TT and CC isogenic endothelial cell lines were plated in 96 well plates at 3,000 cells/well in EGM-2 MV medium (Lonza). After 8 h, cells were quiesced overnight in EBM-2 medium (Lonza) with 1% FBS (Omega Scientific) in addition to hydrocortisone, and ascorbic acid (Lonza) at the same concentration as EGM-2 MV medium. For experiments with sorafenib, media was removed and replaced with EGM-2 MV containing sorafenib 0–10 µM in DMSO (Biotang, Inc.) after 24 h. Untreated cells contained DMSO alone at 1:1000. After 72 h of proliferation at 37 oC 5% CO2, and 95% humidity, viable cells were stained using Cyquant Direct Cell Proliferation Assay (Life Technologies). Images were acquired using the EVOS FLc microscope (Life Technologies) using a 10x objective and counts were accomplished with FIJI particle analyzer. A two-tailed, unpaired t-test was used to analyze differences in cell proliferation and cytotoxicity between TT and CC cells.
Egm 2mv medium
Manufactured by Lonza
Sourced in Switzerland, United States, United Kingdom, France, Belgium
EGM-2MV medium is a cell culture medium used for the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors to support the in vitro expansion of endothelial cells.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Huvecs, by Lonza (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Hmvec l, by Lonza (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Rat tail collagen type 1, by Merck Group (4 mentions)
Gelatin, by Merck Group (4 mentions)
Ebm 2 medium, by Lonza (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Fibronectin, by Merck Group (3 mentions)
Egm 2 mv singlequots, by Lonza (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Hmvec dly, by Lonza (2 mentions)
Fibronectin, by Thermo Fisher Scientific (2 mentions)
Tissue culture plasticware, by Thermo Fisher Scientific (2 mentions)
Polymerized collagen 1, by BD (2 mentions)
Egm 2 medium, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Ebm 2, by Lonza (2 mentions)
Hpaec, by Lonza (2 mentions)
100 protocols using egm 2mv medium
1
Sorafenib Impacts Endothelial Cell Proliferation
2
Lymphatic Endothelial Cell Culture
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Human adult dermal microvascular lymphatic endothelial cells (LEC) were purchased from Lonza (HMVEC-dLy; Braine-l′Alleud, Belgium) and used at passages 3 to 5. Cells were cultured at 37°C in a 5% CO2 atmosphere in endothelial growth microvascular (EGM2-MV) medium (Lonza) composed of EBM2 medium with 5% FBS, hydrocortisone, h-FGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF and GA 1000 [9] (link). For drug treatments, cells were washed and cultured in EGM2-MV medium supplemented with 2% FBS, and half of the medium was renewed every 24 h. Adenosine (Sigma-Aldrich) was used at concentration ranging from 0.1 μM to 10 μM and the Adenosine deaminase inhibitor EHNA (Sigma) was used at a concentration of 10 μM to increase Adenosine half-life. CGS21680 and NECA (Sigma-Aldrich) were used as preferential A2a and A2b agonists, respectively.
Primary macrophages were isolated from peritoneal lavage of C57BL/6 mice intraperitoneally injected with 4% thioglycollate (Sigma-Aldrich, St. Louis, MO) 5 days earlier. After washing off non-adherent cells, macrophages were cultured in serum-free medium (RPMI-1640, Lonza) and conditioned medium was collected.
Primary macrophages were isolated from peritoneal lavage of C57BL/6 mice intraperitoneally injected with 4% thioglycollate (Sigma-Aldrich, St. Louis, MO) 5 days earlier. After washing off non-adherent cells, macrophages were cultured in serum-free medium (RPMI-1640, Lonza) and conditioned medium was collected.
3
Mesenchymal Stem Cell Culture and Lentivirus Transduction
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M-MSCs differentiated from human H9 ESCs [21 (link)] were maintained in EGM2-MV medium (Lonza, San Diego, CA, USA) on rat tail collagen type I (Sigma-Aldrich, St. Louis, MO, USA)-coated plates in a humidified and heated atmosphere of 5% CO2 and 37 °C as previously described [24 (link),26 (link)]. Human UC-MSCs were cultured in low-glucose DMEM containing 10% heat-inactivated FBS, 5 ng/mL human epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL basic fibroblast growth factor, and 50 ng/mL long-R3 insulin-like growth factor-1 (ProSpec, Rehovot, Israel) as previously described [27 (link)]. Both M-MSCs and UC-MSCs were positive for the expression of CD29, CD73, and CD105 surface molecules but lacked CD14, CD34, and CD45 hematopoietic lineage marker expression (Supplementary Figure S1 ). All M-MSCs were expanded for less than ten passages to ensure multipotency. To induce stable GFP expression, M-MSCs were infected with a GFP-expressing lentivirus, which was generated as described previously [28 (link),29 (link)]. M-MSCs were infected with concentrated lentivirus containing a GFP expression construct using 6 µg/mL polybrene (Invitrogen, Carlsbad, CA, USA), and infected cells were selected using 6 µg/mL blasticidin (Invitrogen).
