Primary Human Brain Microvascular Endothelial Cells (ACBRI 376) - Cell Systems
Primary Human Brain Microvascular Endothelial Cells (ACBRI 376) were initiated by elutriation of dispase-dissociated normal human brain cortex tissue.
Primary Human Brain Microvascular Endothelial Cells can provide a useful tool for adherence, transport and permeability studies of the blood-brain-barrier (BBB). ACBRI 376 cells demonstrate particular markers of differentiation (interdigitated cell contact, desmosomes, Z0-1 protein epitopes) similar to those observed in vivo. The morphological and immunofluorescent markers detailed for ACBRI 376 are suggestive of a differentiated cell monolayer that can be used to study properties of the human BBB.
ACBRI 376 cells 3 days after plating, plated with Attachment Factor™, cultured in Cell Systems Complete Medium Kit With Serum and CultureBoost-R™ (4Z0-500-R).
ACBRI 376 cells express endothelial cell markers including CD31 and von Willebrand Factor. They also take up DiI-Ac-LDL in an endothelial cell functional assay.
ACBRI 376 cells express ZO-1 indicating the formation of tight junctions.
These cells were originated using Cell Systems Complete Serum-Free Medium (SF-4Z0-500), and subsequently grown and passaged in Cell Systems Complete Medium (4Z0-500). They are available at Passage 3 [< 12 cumulative population doublings] cryopreserved in Cell Systems Cell Freezing Medium™ (4Z0-705). This vial will initiate a Passage 4 cell culture in a 75cm2 flask.
In addition to cryopreserved vials, these cells also are available in 25cm2 and 75cm2 proliferating cell culture flasks (US domestic market only).
Each vial of cells is shipped with vials of Bac-Off® (antibiotic) and CultureBoost™ (animal derived growth factors) or CultureBoost-R™ (human recombinant growth factors) at no additional cost.
These cells are qualified for use with:
HIV Serologic Test (donor level HIV AB EIA)
HIV PCR Test (frozen cell pool by CLIA Licensed Clinical Lab)
Hepatitis B (HBV) and Hepatitis C (HCV) PCR Test (at frozen cell level)
|Test of frozen cells for Mycoplasma spp. (ATCC method by CLIA Licensed Clinical Lab)||Negative|
Cell Markers and Functional Tests
|CD31||> 98% positive by immunofluorescence|
VWF / Factor VIII
> 95% positive by immunofluorescence
Uptake of Di-I-Ac-LDL
> 95% positive by immunofluorescence
Characterization in Peer-Reviewed Literature
Reprinted with permission from Smits and Mir et al. PLoS ONE (2011) 6:e16282. Figure 5. Inhibition of EZH2 reduces endothelial tubule formation in vitro. (A) HBMVECs (ACBRI 376) were cultured on Matrigel coated plates in EBM, EGM, or EBM supplemented with U87-GFP cells. Scale bar = 450 µm. Inhibition of EZH2 in HBMVECs, either by transfection with pre-miR-101, EZH2 siRNA, or treatment with DZNep significantly reduced tubule formation as compared to control cells. Tubule formation was assessed as tubule length and branching. (B) Quantitation of tubule length and branching using ImageJ software. (n = 3) Error bars indicate s.d. *p<0.05, ***p<0.001, t test.
Reprinted with permission from Smits and Mir et al. PLoS ONE (2011) 6:e16282. Figure 6. Inhibition of EZH2 reduces endothelial migration and invasion in vitro. (A) HBMVEC (ACBRI 376) monolayer cultures were scratched. Images were acquired directly after scratching (t = 0) and 24 h later (t = 24). The migration front is indicated by the dashed lines. Scale bar = 450 µm. Inhibition of EZH2 in HBMVECs, either by transfection with pre-miR-101, EZH2 siRNA or treatment with DZNep significantly reduced migration as compared to control. (B) Quantitation of endothelial cell migration into the scratched area using ImageJ software. (C and D) HBMVECs were transfected with pre-miR-101, EZH2 siRNA, non-related control molecules, or treated with DZNep, incubated on a Transwell system and subsequently analyzed for invasion capability. EZH2 inhibition, either through pre-miR-101, EZH2 siRNA or DZNep, significantly decreased invasion as shown by Hoechst staining. Scale bar = 225 µm. (n = 3) Error bars indicate s.d. *p<0.05, ***p<0.001, t test.
Reprinted with permission from Niego et al. PLoS ONE (2017) 12: e0177332.
Figure 5. Only KD025, but not fasudil, protects brain endothelial cells (ACBRI 376) from rt-PA and plasmin. (A) Small molecule KD025 concentration-dependently blocks permeability changes in primary human brain microvascular endothelial cells (ACBRI 376 BECs; cultured without astrocytes) treated for 6 h with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (2 and 20μM). n = 4. (B) Comparison of selective ROCK-2 inhibition by KD025 (20μM) versus non-selective ROCK inhibition by fasudil (HA1077; 2, 20 and 100μM) against rt-PA+plasminogen (25nM+100nM, respectively) in BECs, as assessed 6 h post stimulation. HA1077 displays no protective capacity in BECs. n = 3. Bars represent mean±SEM. **P<0.01 compared to all other groups, ***P<0.001 compared to t-PA+Plgn by one-way ANOVA with Tukey’s post hoc analysis. ## P<0.01 compared to DMSO control and P = 0.06 by two-tailed paired t-test. (C) Representative phase-contrast micrographs (top panels) and double immunofluorescence images of zonula occludens 1 (ZO-1, representing tight junctions (TJs); white) and nuclei (Hoechst; red) (bottom panels) of ACBRI 376 BECs stimulated for 6 h with DMSO (control) or with rt-PA+plasminogen (25nM+100nM, respectively), in the presence or absence of KD025 (20μM). KD025 attenuates morphological changes (arrows) and preserves TJs in endothelial cells. Asterisks depict areas with degraded TJs. Scale bars = 200μm.
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