hbec 5i human brain endothelial cells  (ATCC)

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. Cells were seeded in equal proportion (200,000 each, ~ 30% confluence) in a 25 cm 2 culture flask and allowed to evolve for ~30 days. Self-organization of endothelial cells in 3D aggregates and accumulation of cancer cells around these structures was observable after ~7 days (80-90% confluence). After ~21 days, clearly delineated 3D networked constructs incorporating the vast majority of cancer cells were observable.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. Cells were seeded in a proportion of HBEC-5i (180,000 cells)/cancer cells (~20,000) in a 25 cm 2 culture flask and allowed to evolve for ~30 days. Self-organization of endothelial cells in 3D aggregates and accumulation of cancer cells in these structures was observable after ~7 days. After ~21 days, clearly delineated 3D networked constructs were observable.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. HBEC-5i cells (~200,000) were seeded in a 25 cm 2 culture flask and allowed to evolve until they formed 3D networked structures (~10 days), time when ~200,000 cancer cells were added to the flask. The migration of SK-OV-3 and MDA-MB-231 cells toward HBEC-5i structures was observable already after 2 days of co-culture. The evolution of endothelial/cancer constructs was monitored for an additional 3 weeks.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Migration, Co-Culture Assay, Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: (A,D) Control wells with red fluorescent SK-BR-3 cells seeded in the upper inserts; ( B,C,E,F ) Experimental wells with red fluorescent SK-BR-3 cells seeded in the upper inserts and HBEC-5i in the bottom wells; (G,J) Control wells green fluorescent SK-OV-3 cells seeded in the upper inserts; ( H,I,K,L ) Experimental wells with green fluorescent SK-OV-3 cells seeded in the upper inserts and HBEC-5i in the bottom wells. The images were taken from the bottom wells after 5 days incubation.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Control, Incubation

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel: HBEC-5i/SK-OV-3 (turbo GFP/green) model; (A) Green fluorescent SK-OV-3 cells (FITC filter); (B) Dead cells (red) visualized by PI staining (TRITC filter); (C) Merged image of transmitted, FITC- and TRITC-filtered images of co-cultured cells. Bottom panel: HBEC-5i/MDA-MB-231 (turbo FP602/red) model; (D) Red fluorescent MDA-MB-231 cells (TRITC filter); (E) Dead cells (green) visualized by staining with EasyProbe dye (FITC filter); (F) Merged image of transmitted, TRITC- and FITC-filtered images of co-cultured cells. Images were acquired after 28 days of co-culture.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Staining, Cell Culture, Co-Culture Assay

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : monoculture controls of HBEC-5i and SK-BR-3 cells treated with Lapatinib (10 µM); (A,B) Dead cells (green) in HBEC-5i monocultures; (C,D) Dead cells (yellow) in red fluorescent SK-BR-3 monocultures. Bottom panel : Co-culture HBEC-5i/SK-BR-3 (turbo FP602/red) cell model treated with Lapatinib (1 and 10 µM). (E-H) Dead cells (yellow) in the endothelial/cancer co-culture system. EasyProbe green dye (FITC filter) was used to visualize the dead cells in all cultures.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Co-Culture Assay

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A , Experimental timeline for inflammatory insult with TNF-α and FGF2 co-treatment. Once confluent (4 days), HBEC-5i or bEnd.3 were pretreated with FGF2 or vehicle for one hour and then stimulated with TNF-α or vehicle for up to 7 days. FGF2 alters mouse ( B ) and human ( C ) endothelial transcription in response to acute TNF-α stimulation. FGF2 promotes faster restoration of TNF-α-induced Cldn5 loss in mouse ( D ) and human ( E ) endothelial cells. Chronic stimulation with TNF-α leads to a reduction in endothelial monolayer integrity measured by trans-endothelial electrical resistance (TEER) in mouse ( F ) and human ( G ) endothelial cells. FGF2 co-treatment preserves normal TEER despite TNF-α. H 7 days of TNF-α treatment promotes spikes and discontinuities in Cldn5 tight junction strands in bEnd.3, which is reversed by FGF2 treatment (scalebar = 20 μm). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with two-way ANOVA followed by Bonferroni’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques:

