Brain-derived endothelial (bEND.3) cells were provided by the Bioresource Collection and Research Center (Hsinchu, Taiwan). These cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Human cerebral microvascular endothelial (hCMEC/D3) cells were purchased from Merck Millipore (Burlington, MA, USA) and maintained in EndoGRO-MV Complete Media Kit (Merck Millipore) supplemented with 1 ng/mL fibroblast growth factor (FGF)-2 (Merck Millipore). All cells were cultured under standard conditions (i.e., 5% CO2 in air in a humidified environment at 37 °C) for 7 days until the cell–cell junctions had been well established before being subjected to subsequent experiments.
Hcmec d3 cells
Manufactured by Merck Group
Sourced in United States, Germany
HCMEC/D3 cells are a human brain-derived cell line that exhibits characteristics of brain microvascular endothelial cells. These cells can be used for in vitro studies related to the blood-brain barrier.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast growth factor 2 (fgf2), by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Ebm 2 medium, by Lonza (2 mentions)
Endogro mv complete culture media, by Merck Group (2 mentions)
Endogro mv complete media kit, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Dmem culture media, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Pmbmecs, by Cell Biologics (1 mentions)
Huvecs, by Lonza (1 mentions)
Egm 2 endothelial cell growth medium 2 bulletkit, by Lonza (1 mentions)
Hcmec d3 cells, by Lonza (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
6 well plate, by Sarstedt (1 mentions)
Stemflex medium, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen 1, by Thermo Fisher Scientific (1 mentions)
27 protocols using hcmec d3 cells
1
Culturing Brain Endothelial Cells
2
Visualizing Vesicle Uptake in Endothelial Cells
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hCMEC/D3 cells (Merck) were grown on pre-coated coverslips in a 12-well plate until 70% confluent. The medium was removed and the cells were stained by WGA640 (Biotum, 20ug/mL, diluted in HBSS) for 10 minutes and washed three times in HBSS. 0.5 mL pre-stained vesicles, or buffer alone, were added to each well and incubated with the cells for 20 minutes. The coverslip was washed twice with 4% paraformaldehyde (PFA) and left to dry in order to fix the cells. 8 μl mounting medium (Vectashield, Antifade Mounting Medium with DAPI) was added to the microscope slide and the coverslip was placed cell side down on the slide. Mounted slides were stored in the dark at 4°C until use.
Images were acquired using an inverted Nikon A1R confocal laser scanning microscope with a 60X Plan-Apo/1.20 NA oil objective. Excitations used laser lines at 408 nm, 488 nm, or 561 nm, and images were recorded at 425/475 nm, 500/550 nm, or 570/620 nm, respectively. Laser intensities/detection settings were kept constant between parallel images to enable sample comparisons. NIS-Elements imaging software 4.0 (Nikon, Japan) was used for image capture. Stacks of images were acquired with a 0.5 μm confocal slice. The slices view was used to display orthogonal XY, XZ and ZY projections of the image sequence.
Images were acquired using an inverted Nikon A1R confocal laser scanning microscope with a 60X Plan-Apo/1.20 NA oil objective. Excitations used laser lines at 408 nm, 488 nm, or 561 nm, and images were recorded at 425/475 nm, 500/550 nm, or 570/620 nm, respectively. Laser intensities/detection settings were kept constant between parallel images to enable sample comparisons. NIS-Elements imaging software 4.0 (Nikon, Japan) was used for image capture. Stacks of images were acquired with a 0.5 μm confocal slice. The slices view was used to display orthogonal XY, XZ and ZY projections of the image sequence.
3
Cell Culture Protocols for Cancer and Blood-Brain Barrier Studies
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All cells were cultured in T75 tissue culture (TC) flasks (Sarstedt #83.3911.002) and 15 cm TC dishes (Sarstedt #83.3903) at 37 °C, 5% CO2, 100% humidity. MDA-MB231 (ATCC #HTB-26) and MDA-MB468 (ATCC #HTB-132) cells required DMEM culture media (Gibco, #41965–035) containing constantly 10% fetal calf serum (FCS) (Gibco), penicillin-streptomycin (100 U/ml) (Gibco) and puromycin (2 μg/ml) (Gibco).
hCMEC/D3 cells (Merck, #SCC066) were grown in EBM-2 medium (Lonza #CC-3156) containing, 5% FCS (Gibco), 1x chemically defined lipid concentrate (Gibco #11905–031), 5 μg/ml ascorbic acid (Sigma #A4544) and 1.4 μM hydrocortisone (Sigma # H0888).
hCMEC/D3 cells (Merck, #SCC066) were grown in EBM-2 medium (Lonza #CC-3156) containing, 5% FCS (Gibco), 1x chemically defined lipid concentrate (Gibco #11905–031), 5 μg/ml ascorbic acid (Sigma #A4544) and 1.4 μM hydrocortisone (Sigma # H0888).
