primary human brain microvascular endothelial cells hbmecs  (Innoprot Inc)

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase contrast images of HBMECs after 24 hours of treatment with 100 μM atorvastatin (ATV). Scale bars 50 μm. ( B ) Cell survival after ATV treatment for 24 hours in both stationary and rotating culture conditions ( n =4 independent repeats). Scale bars 100 μm. ( C ) qPCR analysis of marker expression after ATV treatment in rotating cells shows an increase in VE-Cadherin and NG2 gene expression (non-significant, One-Way ANOVA). ( D ) Fluorescent images of CD31 (green) and ZO-1 (red) cellular expression after 24 hours with ATV and DMSO control. ( E ) Total expression of ZO-1 after treatment ( n =4 independent repeats, two-way ANOVA *** P =0.0009, **** P <0.0001) ( F ) Analysis of ZO-1 co-localised with CD31 on the cell surface when treated with ATV ( n =4 independent repeats, two-way ANOVA, *** P =0.0002). ( G ) Fluorescent images of CD31 (green) and VE-Cadherin (red) cellular expression after 24 hours of ATV treatment in both stationary (top panels) and rotating culture (bottom panels). Zoom inset shows the internalisation of both CD31 and VE-Cadherin from the cell surface when treated with ATV. Scale bars 20 μm. ( H ) Analysis of VE-Cadherin co-localised with CD31 on the cell surface when treated with ATV ( n =3 independent repeats, two-way ANOVA, * P <0.04). ( I ) Total area expression of CD31 and VE-Cadherin as a percentage area of DAPI in both stationary and rotating cells, ( n =3 independent repeats, no significance from analysis with a two-way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Marker, Expressing, Gene Expression, Control

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative images of filipin-stained HBMECs, after 24hours of drug treatment. Scale bars 50 μm. ( B ) Filipin expression as a percentage of area from 6 ROIs in 2 wells across 2 independent repeats after 24 hours with ATV, ** P =0.0081, t -test. ( C ) qPCR analysis of rotated HBMECs after 24 hours of drug treatment shows a significant increase in HMGCR expression ( n =5 independent repeats, * P =0.0323 One-Way ANOVA).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Phase images of tube formation in untreated and ATV-treated HBMECs. Scale bars 20 μm. ( B ) Analysis of the network parameter average vessel length shows a significant reduction with ATV treatment, both pre-tube formation and post (One-way ANOVA with Tukey’s post hoc multiple comparisons test ** P =0.003, *** P =0.0007, **** P <0.0001). ( C ) Cytotoxicity analysis of ATV treated tubes showed no difference from controls ( n =3, t -test).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques:

Journal: bioRxiv

Article Title: Using atorvastatin-induced vascular weakness to model brain haemorrhage in vascularised cerebral organoids

doi: 10.64898/2026.04.20.719465

Figure Lengend Snippet: ( A ) Representative confocal images of whole organoids treated with ATV for 24 hours and the loss of VE-Cadherin expression from the surface. Scale bars 500 μm. ( B ) VE-Cadherin, not CD31 ( P =0.091), expressed as a percentage of DAPI was significantly reduced with ATV treatment compared to DMSO controls (two-tailed t -test, * P =0.011). ( C ) Size progression for 4 batches of organoids that were treated with ATV at day 40. ( D ) Vascular metrics were unchanged for CD31 ( n =15 organoids from 4 batches, non-significant t- test) with slightly more endpoints, signifying single cells. ( E ) Angiotool analysis of VE-Cadherin staining revealed shorter overall vessel length ( n =15 organoids from 4 batches, t- test, * P =0.0389)). ( F ) qPCR analysis of ATV-treated organoids showed a reduction in VE-Cadherin RNA expression; however, other markers of endothelial function are unchanged (One-Way ANOVA, n =5 organoids from 2 batches). ( G ) ATV treatment increased the expression of some cholesterol biosynthesis markers compared to DMSO controls, opposite to what was observed in HBMECs in 2D (One-Way ANOVA, n =5 organoids from 2 batches).

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Innoprot P10361) were maintained on 1% gelatin coated flasks in endothelial cell growth medium MV (PromoCell C-22020).