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A 1 h pretreatment with Fgf2 increases serine-9 phosphorylation of GSK3β in HBEC-5i when compared to no treatment (CTRL 0 h). TNF-α treatment induces rapid, transient dephosphorylation of GSK3β, but this effect is not reversed by Fgf2 coadministration. Each dot represents a replicate ( n = 3). B 1 h Fgf2 pretreatment diminishes basal β-catenin phosphorylation when compared to no treatment (CTRL 0 h). Further, while TNF-α induces a rapid reduction in phosphorylated β-catenin, Fgf2 reverses this dynamic upon inflammatory activation ( n = 3). C In health control endothelial cells (top), β-catenin interacts with VE-Cadherin at the cell membrane, and this complex inhibits Cldn5 transcriptional suppression by FOXO1. Excess cytosolic β-catenin is phosphorylated by GSK3β, targeting it for degradation. When stimulated with TNFα, unbound β-catenin complexes with FOXO1, leading to suppression of Cldn5 expression (bottom, red arrow), while a small amount is targeted for degradation. Meanwhile, when FGF2 is co-administered with TNF-α (bottom, blue arrow), our results suggest that unbound β-catenin is strongly redirected toward GSK3β-mediated phosphorylation. D 30 min of TNF-α is sufficient to induce β-catenin distribution at tight junctions ( n = 4 replicates) with representative images on the right ( E ) (scalebar = 20 μm). F Fgf2 attenuates TNF-α-induced reductions in the wound healing capacity of HBEC-5i ( n = 4 replicates) (**** p < 0.0001). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with one or two-way ANOVA followed by Bonferroni’s post hoc tests or two-tailed t-tests with Welch’s correction when appropriate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control, Membrane, Expressing, Two Tailed Test

Journal: Brain

Article Title: Misdirected yet intact TREX1 exonuclease activity causes human cerebral and systemic small vessel disease

doi: 10.1093/brain/awaf085

Figure Lengend Snippet: Endothelial disease is an important component of the microvascular disease observed in RVCL-S. ( A ) Electron micrograph of microvessel (renal) showing endotheliopathic features such as loss of endothelial cell fenestration (green arrow) and subendothelial lucency (blue arrow). Scale bar = 5 μm. ( B ) For comparison, electron microscopy of normal endothelial cell with fenestrations (red arrows). ( C ) PRISMA pipeline for systematic literature review of RVCL-S pathology. Created in BioRender. McGlasson, S. (2025) https://BioRender.com/x56u663 . ( D ) Schematic of TREX1-dsRed transgenic reporter. These mice express TREX1 and dsRed under the control of the endogenous TREX1 promoter. 8 Created in BioRender. McGlasson, S. (2025) https://BioRender.com/f91o214 . ( E ) Flow cytometry histogram overlaying WT CD31 + cells and TREX1-dsRed CD31 + cells. ( F ) Quantification of median fluorescent intensity (MFI) of dsRed from independent samples ( n = 4 per condition). Data-points show individual experiments, columns show mean with standard error of the mean (SEM). P = 0.0079, unpaired t -test. ( G ) Representative flow cytometry plots of the frequency of vascular endothelial cells (VECs, CD31 + popoplanin − ) and lymphatic endothelial cells (LECs, CD31 + popoplanin + ) in the spleen of Tie2-Cre LSL hTREX1 WT or Tie2-Cre LSL hTREX1 V235Gfs mice. ( H ) Quantification of the frequency and number of VECs from ( H ). Points indicate data from individual mice ( n = 3 per group). Columns show mean. Error bars show SEM. ( I ) Quantification of the frequency and number of LECs from ( H ). Points indicate data from individual mice ( n = 3 per group). Columns show mean. Error bars show SEM. ( J ) Representative flow cytometry plots showing the frequency of hTREX1 + vascular endothelial cells via staining the HA tag. ( K ) Quantification of the frequency of hTREX1 + VECs from ( J ). Points indicate data from individual mice ( n = 3 or 4 per group). Columns show mean. Error bars show SEM. P = 0.0167 by unpaired t -test. RVCL-S = retinal vasculopathy with cerebral leukoencephalopathy with systemic manifestations; PRISMA = Preferred Reporting Items for Systematic reviews and Meta-Analyses; WT = wild-type.