4
Culturing hCMEC/D3 Cells on Collagen
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A T75 flask was coated with collagen (10 ng/cm2) and incubated at 37°C in a 5% CO2 humidified incubator for 1 hour prior to use. Endo-GRO Basal medium was prepared according to the manufacturers instructions (Merck Millipore, SCME004) by addition of 10 mM L-Glutamine, 1 μg/mL Hydrocortisone Hemisuccinate, 0.75 U/mL Heparin Sulfate, 50 μg/mL Ascorbic acid, 5% FBS, 0.2% 5 ng/mL Endo-GRO-LS Supplement, 5 ng/mL Rh EGF and 1 ng/mL FGF-2). A vial of hCMEC/D3 cells (Merck) was thawed in a 37°C water bath, transferred to a sterile 15 mL conical tube followed by a drop-wise addition of Endo-GRO complete medium. Cells were centrifuged at 300 xg for 3 min at RT and the pellet was resuspended in 10 ml of pre-warmed hCMEC/D3 complete medium. Finally the cells were plated on the pre-coated T75 flask and incubated at 37°C in a 5% CO2 humidified incubator until 80% confluence was reached.
5
Culturing Primary Mouse and Human Brain Cells
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Primary mouse brain microvascular endothelial cells (pMBMECs), pericytes and astrocytes originated from CD-1 mice. The pMBMECs (CD-1023) and astrocytes (CD-1285) were obtained from CellBiologics (Chicago, IL, USA). The pMBMECs were maintained in complete mouse endothelial cell medium from the CellBiologics W Kit (M1168), whereas astrocytes were maintained in phenol-free astrocyte medium–animal (1831-prf) from ScienCell (Carlsbad, CA, USA). Mouse brain vascular pericytes (ScienCell; M1200) were maintained in phenol-free pericyte medium–mouse (ScienCell; 1231-prf). All cells were maintained up to passage 4.
Primary human brain cortical pericytes (ScienCell; 1200) were maintained in phenol-free pericytes medium (ScienCell; 1201-prf), whereas primary human brain cortical astrocytes (ScienCell; 1800) were maintained in phenol-free astrocyte medium (ScienCell, 1801-prf). Informed consent agreements were obtained from all donors according to Sciencell policies. Brain microvascular endothelial cells, hCMEC/D3 cells [39 (link)] (Merck, Darmstadt, Germany; SCC066) were cultured in EGM-2 endothelial cell growth medium-2 from the BulletKit (Lonza, Basel, Switzerland; CC-3162). Pericytes and astrocytes were used for experiments up to passage 4, whereas hCMEC/D3 cells were used up to passage 40.
Primary human brain cortical pericytes (ScienCell; 1200) were maintained in phenol-free pericytes medium (ScienCell; 1201-prf), whereas primary human brain cortical astrocytes (ScienCell; 1800) were maintained in phenol-free astrocyte medium (ScienCell, 1801-prf). Informed consent agreements were obtained from all donors according to Sciencell policies. Brain microvascular endothelial cells, hCMEC/D3 cells [39 (link)] (Merck, Darmstadt, Germany; SCC066) were cultured in EGM-2 endothelial cell growth medium-2 from the BulletKit (Lonza, Basel, Switzerland; CC-3162). Pericytes and astrocytes were used for experiments up to passage 4, whereas hCMEC/D3 cells were used up to passage 40.
6
Culturing HUVECs and hCMEC/D3 Cells
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Pooled human umbilical vein endothelial cells (HUVECs) and a human cerebral microvascular endothelial cell line (hCMEC/D3) were cultured in EGM™-2 MV consisting of EBM™-2 and EGM™-2 MV SingleQuots™ Kit with 2.4% FBS, and 1% penicillin and streptomycin. HUVECs and cell culture reagents were purchased from Lonza (Walkersville, MD, United States). hCMEC/D3 cells were purchased from EMD Millipore (Temecula, CA, USA). HUVECs and hCMEC/D3 cells were detached by TrypLE and seeded on samples at a density of 2 × 104 cells/cm2. Cells were used between passages 4 − 6.