Techniques: Expressing, Two Tailed Test, Staining, RNA Expression

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Vessel formation with HBMVECs is enhanced at lower Geltrex TM concentrations and by mixed endothelial‐organoid media in the presence of VEGF. A) Graphical representation of 100 000 HBMVECs encapsulated in different Geltrex TM concentrations and grown for 10 days using different media and VEGF doses. Created with Biorender.com. B) Representative confocal images of Calcein AM‐stained endothelial networks formed in 80%, 60%, and 40% Geltrex TM supplemented with 50 ng mL −1 VEGF for 10 days. Scale bars: 500 µm. (C) Percentage of vessel area (%), total vessel length (mm), and lacunarity values of endothelial networks formed in 80%, 60%, and 40% Geltrex TM , with or without 50 ng mL −1 VEGF. Mean ± SD, N = 6 independent wells, two repeats. Two‐way ANOVA; asterisks showing Tukey's post hoc comparisons; ns = not significant ( p > 0.05); * p < 0.05, *** p < 0.001, **** p < 0.0001. D) Representative confocal images of Calcein AM‐stained endothelial networks grown in 50% Geltrex TM with different media (endothelial cell growth media (EC), organoid maturation media (CO), or mixed media at 1:1, 1:3, and 1:7 EC:CO ratios), all supplemented with 50 ng mL −1 VEGF for 10 days. Scale bars: 500 µm. E) Total vessel length (mm), number of junctions per mm 2 , and end points per mm 2 of endothelial networks grown in different media compositions. Mean ± SD, N = 6 independent wells, two repeats. One‐way ANOVA; asterisks showing Tukey's post hoc comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001. F) Representative confocal images of Calcein AM‐stained endothelial networks formed in 50% Geltrex TM with 1:7 mixed media, supplemented with 25 or 50 ng mL −1 VEGF, with media exchanges every 2 or 4 days for 10 days. Scale bars: 500 µm. G) Percentage of vessel area (%), number of junctions per mm 2 , and lacunarity values of endothelial networks formed at different VEGF concentrations (25, 50 ng mL −1 ) and media exchange frequency (every 2 or 4 days). Mean ± SD, N = 6 independent wells, two repeats. Two‐way ANOVA; asterisks showing Tukey's post hoc comparisons; * p < 0.05.

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Staining

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Promoting vascularization of the organoid accelerates early organoid growth and spherical morphology without impacting on the size or tissue stiffness by day 40. A) Brightfield images of standard COs grown according to the Lancaster protocol [ ⁹ ] (CTRL) compared to COs grown in the modified protocol, where HBMVECs were encapsulated in a Geltrex TM droplet and grown in mixed media supplemented with VEGF (BECs MM+VEGF). Representative images were captured on days 8, 11, 16, 22, and 40. Scale bar: 1 mm. B) Organoid size evolution from encapsulation until day 40 across the six tested conditions: CTRL (red), +VEGF (orange), MM+VEGF (yellow), BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple). Organoid size was measured as the area in the brightfield images (mm 2 ). Mean ± SD, N = 16‐25, five independent batches, two different iPSC lines. Mixed‐effect model, followed by Tukey's post hoc comparisons; on day 16, BECs MM+VEGF were significantly different (&) from CTRL (p = 0.001), MM+VEGF (p = 0.0013), BECs CTRL (p = 0.0002), and BECs +VEGF (p = 0.0085). On day 20, BECs MM+VEGF were significantly different ($) from CTRL (p = 0.0143), +VEGF (p = 0.0128), MM+VEGF (p = 0.0027), BECs CTRL (p = 0.0016) and BECs +VEGF (p = 0.0007). C) Largest longitudinal area (mm 2 ) of day 40 organoids across all conditions (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 19–31, 5 independent batches, two different iPSC lines. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05. D) Organoid circularity at day 40 across all conditions (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 14‐27, five independent batches, two different iPSC lines. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05, ** p < 0.01, **** p < 0.0001. E) Nanoindentation measurements of tissue stiffness (Young modulus, in KPa) of CTRL and BECs MM+VEGF organoids. Mean ± SD, N = 10–13, two independent batches, two different iPSC lines. Unpaired T ‐test; ns = not significant ( p > 0.05). F) Degradation of the Geltrex TM droplet area (mm 2 ) over time (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs; dashed lines represent organoids generated with the A‐iPSC line; uninterrupted lines represent organoids generated with the S‐iPSC line). Mean ± SD, N = 9–15, two or three independent batches per each of the two IPSC lines. G) ELISA quantification of VEGF in culture supernatants from CTRL and BECS MM+VEGF organoids. The red line represents the 50 ng mL −1 VEGF dose added in the MM+VEGF media. Mean ± SD, N = 4, two independent batches for each of the two iPSC lines. Two‐way ANOVA, asterisks showing Sidak's post hoc comparisons; **** p < 0.0001. H) ELISA quantification of matrix metalloproteinase 9 (MMP‐9) in culture supernatants from CTRL and BECS MM+VEGF organoids. Mean ± SD, N = 4, two independent batches for each of the two iPSC lines. Two‐way ANOVA, asterisks showing Sidak's post hoc comparisons; **** p < 0.0001.

