Journal: Advanced Science
Article Title: Subventricular Zone‐on‐a‐Chip: A Model to Study Neurogenesis Disruption in Neonatal Intraventricular Hemorrhage
doi: 10.1002/advs.202502145
Figure Lengend Snippet: Overview of the SVZ‐on‐a‐chip. A) The SVZ‐on‐a‐chip structure 24 h post‐seeding, showing distinct cellular compartments visualized via staining. The entire chip structure is shown (scale bar = 3000 µm) alongside a magnified maximum Z‐projection view (scale bar = 100 µm). Arrows indicate neural stem cells (NSCs), and triangles highlight human fetal astrocytes (HFAs) embedded in Matrigel, occupying the central channel. The right channel is lined with human brain microvascular endothelial cells (HBMECs), while the left channel contains human choroid plexus epithelial cells (HCPEpiC). B) Neural channel with HCPEpiC seeded along the central channel (scale bar = 200 µm). C) The central channel, highlighted by triangles, is shown in closer detail (scale bar = 200 µm). D) 3D view of the inner channel, showing cells stained with Nestin (gray). Astrocytes, marked by triangles, are stained red but lack Nestin expression (scale bar = 70 µm). E) Inner channel viewed from a different angle (scale bar = 70 µm). F) Vascular channel lined with HBMECs (scale bar = 70 µm). G) HBMECs fully covering the vascular channel (scale bar = 70 µm). H) A magnified maximum Z‐projection view of the SVZ‐on‐a‐chip 7 days after complete seeding, showing significant cellular growth (scale bar = 100 µm). Clusters of NSCs are observed invading both the neural and, predominantly, the vascular channel. I) Maximum Z‐projection view of Nestin staining in the middle channel (scale bar = 100 µm). J) Close‐up of DCX‐positive neural stem cells invading the vascular channel. DCX staining was used instead of Nestin because HBMECs also express Nestin (scale bar = 50 µm). SVZ: Subventricular Zone, NSCs: Neural Stem Cells, HFAs: Human Fetal Astrocytes, HBMECs: Human Brain Microvascular Endothelial Cells, HCPEpiC: Human Choroid Plexus Epithelial Cells, DCX: Doublecortin.
Article Snippet: DPBS minus Ca ++ and M g++ (14 190 144, Thermo Fischer, Waltham, Massachusetts, USA), DPBS with Ca ++ and Mg ++ (14 040 091, Thermo Fischer), Astrocyte Media (AM1801, ScienCell, Carlsbad, California, USA), TrypLE (12 604 013, Thermo Fischer), Attachment factor protein 1× (S006100, Thermo Fischer), EGM ‐2 MV Microvascular Endothelial Cell Growth Medium‐2 BulletKit (CC‐3202, Lonza, Basel, Switzerland), Endothelial Cell Growth Medium MV2 (C‐22022, PromoCell, Heidelberg, Germany), Epithelial Cell Medium (Innoprot, P60104 , Derio, Spain), poly‐L‐lysine (PLL) (Innoprot), DMEM: F12 Glutamax (31331‐028, Thermo Fischer), N2 supplement ( 17502‐048, Thermo Fischer), B27 serum free (11 530 536, Thermo Fischer), FGF (233‐FB, R&D Systems, MN, USA), EGF (E9644, Sigma Aldrich, Burlington, Massachusetts, USA), PLO (P3655, Sigma Aldrich), Laminin (L2020, Sigma Aldrich), Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV‐free (354 230, Corning, Corning, New York, USA), Antibiotic‐Antimycotic (15 240 062, Themo Fischer), idenTx 3 Chip (Aim Biotech, Singapore), Transwells 0.4 μM microporous membrane (833 932 041, Sarstedt, Nümbrecht, Germany), High Pure RNA Isolation Kit (11 828 665 001, Roche, Basel, Switzerland), Clariom S Affymetrix Assay (902 927, Thermo Fischer), High‐capacity RNA‐to‐cDNA kit (4 387 406, Thermo Fischer), TaqMan probes (Applied Biosystems, California, USA), Fast Advanced Master Mix (4 444 557, Applied Biosystems), TRIzol (15 596 026, Thermo Fischer), Chloroform (194 002, MP Biomedicals, Santa Ana, California, USA), Cell recovery solution (354 253, Corning), Anti‐Adherence Rinsing Solution (0 7010, STEMCELL Technologies, Vancouver, Canada), AggreWell800 24‐well plates (34 811, STEMCELL Technologies), Goat serum (G9023, Sigma Aldrich), Trypan Blue (1 450 021, Bio‐Rad, Hercules, California, USA), LEGENDplex Human Inflammation Panel 1 (13‐plex) assay using a V‐bottom plate (BioLegend, San Diego, California, USA), CellROX Green reagent ( C10444 ,Invitrogen), JC‐1 dye (T3168, Invitrogen) IL1receptor antagonist (SRP3327, Sigma Aldrich).
Techniques: Staining, Expressing
Innoprot Inc is a verified supplier 
Innoprot Inc manufactures this product 
![Drug screening and validation of Niclosamide using CCA cell lines and primary biliary <t>epithelial</t> cells. ( A ) A library of 104 off-patent drugs was screened using the CCLP CCA cell line. CCLP cells were plated at 1 × 10 4 cells per well in a 96 well plate and treated with each drug at its clinically relevant peak serum concentration for 96 h. Cell viability was then determined using an MTT assay and normalised to cells treated with the corresponding vehicle control for each drug. N = 3 biological repeats with a paired, two-tailed T-test followed by Benjamini–Hochberg multiple corrections. The inhibition of viability (%) is plotted against the reciprocal of the p value for each drug and Niclosamide is labelled. ( B – E ) Niclosamide dose–response curves for CCA cell lines (CCLP, RBE, and KKU-M055) and primary biliary epithelial cells (BECs) plated as in ( A ) and treated with Niclosamide [10 nM–100 μM] or vehicle control for 72 h. Cell viability was measured using an MTT assay and normalised to vehicle control. N = 3 biological repeats each performed in triplicate. Error bars represent SEM. When error bars cannot be seen they are smaller than the symbols. ( F ) The graph shows the relative EC50 values for each cell type calculated using GraphPad Prism. Error bars represent SEM (* = p < 0.05 unpaired t -test). The inserted Western blot shows PRH levels in the CCA cell lines prior to treatment and with β-actin as a loading control.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1616/pmc12651616/pmc12651616__cancers-17-03721-g001.jpg)