7
Culturing hCMEC/D3 Brain Endothelial Cells
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Human brain capillary endothelial hCMEC/D3 cells were obtained from Merck (Darmstadt, Germany). hCMEC/D3 cells were seeded in the collagen-I coated flasks and cultured in EGM-2 Endothelial Cell Growth Medium-2 Bullet Kit (Lonza, Verviers, Belgium) containing growth factors and 5% heat-inactivated fetal bovine serum (AusGene X, Brisbane, Australia) at 37 °C in 5% CO2.
8
Human iPSC and Meningioma Cell Culture Protocols
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Human induced pluripotent stem cell (iPSC) line IMR90-4 (WiCell) was maintained on Matrigel (Corning) coated 6-well plates (Sarstedt) in StemFlex medium (Gibco), passaging twice a week and changing medium daily as previously described [33 (link), 44 (link)–46 , 48 (link)]. Meningioma cells of the meningothelial histological subtype were derived from tumor biopsies and characterized as previously described [36 (link)]. Meningioma cells were grown in DMEM + GlutaMAX (Gibco) with 10% FCS (Gibco) and 1% Pen/Strep (Gibco) and passaged twice a week with a seeding density of 4 × 103 cells/cm2. The cells were expanded and used for experiments up to passage 10 or until showing signs of senescence. hCMEC/D3 cells (Sigma, SCC066) were cultured in EndoGRO-MV Complete Culture Media (Sigma) splitting as needed. hCMEC/D3 and meningioma cells were grown on 30 µg/ml collagen 1 (rat tail, Gibco) coated tissue culture flasks.
9
In Vitro Drug Transport Model Validation
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The six drugs used for constructing the mathematical model (amitriptyline, caffeine, carbamazepine, fleroxacin, pefloxacin and zolpidem) and HPLC-grade solvents (acetonitrile, methanol and water) were purchased from Sigma-Aldrich (Barcelona, Spain). The MDCK cell line was purchased from ATCC (USA), MDCK-MDR1 cells were provided by Dr. Gottessman, and the MM (National Institutes of Health, Bethesda, MD, USA) and hCMEC/D3 cell lines were purchased from Cedarlane (Burlington, ON, Canada). Pig brain homogenate was kindly supplied by a local slaughterhouse.
Dulbecco’s modified eagle’s medium (DMEM) with a high content of glucose, L-glutamine, HEPES, MEM non-Essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich (Barcelona, Spain).
The products needed for the culture of hCMEC/D3 cells were purchased from Sigma-Aldrich (Barcelona, Spain) (hydrocortisone, ascorbic acid, HEPES, Triton X-100 and bFGF), Gibco (Barcelona, Spain) (FBS, penicillin–streptomycin, chemically defined lipid concentrate, HBSS, collagen I rat protein and trypsin-EDTA), Lona (Barcelona, Spain) (EBM-2 medium).
Dulbecco’s modified eagle’s medium (DMEM) with a high content of glucose, L-glutamine, HEPES, MEM non-Essential aminoacid, penicillin−streptomycin, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and fetal bovine serum (FBS) for the cell culture of MDCK and MDCK-MDR1 cell lines were purchased from Sigma-Aldrich (Barcelona, Spain).
The products needed for the culture of hCMEC/D3 cells were purchased from Sigma-Aldrich (Barcelona, Spain) (hydrocortisone, ascorbic acid, HEPES, Triton X-100 and bFGF), Gibco (Barcelona, Spain) (FBS, penicillin–streptomycin, chemically defined lipid concentrate, HBSS, collagen I rat protein and trypsin-EDTA), Lona (Barcelona, Spain) (EBM-2 medium).
10
In Vitro Blood-Brain Barrier Model
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The in vitro BBB models were prepared according to the method previously reported.[10 ] Briefly, the insert of 24‐Trans‐well plate (Corning, Corning, NY) was coated with Collagen I (Corning) for 1 h so that endothelial cells could attach to the surface. Afterward, the insert cultured with 10 000 cells of hCMEC/D3 cells (Sigma‐Aldrich) in 100 µL of a EndoGRO‐MV complete culture media (Sigma‐Aldrich) was placed in the Trans‐well plate containing 500 µL of the same culture media in the bottom well. Once the cells reached to 90% of confluency (after ≈2 days), they were serum‐starved for 3 days by incubation in a serum‐free EndoGRO‐MV complete culture media to enhance the tight junction formation between adjacent endothelial cells and prevent multilayered paracellular barriers on the surface of Trans‐well insert. The BBB models were accepted if the effective permeability coefficient of a fluorescent‐labeled 40 kDa dextran (Thermo Fisher Scientific) was less than ≈1.0 × 10−5 cm s−1, which validated the reasonable integrity of BBB tight junctions. Details were described in the “Assessment of BBB Permeability” subsection.
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