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Modification, Encapsulation, Generated, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Abundant multicellular endothelial networks are formed on the surface of COs, with morphological features dependent on media conditions or Geltrex TM concentration. A) Confocal images of A and S‐COs grown under tested vascularization conditions, wholemount stained for CD31 (green) and βIIITUB (magenta). Dashed lines separate the different conditions: media used (CTRL, +VEGF or MM+VEGF, vertical display), presence or absence of encapsulated HBMVECs (horizontal display). Within each condition, a comparison was made between organoids generated with the A‐and S‐iPSCs lines. Scale bars: 500 µm. At the bottom, higher magnification confocal images showing endothelial networks on the surface of BECs MM+VEGF organoids, for both iPSCs lines. Scale bars: 100 µm. B) Percentage of CD31+ surface in A‐COs. Control (red), +VEGF (orange), MM+VEGF (yellow), BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple); no HBMVECs, white filling; +HBMVECs, grey filling. Violin shape represents data distribution, Median (thick line), interquartile range (thin lines), N = 6–12, three independent batches. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05, ** p < 0.01. C) Percentage of CD31+ surface in S‐COs. Same color system as above. The violin shape represents data distribution, median (thick line), interquartile range (thin lines), N = 6–8, two independent batches. Kruskall‐Wallis test, asterisks showing Dunn's post hoc comparisons; *p < 0.05, ** p < 0.01, *** p < 0.001. D) Quantification of network end points per mm 2 , average vessel length (mm), and junctions per mm 2 of the HBMVECs‐containing organoids, separated by iPSCs origin. BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 5–12, three or two independent batches for each line. Two‐way ANOVA test; media and iPSC line factor significance expressed on top of each graph. Tukey's post hoc comparisons are represented by brackets over relevant bars; ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. E) Degradation rate of different concentrations of Geltrex TM droplets in BECs MM+VEGF conditions, measured by the area change (mm 2 ) over time. Light blue (60%), black (80%), Bordeaux (95%). Mean ± SD, N = 5, one batch of organoids. F) Representative confocal images of BECs MM+VEGF A‐COs grown at different Geltrex TM concentrations (60%, 80% and 95%), wholemount stained for CD31 (green) and βIIITUB (magenta). Scale bars: 500 µm. On the right, higher magnification images of the endothelial networks are shown. Scale bars: 100 µm. G) Vessel diameter (µm) of the superficial endothelial networks formed under different Geltrex TM concentrations. Light blue (60%), black (80%), Bordeaux (95%). Mean ± SD, N = 9–22 (individual organoids) and >5 areas of interest per organoid analyzed, five different batches, same A‐iPSC line. Kruskal‐Wallis test, asterisks showing Dunn's post hoc comparisons; **** p < 0.0001. H) End points per mm 2 of the superficial endothelial networks formed under different Geltrex TM concentrations. Light blue (60%), white (80%), and Bordeaux (95%) colors were used. The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 9–22, five different batches, and the same A‐iPSC line. One‐way ANOVA test, Tukey's post hoc comparisons; not significant ( p > 0.05).

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Concentration Assay, Staining, Comparison, Generated, Control