixed gel, respectively. Cells were fixed on day 21 and stained for neurons (Tuj1, green) and nuclei (DAPI, blue). Scale bar, 100 µm. (E–F) Quantitative analysis of the percentage of migrating cells in a 3D manner. The number of NSCs migrating into the 3D gel region. Data are shown as the mean ± s.e.m. (N = 3, n ≥ 12). *p < 0.05 vs. 2–2 mg/ml.

Figure 5

The process of neurogenesis in 21-day culture. (A) Immunofluorescence projection images of NSC-derived neurons in 2–8 mg/mL fibrin-Matrigel mixed gel. Cells were fixed on day 21 and stained for neurons (Tuj1, green) and nuclei (DAPI, blue). Scale bar, 100 µm. (B) A histogram of neurite length on days 7, 14, and 21. (C) Time course of average neurite length in 21-day culture. Data are shown as the mean ± s.e.m. (N = 3, n ≥ 12).

Figure 6

Triculture of NSCs, BMECs and MSCs for constructing neurovascular tissues. (A) Representative phase-contrast images on days 3, 7 and 10, and corresponding immunofluorescence images of neurons (Tuj1, green), BMECs (PECAM-1, red) and cell nuclei (DAPI, blue) on day 10. Arrowheads and areas surrounded by dotted lines in immunofluorescence images represent the gel shrinkage. Scale bar, 200 μm. (B) Quantitative analysis of the gel shrinkage in different seeding conditions on day 10. Data are the means ± s.e.m. (N = 3, n ≥ 9). *p < 0.05 vs. “Same day” and “BMEC-MSC pre-seeding”. (C) Enlarged views of neurons and BMECs in NSC pre-seeding condition. Scale bars, 100 μm.