Human Cortex Spheroid with a Functional Blood Brain Barrier for High-Throughput Neurotoxicity Screening and Disease Modeling

Abstract

The integral selectivity characteristic of the blood brain barrier (BBB) limits therapeutic options for many neurologic diseases and disorders. Currently, very little is known about the mechanisms that govern the dynamic nature of the BBB. Recent reports have focused on the development and application of human brain organoids developed from neuro-progenitor cells. While these models provide an excellent platform to study the effects of disease and genetic aberrances on brain development, they may not model the microvasculature and BBB of the adult human cortex. To date, most in vitro BBB models utilize endothelial cells, pericytes and astrocytes. We report a 3D spheroid model of the BBB comprising all major cell types, including neurons, microglia and oligodendrocytes, to recapitulate more closely normal human brain tissue. Spheroids show expression of tight junctions, adherens junctions, adherens junction-associated proteins and cell specific markers. Functional assessment using MPTP, MPP+ and mercury chloride indicate charge selectivity through the barrier. Junctional protein distribution was altered under hypoxic conditions. Our spheroid model may have potential applications in drug discovery, disease modeling, neurotoxicity and cytotoxicity testing.

Figure 1

Assessment of cell localization, tight junction protein expression, and barrier properties in self-assembled spheroids. Spheroids in (A) show self organisation of endothelial cells and pericytes to the outermost sphere whereas the astrocytes and neurons (violet) are encapsulated by the endothelial cells and pericytes. (B) Spheroids containing HBMEC, astrocytes, and pericytes were formed through self assembly. Shown is ZO-1, a tight junction protein that prevents free paracellular transport of molecules across the vascular-type barrier. Dapi (blue) shows nuclear staining. Also shown in the figure are empty pockets where there is no ZO-1, this indicates that the HBMECs are likely not completely covering the spheroid. (C) Permeability assessment was done through incubating the spheroids containing HBMEC, pericytes and astrocytes with FITC labeled IgG. The upper panel represents spheroids with an uncompromised vascular-type barrier (control) and the lower panel shows spheroids that were treated with histamine to transiently open the vascular-type barrier. Scale bar, 300 μm.

Figure 2

Six cell-type spheroid Characterization. Live-Dead Staining was conducted on days 5 (A), 7 (B), 10 (C), and 21 (D) to assess cell viability. Green represents live cells and red indicates dead cells; In each case, 8 randomly selected spheroids were assed for cell viability. (E) Green florescence for live cells was quantified and expressed as a percentage of the total fluorescence (red and green). Immunohistochemistry was conducted on spheroids to determine phenotypic protein expression. CD31 expression (F) shows localization of HBMEC in self-assembled spheroids. Even though the cells migrate to the outer sphere, the endothelial cells do not cover the spheroid. Expression of claudin-5 (G, red) was established through immunofluorescent staining. Expression of P-gp in G and Q (an efflux protein responsible for the efflux of xenobiotic substances out of the brain tissue) was established in self –assembly and staged self-assembly spheroids, respectively. Self-assembly spheroids were stained for ZO-1 (H) and DAPI (blue). Selected area was imaged at higher magnification in order to visualize the cell border connections formed by ZO-1 (I). Specific cell markers were used to identify normal protein expression in individual cell type incorporated into the spheroid made through a staged self-assembly: (J) showing CD31 staining signifying that the HBMEC cover the spheroid in a staged self-assembly, PDGFR (K) an pericyte marker, GFAP (L) an astrocyte marker, Iba1 (M) a microglial marker, N O4 and MBP markers of oligodendrocyte progenitors and mature oligodendrocytes respectively. MAP2 (O) is a neuronal marker. Neuronal spheroids were stained for Tyrosine Hydroxylase (P) to identify the presence of dopaminergic neurons. Transmembrane glucose transporter and efflux transporter protein expression and localization were established by staining for P-gp (Q) and GLUT1 (R) respectively. All markers were found to have a thorough distribution. Images were obtained at 10x magnification. Scale bars are depicted in white. Scale bars, 200 μm and 30 μm (for L only).

Figure 3

Blood-brain barrier marker expression and effect of hypoxia on tight and adherens junction protein distribution. The presence of tight junction markers Zona occludens-1 (ZO-1) (A), claudin-5 (B), and adherens junction proteins β-catenin (C) and VE-Cadherin (D) were confirmed via immunofluorescent staining and confocal microscopy at 10x. The lower panels in A′–D′ are parts of spheroids in A–D taken at higher magnification. Immunofluorescent staining was conducted on spheroids consisting of all types for junction markers claudin-5, ZO-1, β-catenin, and Ve- cadherin under normoxic (upper panel, E, G, I, & K). The lower panels in (F, H, J, & L) show spheroids that were cultured under hypoxic conditions at 37 °C, 0.1% O₂ for 24 hrs prior to immunofluorescent staining and imaging. Spheroids under hypoxic condition exhibited disrupted intercellular junction markers compared to their normoxic counterparts. Images were taken at higher magnification. (M) Fluorescence for claudin-5, ZO-1, β-catenin and VE-cadherin was quantified and normalized to DAPI fluorescence per section. Student T-Test, two tailed hypothesis **P $<$ 0.05 with normoxic condition, n_organoids = 3. Data represented as mean ± s.e.m. Scale bars 250 μm (A–D) and 50 μm (E–L).

Figure 4

BBB Integrity and selectivity using FITC labeled IgG, Mercury, MPTP and MPP+. (A) Shows IgG permeability in untreated (upper panel) and Histamine treated (lower panel) spheroids. (B–D) Results from 4 separate experiments were pooled and normalized to the ATP production in untreated spheroids. Neuronal (BBB−) spheroids treated with mercury (II) chloride (B) exhibited low ATP production. Spheroids consisting of all six cell types (BBB+) that were treated with mercury (II) chloride exhibited higher viability and were statistically significant with respect to their untreated counterparts. In C, there was no significant in ATP production in untreated versus MPTP treated BBB− spheroids. MPTP treated BBB+ spheroids exhibited significantly low ATP production compared to the untreated group. MPP+ (D) BBB− spheroids exhibited significantly lower ATP production compared to the untreated neuronal spheroids. BBB+ spheroids that were treated with MPP+ exhibited no significantly different cell viability compared to the untreated group. In E, BBB+ spheroids were treated with Histamine to allow MPP+ and Mercury ion to penetrate into the organoids. Both MPP+ and mercury (II) chloride demonstrated significantly lower ATP production compared to untreated control. Student T-Test, two tailed hypothesis, *P < 0.05, ****P < 0.0001, with untreated spheroids, n_organoids = 64 (B–D), n_organoids = 32 (E, experiment only perfomed once). Data represented as mean ± s.e.m.