primary human astrocytes  (Innoprot Inc)

Journal: Bioactive Materials

Article Title: Human in vitro models of neurovasculature and the application to pre-clinical intracerebral haemorrhage research

doi: 10.1016/j.bioactmat.2025.10.018

Figure Lengend Snippet: Transwell models and the recreation of the neurovascular unit. A. Schematic representation of the configurations for in vitro static human primary cell-based blood-brain barrier models. A monoculture model contains HBMEC seeded on the apical side of the well-insert porous membrane. A non-contact co-culture contains HBMEC seeded on the upper surface of the well-insert support and HA seeded at the bottom of the culture well. A contact co-culture model includes HBVP seeded on the lower surface of the well-insert support with HBMEC on the upper surface. For the triple culture model, HBMEC are seeded on the upper surface of the support with HBVP seeded on the lower surface and HA seeded on the bottom of the culture wells. Adapted from Fattakhov et al. (2022) . HBMEC = human brain microvascular endothelial cells; HA = human astrocytes; HBVP = human brain vascular pericytes. B. in vitro model of subarachnoid haemorrhage. The blood–brain barrier divides the system into two compartments (vascular side: upper compartment, brain parenchymal side: lower compartment). Medium was added to both compartments to prevent the cells from drying out. To simulate subarachnoid haemorrhage, the medium in the lower compartment was replaced by human serum to facilitate contact with the medium from above and the serum from below. Since the compartments are separated by a microporous membrane, the diffusion of small molecules (e.g., from the serum) is possible. Adapted from Thal et al. (2022) . BMEC = human brain microvascular endothelial cells. C. Utilising the transwell model system to determine the effects of TNFα/IFNγ stimulation on the AD patient-derived BBB model, AD iBEC were cultured on Transwell inserts and TNFα/IFNγ were added to the top chamber of the Transwell insert. Following 24 h treatment, TEER measurement and 5 kDa dextran permeability assays were performed. Cell pellet and supernatant samples were collected for subsequent analysis with qPCR, ELISA, and LDH assays. Adapted from Wasielewska et al. (2024) . BBB = blood-brain barrier; TNFα = tumour necrosis factor α; IFNγ = interferon gamma; ELISA = Enzyme-Linked Immunosorbent Assay; LDH = lactate dehydrogenase; TEER = transendothelial electrical resistance; AD = Alzheimer's Disease; iBEC = induced brain endothelial-like cell; qPCR = Quantitative PCR. D. i. Cells were thawed in petri dishes and let grown for 2 days. Then, cocultures were settled by seeding human ECs in the filters and brain pericytes in the bottom compartment either for 12-well plates or miniaturized 96 TW systems. ii. In the original 12 TW model, filters were cut and placed in coverslips, then returned to acquire the pictures over the luminal faces of the ECs. iii. Filters from the miniaturized systems were directly placed on a 3D frame developed and adapted to the microscope. Adapted from Moya et al. (2021) . TW = transwell.

Article Snippet: , • Human primary astrocytes (Sanbio) • Human cerebral microvascular endothelial cells (hCMVEC/D3) • Peripheral blood mononuclear cells (PBMCs) , • Triple B slides designed in house • Endothelial cells seeded onto filter and cultured under flow • Astrocytes cultured on underside of the filter • PBMCs injected into flow and transmigration into lower chamber analysed , • TEER • BBB diffusion permeability • Tight junction protein expression • Immune cell transmigration , • Inflammatory profiling • Patient cells/blood • BBB impairment • Cell death • Transcytosis of immune cells • Hypertension , • Patient-derived cells • Adjustable chip design for 2D and 3D , • No neuronal culture • Synthetic filter barrier , .

