Fig 1.
Reproducible 3D-vascularized spheroid tissue construction process of high content/high throughput platform.
(A) structure of OrganoPlate Graft in 384 well plate format, (B) experimental procedure for constructing 3D-vascularized spheroid tissue, and (C) represented images of each brain tissue construction process step; (a) collagen-I loading, (b) endothelial cell seeding in the perfusion channels and vessels formation under flow, (c) vascular network formation using an angiogenic cocktail, and (d) cortical spheroid placement and maturation onto the pre-formed vascular network.
Fig 2.
Angiogenic-derived microvascular network.
(A) Microvasculature morphology in the OrganoGraft: Region A (perfusion channel) and Region B (new angiogenic vasculature), effects of shear stress on endothelial cell alignment, and polar histogram quantification of cell alignment with respect to bidirectional flow using a rocker (Right Figures). (B-C) Representative images of vascular network acquired from F-actin staining (B) 2D and (C) 3D images with cross-section view of vascular network. (D) Permeability of vascular network acquired as time lapsed images after the addition of the FITC-dextran of collagen-I matrix only (control) and vascular network with collagen-I matrix (pictures were taken at every 2 minutes). (Significance was calculated using unpaired two-tailed student t-test, *** P<0.001). (E) Represented angiogenesis of endothelial cells and quantification of angiogenesis in number and length of micro-vasculature with and without PMA in angiogenic cocktail (pink color shows sprouted vessels using Image J, n = 3). (F) Represented angiogenesis with collagen density of 6 mg/mL, 7 mg/mL, and 8 mg/mL. Microvessels were pre-formed for 3 days in perfusion environment at both perfusion channels and induced gradient driven sprouting (Day 0) using angiogenesis cocktail of containing with VEGF (37.5 μg/mL), FGF-2 (10 μg/mL), MCP-1 (37.5 μg/mL), HGF (37.5 μg/mL), and S1P (250 nM), without and with 2 ng/mL PMA addition. (Significance was calculated using unpaired two-tailed student t-test, * P<0.1, ** P<0.01, *** P<0.001).
Fig 3.
(A) Representative fluorescent images of cortical neural markers and adhesion markers expressed in derived cortical spheroids. (a) White: beta III-tubulin, (b) Red: SATB2, (c) Green: TBR1, (d) Blue: Nuclei (Hoechst), (f) White: β1-integrin, (g) Red: β3- tubulin, (h) Green: N-cadherin, (i) Blue: Nuclei (Hoechst). Scale Bar: 50 μm. (B) (a) Schematic diagram of cell mass and neurite spared, (b) represented images of cortical spheroid in Collagen and Matrigel as well as (c) quantitative data of spheroid diameter and (d) neurite outgrowth of cortical spheroid in Collagen and Matrigel in days.
Fig 4.
Cortical spheroid on vascular network.
(A) 3D and 2D confocal images of spheroid on vascular network (Red (CD31) is microvessel and green (beta III tubulin) is cortical spheroid), (B) represented images of cortical spheroid growth and spreading on vascular network and quantitative data for spheroid diameter and neurite outgrowth of cortical spheroid in Collagen (7 mg/mL) and (8 mg/mL), (C) fluorescent images of cortical neural markers from the middle (upper one) and outer (lower one) regions after extracted from vascular network. White: beta III-tubulin, Red: SATB2, Green: TBR1, and Blue: Nuclei (Hoechst). Scale Bar: 50 μm, and (D) 2-Week culture of cortical spheroid on vascular network and cross-section views of neurites-wrapped vascular network (green is beta III tubulin and red is CD31).