Article Snippet: Human brain endothelial cells (hBEC-5i, ATCC-CRL-3245) were maintained in DMEM/F12 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine solution (Gibco) and 40 μg/ml endothelial cell growth supplement (Millipore) and cultured in 5% CO 2 and normoxic conditions at 37°C. hBEC-5i endothelial cells were grown on vessels that had been coated with 0.1% gelatin for at least 1 h in a 37°C incubator.

Techniques: Comparison, Electron Microscopy, Transgenic Assay, Control, Flow Cytometry, Staining

Journal: Brain

Article Title: Misdirected yet intact TREX1 exonuclease activity causes human cerebral and systemic small vessel disease

doi: 10.1093/brain/awaf085

Figure Lengend Snippet: Human endothelial cells with RVCL-S patient mutations show increased DNA damage, cell cycle defects and increased chromosomal abnormalities . ( A ) Human brain endothelial cells were transformed using lentivirus to express TREX1 with a patient mutation. The MRI brain scan from a patient with this mutation is shown. Created in BioRender. McGlasson, S. (2025) https://BioRender.com/l89e736 . ( B ) Lentiviral expression of EGFP-TREX1 in human brain endothelial cells (hBEC-5i), co-stained with 4′,6-diamidino-2-phenylindole and 53BP1. Illustrative image taken by confocal microscope at ×20 magnification, images shown with digital zoom. ( C ) Quantification of 53BP1 foci in response to lentiviral expression of EGFP-TREX1 WT , EGFP-TREX1 V235fs or an empty EGFP vector. Data-points show three independent experiments and columns show mean. Error bars are standard error of the mean (SEM), P = 0.0315, unpaired t -test. ( D ) Representative image of chromatin bridge seen in human brain endothelial cells expressing EGFP-TREX1 V235fs . Confocal images taken at ×63 magnification. Quantification of percentage of cells with chromatin bridges. Data-points show three independent experiments and columns show mean. Error bars are SEM, P < 0.0001, unpaired t -test. ( F ) Quantification of cells in G2/M in response to lentiviral expression of EGFP-TREX1 V235fs , EGFP-TREX1 WT or an empty EGFP vector. Data-points show three independent experiments and columns show mean. Error bars are SEM, P = 0.0248, unpaired t -test. Clonal expression of EGFP-TREX1 V235 in hBECs. Top row : ×20 magnification; bottom row : ×63 magnification. Line drawn to indicate GFP + versus GFP − cell clones. ( H ) Quantification of the correlation (Pearson's r ) between corrected total cell fluorescence (CTCF) of EGFP and 53BP1 foci. n = 2 per group, with at least 30 cells counted per experimental group (for full breakdown see and ). Columns show mean. Error bars show standard deviation. P = 0.0117, unpaired t -test. ( I ) Quantification of the correlation (Pearson's r ) between nuclear EGFP-TREX1 and 53BP1 foci. n = 3 WT, n = 4 V235. Points show independent experiments with mean (for full breakdown see and ). Error bars show SEM. P = 0.0158, unpaired t -test. RVCL-S = retinal vasculopathy with cerebral leukoencephalopathy with systemic manifestations; WT = wild-type.

Article Snippet: Human brain endothelial cells (hBEC-5i, ATCC-CRL-3245) were maintained in DMEM/F12 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine solution (Gibco) and 40 μg/ml endothelial cell growth supplement (Millipore) and cultured in 5% CO 2 and normoxic conditions at 37°C. hBEC-5i endothelial cells were grown on vessels that had been coated with 0.1% gelatin for at least 1 h in a 37°C incubator.

Techniques: Transformation Assay, Mutagenesis, Expressing, Staining, Microscopy, Plasmid Preparation, Clone Assay, Fluorescence, Standard Deviation