Techniques: In Vitro, Membrane, Co-Culture Assay, Diffusion-based Assay, Derivative Assay, Cell Culture, Permeability, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Microscopy

Journal: Bioactive Materials

Article Title: Human in vitro models of neurovasculature and the application to pre-clinical intracerebral haemorrhage research

doi: 10.1016/j.bioactmat.2025.10.018

Figure Lengend Snippet: Utilising hydrogels to add 3D complexity to models of the blood-brain barrier . A. The biofabrication process of the in vitro BBB model. (a) Step 1, gelatin and sodium alginate were extruded by coaxial needle, which contained growth factors. (b) Step 2, heparin-chitosan electrostatic self-assembled coating, this core–shell scaffold structure played a role in controlled release of the growth factors. (c) Wrapped endothelial cell layer. (d) Wrapped astrocyte layer, (e) after culture and induction, the internal scaffold gradually degraded and tight junctions were formed between endothelial cells. ii. (a) Overall optical photograph, (b) Haematoxylin and eosin staining of the cross-section, (c) Local magnification. Adapted from Liu et al. (2020) . BMEC = human brain microvascular endothelial cells; RA = rat astrocytes. B. A 3D BBB model consists of EC arranged in a cylindrical monolayer, forming a tight seal and separating a “blood” compartment from an array of capillaries presenting chemotactic gradients. The 3D BBB model is constructed by ( 1 ) coating an adhesive molecule, PDL and a gel on PDMS microstructures and a glass substrate, ( 2 ) plating brain EC first on the top and ( 3 ) later on the bottom surfaces and ( 4 ) culturing to form tight monolayers. Adapted from Cho et al. (2015) . PDL = Poly-D-lysine; PDMS = polydimethylsiloxane; EC = endothelial cells; BBB = blood-brain barrier. C. i. The BTI Chip. A blood vessel endothelium contacts a cell-embedded GelMA-PEG hydrogel . ii . Parallel laminar flow profiles can be established simultaneously in multiple Y channels within the same PDMS housing. iii. Immunofluorescence micrographs of HDFn-embedded gels and hBMVEC endothelium demonstrate successful fabrication of an array of blood-tissue interfaces. Staining for VE-Cadherin (red) distinguishes hBMVEC from HDFn (phalloidin-stained actin, green; DAPI-stained nuclei, blue). Adapted from Young et al. (2023) . BTI = blood-tissue interface; PDMS = polydimethyl-siloxane; GelMA-PEG = gelatin methacryloyl polyethylene glycol; HDFn = human dermal fibroblasts, neonatal; hBMVEC = human brain microvascular endothelial cells; DAPI = 4′,6-diamidino-2-phenylindole. D. i. 3D artist impression of the NVU on-a-chip model in the OrganoPlate 3-lane culture platform. ii. 3D reconstruction of the human NVU model showing a vessel of brain endothelial cells (PECAM-1, magenta) grown against an extracellular matrix gel, in co-culture with networks of astrocytes (s100β, green) and neurons (TUBB3, red). iii. Astrocytes (s100β, green) and neurons (TUBB3, red) are present in the bottom lane of the chips and form networks. All images were acquired from 14-day old cultures. Scale bars are 50 μm. Adapted from Wevers et al. (2021) . E. i. A cross-section through the chip showing the PDMS channel containing the collagen gel made with viscous fingering and a central lumen. ii. Time-lapse images of the fingering method (bar, 500 μm). iii. Second harmonic generation image of the collagen distribution in the 3D BBB chip, an intensity generated voxel illustration of the lumen based on this information (bar, 100 μm), and a high magnification of the second harmonic generation image showing of collagen microstructure in the cylindrical gel within the 3D BBB chip (bar, 50 μm). iv. fluorescence confocal micrographs of the engineered brain microvessel viewed from the top, and cross-section at either low ( v. ) or high ( vi. ) magnification. Green indicates F-actin staining, blue represents Hoechst-stained nuclei, and magenta corresponds to VE-Cadherin staining. Adapted from Herland et al. (2016) .

Article Snippet: , • Human primary astrocytes (Sanbio) • Human cerebral microvascular endothelial cells (hCMVEC/D3) • Peripheral blood mononuclear cells (PBMCs) , • Triple B slides designed in house • Endothelial cells seeded onto filter and cultured under flow • Astrocytes cultured on underside of the filter • PBMCs injected into flow and transmigration into lower chamber analysed , • TEER • BBB diffusion permeability • Tight junction protein expression • Immune cell transmigration , • Inflammatory profiling • Patient cells/blood • BBB impairment • Cell death • Transcytosis of immune cells • Hypertension , • Patient-derived cells • Adjustable chip design for 2D and 3D , • No neuronal culture • Synthetic filter barrier , .

