Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation

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TL;DR

This study developed a microfluidic model of the human neurovascular niche (NVN) containing perfused brain microvascular networks (BMVNs) composed of primary human brain endothelial cells, pericytes, and astrocytes. When neural progenitor cells (NPCs) or neuronal precursor cells (NPreCs) were introduced into these BMVNs, their survival, proliferation, neurogenesis, and maturation were significantly enhanced compared to cells cultured alone. The application of interstitial fluid flow further improved neuron differentiation and maturation, demonstrating the critical role of both vascular cells and physical stimuli in recapitulating the human NVN in vitro.

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**Background:** Neural progenitor cells (NPCs) reside near brain microvascular networks (BMVNs) in the neurovascular niche (NVN), where blood vessels regulate NPC self-renewal and neurogenesis. Existing in vitro NVN models often lack proper cellular composition (brain endothelial cells, pericytes, astrocytes) and physical stimuli such as interstitial fluid flow (IF) and luminal flow. This study aimed to develop a perfused, three-dimensional microfluidic model of the human NVN that recapitulates these features and evaluate its effects on NPC survival, proliferation, neurogenesis, and maturation.

**Methods:** Primary human brain endothelial cells (BECs), pericytes (PCs), and astrocytes (ACs) were cocultured in fibrin hydrogels within microfluidic devices (AIM Biotech) to generate self-assembled, perfused BMVNs. Human iPSC-derived NPCs (XCL-1 line) expressing ZsGreen1 were introduced either as dispersed cells or aggregated into neurospheres (100–400 cells per neurosphere). NPC survival was assessed using LIVE/DEAD staining. Neurosphere expansion was quantified by the day 7/day 1 area ratio. Neurogenesis was evaluated by measuring neurite outgrowth (total neurite length and maximum extension) from NPreC neurospheres, expression of stem cell markers (Sox2, nestin) and neuron markers (Tuj1, MAP2) via immunocytochemistry, and spontaneous calcium oscillations using Fluo-8 AM dye. Experiments compared NPC/NPreC solo-culture versus coculture with BMVNs, with individual cell types (BECs, PCs, or ACs alone), and under flow versus static conditions.

**Key Results:** Dispersed NPCs cultured alone in MFDs showed high initial viability (92.71 ± 1.07% on day 1 at 2×10⁶ cells/ml) but nearly all died by day 7 (1.32 ± 0.44% live). Increasing cell density, adding Matrigel, or changing medium did not improve survival. NPC neurospheres also regressed when cultured alone (area ratio 0.10 ± 0.03 for 200 cells/neurosphere). In contrast, NPC neurosphere expansion was significantly enhanced in all coculture conditions: NPC-PC (area ratio 3.61 ± 0.20), NPC-AC (3.69 ± 0.22), NPC-BEC (13.53 ± 1.30), and NPC-BEC-PC-AC (10.15 ± 0.62) compared to NPC alone (P < 0.0001). NPC-BEC and NPC-BEC-PC-AC conditions produced significantly larger expansion than NPC-PC or NPC-AC conditions (P < 0.0001). NPCs in quad-culture expressed Sox2 and Ki67, confirming self-renewal and proliferation. Dispersed NPCs cocultured with BMVNs survived for 1 week, with no significant difference in cell number between flow (87.17 ± 8.57 cells/area) and static (70.50 ± 12.15) conditions. For NPreC neurospheres, total neurite outgrowth was significantly enhanced in all coculture conditions (NPreC-PC: 27.09 ± 3.65 mm, NPreC-AC: 21.80 ± 2.31 mm, NPreC-BEC: 25.84 ± 1.44 mm, NPreC-BEC-PC-AC: 17.52 ± 1.32 mm) compared to NPreC alone (10.10 ± 0.98 mm). Calcium imaging revealed significantly more firing neurons in NPreC-BEC-PC-AC neurospheres (3.077 ± 0.560 per slice) than in NPreC alone (0.231 ± 0.166, P < 0.001), with higher firing rates (1.077 ± 0.176 vs. 0.046 ± 0.033 spikes/min, P < 0.0001). Application of IF during quad-culture significantly enhanced neurosphere core area (18,629 ± 2,997 vs. 14,238 ± 2,902 μm², P < 0.05) and total neurite outgrowth (19.61 ± 0.48 vs. 14.08 ± 0.62 mm, P < 0.0001) compared to static conditions. Dispersed NPreCs cocultured with BMVNs under flow showed significantly more total ZsGreen1⁺ cells (136.55 ± 6.53 vs. 86.91 ± 6.42, P < 0.0001) and MAP2⁺/ZsGreen1⁺ cells (76.17 ± 1.90 vs. 48.83 ± 4.84, P < 0.01) than static conditions, though the percentage of MAP2⁺ cells was similar (61.97 ± 4.68% vs. 63.23 ± 0.65%).

**Clinical Implications:** This microfluidic NVN model successfully recapitulates key cellular and physical features of the human neurovascular niche, demonstrating that perfused BMVNs composed of brain-specific cell types enhance NPC survival, self-renewal, neurogenesis, and functional maturation. The model's ability to support long-term culture (up to 2 weeks) and generate spontaneously active neurons makes it a valuable platform for studying neurodegenerative diseases (e.g., Alzheimer's, Parkinson's) where neurovascular dysfunction plays a role. The finding that interstitial fluid flow enhances neurogenesis highlights the importance of physical stimuli in stem cell fate regulation. This system could be used for preclinical testing of brain therapeutics that rely on vascular delivery and for investigating mechanisms of neurovascular coupling in health and disease.