MenSC and umbilical cord (UC) MSC were isolated from menstrual fluid of the umbilical cord of three different healthy donors each and characterized according to ISCT31 (link) criteria as we have previously described15 (link),22 (link),32 . Human dermal fibroblasts were purchased from Lonza (cat. CC-2511, Lonza, Walkersville, MD USA). Human umbilical cord vein endothelial cells (HUVEC) and human microvascular endothelial cell line (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVEC and fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (15-018-CV, Corning, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Lonza, Walkersville, MD USA), 1% Penicillin/Streptomycin (P/S) (Life Technologies, Carlsbad, CA, USA) and 1 mM L-glutamine (Life Technologies). HMEC-1 cells were cultured in endothelial cells growth media 2 (EGM-2) (cc4147, Lonza). All cells were regularly tested for mycoplasma contamination using a mycoplasma detection kit (G238, Applied Biological Materials Inc., Richmond, BC, Canada). All cells were cultured in humidified incubation chambers at 37 °C with 5% CO2.
Human umbilical vein endothelial cells (huvec)
Manufactured by Corning
Sourced in United States
HUVEC is a primary human cell line derived from human umbilical vein endothelial cells. The cells are used in cell culture applications to study endothelial cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Human umbilical vein endothelial cells (huvec), by PromoCell (4 mentions)
Mda mb 157, by American Type Culture Collection (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (2 mentions)
Hmec 1, by Lonza (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Egm 2, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Hmec 1, by American Type Culture Collection (1 mentions)
Cellbind, by Corning (1 mentions)
Apotome microscope, by Zeiss (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Mda mb 468, by Corning (1 mentions)
Sk br 3, by Thermo Fisher Scientific (1 mentions)
Mda mb 436, by Corning (1 mentions)
Mccoy 5a, by Thermo Fisher Scientific (1 mentions)
Hcc1806, by Corning (1 mentions)
12 protocols using human umbilical vein endothelial cells (huvec)
1
Isolation and Characterization of Mesenchymal Stem Cells
2
Culturing Primary Human Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany) and cultured in a complete endothelial cell growth medium (designated as complete EGM) containing 2% FBS and growth factors supplied by the company’s (cat # C-22010 ready to use kit). HUVEC (passage 2–4) were cultured and maintained on 10 cm culture plates (Cell-bind, Corning) until confluent and grew under standard cell culture conditions (37 °C, 5% CO2). Cells were split into the desired plate one day before the experiments. For Seahorse experiments, cells are attached for at least 24 h before experiments.
3
Angiogenic Potential of 3D Tumor Spheres
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The effect of soluble factors secreted by Luc vs. CD44 kd 3D tumor spheres under normoxic vs. hypoxic conditions on the angiogenic behavior of EC (human umbilical vein ECs, HUVEC; PromoCell) was measured by a tube formation assay. For this purpose, 80 μL of Matrigel® Growth Factor Reduced (Corning) was applied to a 96‐well plate, and 1.5 × 104 HUVEC were added in 100 μL of CM from HT‐29 Luc or CD44 kd tumor spheres grown in Matrigel/HA (see above) and cultivated for 4 h in the presence of 10% FCS. Images were acquired using the Zeiss ApoTome microscope and a 10x A‐Plan Ph1 objective (NA: 0.25, WD (mm): 4.5). Tube formation was assessed by measuring the total tube area per well [μm2] with Fiji imagej software.
4
Neutrophil Transmigration Assay with ANCA
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Migration was tested either in fibronectin-coated transwells or across HUVEC or gMVEC monolayers, (3.0 μm, 6.5 mm from Corning, NY, USA). Neutrophils (1.5 × 106) in HBSS++were added to the upper well and stimulated at 37 °C with HBSS or 10-8 M fMLF (Sigma) to transmigrate to the lower well. In indicated experiments 100 μg/ml ANCA or control IgGs were also added to the upper well. After 90 min (for FN coating) or 3 hrs (for EC monolayers) transmigrated cells were quantified by MPO assay using a standard curve as described19 (link).
