(355c) 3D Mibrain-on-Chip for Modeling BBB Function and Delivery to the Brain Inclusive of All Major CNS Cell Types

Treatment development for neurological disease has been limited by the tight and highly selective human blood brain barrier (BBB). A major bottleneck to developing effective therapeutics is that current models fail to recapitulate human brain tissue and its BBB with sufficient fidelity. While animal models have been a helpful research tool, the rodent BBB is far less selective than human and does not mimic the complexity of human genetics. In vitro models have the advantage of enabling high throughput screening, real-time monitoring at high spatiotemporal resolution, and readily tunable genetic backgrounds in combination with iPSC technology. The brain is comprised of six major different cell types arranged in a stereotyped 3D architecture, each of which affects BBB properties. Microphysiological systems harnessing natural biomaterials have enabled the co-culture of 3 of the relevant cell types into BBB models for a reductionist model. However, these models are missing the contributions of neurons and other cells that strengthen the BBB, have not yet demonstrated selective barrier properties matching human brain tissue on the molecular level, and often use materials that have deleterious effects on neurons and inhibit their co-culture. Engineered biomaterials provide highly tunable 3D scaffolds that can promote the formation of tissue mimics with physiologically relevant stiffness and biochemical compositions. Recently, we have engineered a soft hydrogel biomaterial, Neuromatrix Hydrogel, with brain-specific biochemical cues that supports the self-assembly of all six brain cell types to form a 3D immuno-glial-neurovascular miBrain model with enhanced cell- and tissue-scale phenotypes (Fig. 1A-F). Given that engineered hydrogels have limitations for 3D cell culture in confined microfluidic environments due to their innate swelling properties that introduces deleterious stresses to cells, we engineered a novel microfluidic platform, GelChip, to be compatible with engineered hydrogels and complex 3D cell co-cultures harnessing a 3D Printing strategy (Fig. 1G). GelChips enable the culture of miBrains with 3D perfusable vascular networks within co-cultures of all six major brain cell types arranged in integral BBB and neurovascular units (Fig. 1H), microglial immune cells (Fig. 1I), and myelinated neuronal networks (Fig. 1J), to form the miBrain-on-Chip. This is the first perfusable brain model to our knowledge with iPSC-derived endothelial cells in combination with other iPSC-derived cell types, here with six iPSC-derived cell types. With the miBrain-on-Chip, we have characterized the barrier properties, assessed delivery into the parenchymal brain tissue through the BBB, and harnessed the model to probe the effect of APOE4, the strongest known genetic risk factor for sporadic Alzheimer’s Disease. This 3D miBrain-on-Chip could be harnessed to assess BBB penetration and downstream neurological effects and screen therapeutics towards optimizing delivery to the brain.

Fig. 1: Human iPSC-derived 3D Immuno-Glial-Neurovascular miBrain-on-Chip. (A) Schematic of miBrain formation harnessing patient-specific iPSCs differentiated into each of the resident brain cell types, encapsulated in Neuromatrix Hydrogel, and co-cultured for integral cell network self-assembly in our novel GelChip microfluidic platform to enable perfusable flow through vascular lumens within the miBrain, (B) distribution of (from left to right) iPSC-derived pericytes (cyan: mCherry-pericytes), astrocytes (cyan: mCherry-astrocytes), BMECs (cyan: mCherry-BMECs), neurons (cyan: tubulin neuron label), oligodendroglia (cyan: tdTomato pre-transfected oligodendroglia), and iMG (cyan: membrane pre-labeled iMG) throughout the full 3D miBrain, (C) neuronal phenotypes in miBrains cultured in dextran-based hydrogels fabricated with various brain ECM proteins (cyan: neurofilament, blue: Hoechst; scale bars, 50 µm), (D) example raster plots from MEA recordings of miBrains in Neuromatrix Hydrogel compared to Matrigel, (E) persistence of neurovascular unit phenotypes in miBrains cultured in Neuromatrix Hydrogel versus Matrigel after 5 weeks in culture (red: PECAM, cyan: TUBB3, blue: Hoechst; scale bar, 50 µm), (F) macroscopic view of gel structural integrity for miBrains cultured in VCN-incorporated engineered dextran-based hydrogel named Neuromatrix Hydrogel versus Matrigel after 4 weeks, (G) comparison of vascular networks in GelChips (right) compared to commercially available microfluidic platforms (left) (red: endothelial cells, scale bar, 200 µm); miBrains recapitulate key hallmarks of human brain tissue, inclusive of (H) BBB and neurovascular units with (top) 3D astrocytes distributed throughout BMEC networks throughout the miBrain (red: Imaris surfaces from ZO1-BMECs, green: mCherry-astrocytes; scale bar, 500 µm), (bottom, left) astrocytes with end-feet extending to vessels expressing canonical aquaporin-4 transporter (green: GFAP, gray: AQP4, red: PECAM, blue: Hoechst; scale bar, 30 µm; insert, gray: AQP4), and (bottom, right) anterior view of a miBrain 3D rendering (red: PECAM, cyan: neurofilament, blue: Hoechst; scale bar, 50 µm), (G) microglia: (top) 3D iMG distributed throughout BMEC networks throughout the miBrain (red: Imaris surfaces from mCherry-BMECs, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 500 µm), (bottom, left) visualized at higher magnification (red: Imaris surfaces from mCherry-BMECs, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 100 µm), and (bottom, right) 3D iMG distributed throughout neuronal networks throughout the miBrain (cyan: Imaris reconstruction of tubulin neuron label, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 100 µm), and (H) myelinated neuronal networks: (top) 3D oligodendroglia distributed throughout neuronal networks throughout the miBrain (cyan: tubulin neuron label, green: Imaris surfaces from tdTomato pre-transfected oligodendroglia; scale bar, 500 µm), (bottom, left) myelin dye-labeled neurons and oligodendroglia (red: FluoroMyelin, cyan: tubulin neuron label, green: tdTomato pre-transfected oligodendroglia, scale bar, 100 µm), and (bottom, right) visualized also via Imaris reconstructions for myelin and oligodendroglia (red: Imaris surfaces of FluoroMyelin, green: Imaris surfaces of tdTomato pre-transfected oligodendroglia; scale bar, 100 µm).