Techniques: In Vitro, Staining, Construct, Adhesive, Immunofluorescence, Co-Culture Assay, Generated, Fluorescence

Journal: Bioactive Materials

Article Title: Human in vitro models of neurovasculature and the application to pre-clinical intracerebral haemorrhage research

doi: 10.1016/j.bioactmat.2025.10.018

Figure Lengend Snippet: Approaches utilising microfluidics to add flow and enable shear stress. A. i. Schematic view of a microfluidic device having three types of microfluidic channels. ii. Section view of microfluidic device describing sequential loading and culture progress of BBB. Channel C (red) is the region where the final BBB microenvironment is constructed. Channel F (green) possesses 3D fibroblasts, which acts as a source of angiogenic factors. Channel M1 and M2 (blue) are media channels. iii. Day by day confocal imaging of channel C where HBMEC (anti-CD31, red) sprouts from the left end to right end. Astrocytes (anti-GFAP, white) protrude to generate end-feet and pericytes (anti-αSMA, green) wrap around EC as the day goes by. Nuclei were stained with Hoechst 33342 (blue); scale bar = 100 μm. Adapted from Lee et al. (2020) . BBB = blood-brain barrier; HBMEC = human brain microvascular endothelial cells. B. The developed multi-channel multi-layer BBB chip with integrated electrical impedance sensor array for TEER analysis. Adapted from Jeong et al. (2017) . BBB = blood-brain barrier; TEER = transendothelial electrical resistance; PDMS = polydimethylsiloxane. C. Simplified anatomical illustration of the neurovascular unit, highlighting positions and cell–cell interactions between vascular endothelial cells (pink) that line brain blood microvessels (left, right) and surrounding perivascular pericytes (yellow) and astrocytes (blue) that form the BBB, as well as neighbouring neurons (green) in the brain parenchyma and a schematic of the experimental setup of NVU system. Adapted from Maoz et al. (2018) . BBB = blood-brain barrier; aBlood = artificial blood; aCSF = artificial cerebrospinal fluid. D. Schematic of BBB-Chip seeding paradigm, with iBMECs seeded on the blood side and primary human astrocytes and pericytes seeded on the brain side. Immunocytochemistry 5 days post-seeding shows the blood side is populated with iBMECs that form a monolayer and express phalloidin (magenta). The brain side is seeded with primary human astrocytes that express GFAP (green) and pericytes. Scale bar, 1 mm. High magnification images show expression of membrane ZO1, GFAP and α-SMA. Adapted from Vatine et al. (2019) . iBMECs = induced pluripotent stem cell derived brain microvascular endothelial-like cells; ZO1 = zona occludens 1; GFAP = glial fibrillary acidic protein; α-SMA = α smooth muscle actin. E. The chip design and spatial distribution of the neurovascular unit constituent cells in the chip. Adapted from Lyu et al. (2021) . CSF = cerebrospinal fluid F. Human organs for the Human-Body-on-Chip (HuBoC), with a representative Organ Chip photograph and sectional schematic. Adapted from Novak et al. (2020) . G. Coculture of hCMEC/D3 and primary human astrocytes in μHuB. hCMEC/D3 monolayers (green) were cultured in the vascular (apical) compartments with primary human astrocytes (red) in the tissue (basolateral) compartment (nuclei, blue). Adapted from Brown et al. (2019) . hCMEC/D3 = human brain microvascular endothelial cells.

Article Snippet: , • Human primary astrocytes (Sanbio) • Human cerebral microvascular endothelial cells (hCMVEC/D3) • Peripheral blood mononuclear cells (PBMCs) , • Triple B slides designed in house • Endothelial cells seeded onto filter and cultured under flow • Astrocytes cultured on underside of the filter • PBMCs injected into flow and transmigration into lower chamber analysed , • TEER • BBB diffusion permeability • Tight junction protein expression • Immune cell transmigration , • Inflammatory profiling • Patient cells/blood • BBB impairment • Cell death • Transcytosis of immune cells • Hypertension , • Patient-derived cells • Adjustable chip design for 2D and 3D , • No neuronal culture • Synthetic filter barrier , .

Techniques: Shear, Construct, Imaging, Staining, Immunocytochemistry, Expressing, Membrane, Derivative Assay, Cell Culture