5
Cell Culture Conditions for Cancer Cell Lines
6
Cultivating HUVEC for CDDO-Im Transcriptomics
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Stock cultures of gender-mixed HUVEC (Lifeline Cell Technology, Walkersville, MD, USA) pooled from 10 different donors were cultivated on T75 flasks (Corning Inc., Corning, NY, USA) in MCDB 131 medium at 37°C in a humidified atmosphere of 92% nitrogen, 3% oxygen, and 5% CO2 with medium changes every 2 days until confluence is reached.18 (link),19 (link) MCDB 131 medium, trypsin/EDTA, and antibiotic/antimycotic were obtained from Life Technologies (Carlsbad, CA, USA). Endothelial supplements were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Prior to an experiment, HUVEC were subcultivated with trypsin/EDTA onto Costar 96-well or 12-well multiplates (Corning Inc.) at 5000 cells/cm2, grown to confluence, and kept for 72 hours to produce a quiescent cell layer. Only the second through fifth population doublings of cells were used as described in a study by Wang et al.20 (link) The derivative CDDO-Im was diluted (1000-fold) after dissolving in DMSO (0.1%) with medium to a final concentration of 200 nM. Human umbilical vein endothelial cells were grown to confluence at which time they were pretreated with CDDO-Im for their respective incubation times (0.5, 1, 3, 6, and 24 hours). Gene expression profiling of CDDO-Im–treated HUVEC was compared with vehicle control (0.1% DMSO).
7
HUVEC Angiogenesis Assay with STS
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Human umbilical vein endothelial cells (HUVEC) sourced from Promocell (Heidelberg, Germany) were routinely cultured in complete growth media (EGM-2) (Promocell, Heidelberg, Germany). For angiogenic assays, HUVEC were plated at a density of 1.0 × 104 cells/well in 96-well plates previously coated with reduced growth factor Matrigel (Corning, NY, USA) and let attach for 1 h. Following, media was replaced with CoCl2 (400 μM) and either a non-formulated or liposome formulated STS dissolved in DMEM containing 0.5% FBS. The formation of capillary-like structures was assessed after 6 h. Images captured per well at 4× magnification, using a Nikon Eclipse Ti-E phase-contrast inverted microscope (Nikon Instruments Inc., Melville, NY, USA) and total branching length analysed using Image J Angiogenesis Analyzer tool from three independent experiments performed in duplicate.
8
Culturing Endothelial Cell Lines
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HUVECs (ScienCell, USA), hCMEC/D3 (Cedarlane, Canada), hBMEC (Cell Systems, USA), and hDMEC (Promocell, Germany) were cultured in endothelial cell medium (ScienCell, USA) containing 10% fetal bovine serum (FBS), 1% penicillin‐streptomycin (P/S), and 1% endothelial cell growth supplement (ECGS). Frozen stocks were prepared for all cell types after expanding cells in vendor‐recommended media. Frozen stocks of the following passages were thawed and cultured in T75 culture flasks (Corning, USA), where Passage 1 for all cell type indicates the initial stock received from the vendor; HUVEC: Passage 4, hCMEC/D3: Passage 5–7, hBMEC: Passage 6, hDMEC: Passage 3, 2–3 d prior to cell seeding. All cells were cultured in a humidified 37 °C incubator with 5% CO2.
9
3D Printed Microgel Discs for Angiogenesis
10
Osteogenic Co-culture of Endothelial and Mesenchymal Cells
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HUVECs (Cyagen Biosciences Inc., CA, USA) were cultured in Endothelial Growth Medium-2 (EGM-2; Lonza, MA, USA). Before their integration in a 3D co-culture, sGMSCs and HUVECs were collected by trypsin. The osteogenic induction medium in the GS and GHS systems is composed of a combination of EGM-2 and DMEM osteogenic media tailored to match the cell proportions. The induction medium was renewed every 48 h to maintain optimal nutrition for the cellular spheroids.
As a control, sGMSCs and HUVECs were respectively cultured in ultra-low attachment 6-well plates (Corning, NY, USA) at a density of 1×106 cells per well to assemble GS and HUVEC Spheroids (HS). To form GHS with varying ratios, single-cell suspensions of sGMSCs and HUVECs were arranged at a total cellular density of 1×106 cells per well, with GHR of 5:1, 4:1, 3:1, 2:1, and 1:1. These mixed single-cell suspensions were then propagated in ultra-low attachment 6-well plates and subjected to unbroken osteogenic induction for 7–14 days. The morphology of the generated GS and GHS were inspected under a microscope 12–48 h post-seeding, and their diameters were quantified using ImageJ (n = 3).
As a control, sGMSCs and HUVECs were respectively cultured in ultra-low attachment 6-well plates (Corning, NY, USA) at a density of 1×106 cells per well to assemble GS and HUVEC Spheroids (HS). To form GHS with varying ratios, single-cell suspensions of sGMSCs and HUVECs were arranged at a total cellular density of 1×106 cells per well, with GHR of 5:1, 4:1, 3:1, 2:1, and 1:1. These mixed single-cell suspensions were then propagated in ultra-low attachment 6-well plates and subjected to unbroken osteogenic induction for 7–14 days. The morphology of the generated GS and GHS were inspected under a microscope 12–48 h post-seeding, and their diameters were quantified using ImageJ (n = 3).
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