4
Isolation and Culture of Late Outgrowth EPCs
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The technique to culture EPC subpopulations was conducted as described previously (9 (link),10 (link)). The blood samples were obtained from healthy volunteers subsequent to obtaining informed, written consent (n=20; age, 28±4 years; 10 male, 10 female), and were treated, observed and analyzed individually in independent experiments. The peripheral blood mononuclear cells (PBMNCs) were isolated by density-gradient centrifugation with Ficoll separating solution (Cedarlane Laboratories Ltd., Burlington, Ontario, Canada). The study was approved by the ethics committee of Zhejiang University (Hangzhou, China). PBMNCs were plated into fibronectin-coated six-well plates in 1.5 ml EGM-2MV medium (Lonza Group, Basel, Switzerland) at a density of 2×105/cm2, containing 10% fetal bovine serum, vascular endothelial growth factor, fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor, and ascorbic acid. Adherent MNCs cultured for 7–21 days in the above conditions were used to derive late outgrowth EPCs and the colonies exhibit a cobblestone morphology.
5
Human Endothelial Cell Culture and Stimulation
6
Endothelial Cell Isolation and Culture
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To positively select for the endothelial population, the mixed cell population was resuspended in 60 µl of Endothelial Medium (EGM-2 MV medium, Cat# CC-3202, Lonza, Basel, Switzerland) containing penicillin/streptomycin, per 106 cells and 20 µl FcR blocking reagent. 20 µl of anti-CD31 beads (Cat# 130-091-935, Miltneyi Biotec) were added, mixed and incubated for 15 min at 4 °C. The cell mixture was resuspended in 1 ml of Endothelial Medium and centrifuged at 300 g for 3 min. Beads with positively selected cells and non-labelled cells were isolated using a LS column in a magnetic field. CD31+ cells were plated at 5 × 105 cells per 75 cm2on collagen-coated (0.1 mg/ml rat tail collagen type I in PBS, Cat# 60-30-810, First Link UK, Wolverhampton, UK) cell culture flasks in supplemented Endothelial Medium.
7
Immunofluorescence Assay for TIMAP in ECs
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Primary human glomerular ECs and HUVECs free of mycoplasma contamination were purchased from Angio-Proteomie (Boston, MA) and maintained in EGM-2MV medium (CC3202, Lonza, Walkersville, MD). EGM-2MV consists of EBM-2 basal medium (CC3356) plus supplements (CC4147) from Lonza. Cells were grown on plates coated with Quick Coating Solution cAP-01 from Angio-Proteomie. For all experiments, ECs were used at passages 5–6. COS7 cells were maintained in DMEM containing 10% FBS.
Immunofluorescence studies were done as described previously (57 (link)). Briefly, ECs were cultured in complete medium on glass coverslips. 48 h later, they were washed, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 10% normal goat serum, and then incubated with chicken anti-TIMAP IgY (1:5000 dilution overnight at 4 °C), followed by Alexa Fluor 594 goat anti-chicken IgG (1:4000 dilution).
Immunofluorescence studies were done as described previously (57 (link)). Briefly, ECs were cultured in complete medium on glass coverslips. 48 h later, they were washed, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 10% normal goat serum, and then incubated with chicken anti-TIMAP IgY (1:5000 dilution overnight at 4 °C), followed by Alexa Fluor 594 goat anti-chicken IgG (1:4000 dilution).
8
Airway-on-a-Chip: Modeling Lung Epithelium
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HMVEC-L were obtained from Lonza and maintained in EGM-2 MV medium (Lonza). To prepare the airway-on-a-chip, first, the bottom channel of a polydimethylsiloxane (PDMS) device was precoated with fibronectin (3 μg/ml; Sigma-Aldrich). HMVEC-L were suspended at 5 × 106 cells/ml in EGM-2 MV medium. Suspension medium (10 μl) was injected into the fibronectin-coated bottom channel of the PDMS device. Then, the PDMS device was turned upside down and incubated for 1 hour. After 1 hour, the device was turned over, and EGM-2 MV medium was added into the bottom channel. After 4 days, human airway organoids (AOs) were dissociated and seeded into the top channel. AOs were dissociated into single cells and then suspended at 5 × 106 cells/ml in AO differentiation medium. Suspension medium (10 μl) was injected into the top channel. After 1 hour, AO differentiation medium was added to the top channel. The cells were cultured under a humidified atmosphere with 5% CO2 at 37°C.
9
Culturing primary human endothelial cells
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Immediately after the collagenase step, the freshly isolated cells were seeded together with the matrix debris generated by the action of the collagenase on non-coated plastic dishes and maintained in EGM-MV (containing endothelial cell growth complement, EGF 10 ng/ml, heparin 90 μg/ml and hydrocortisone 1 μg/ml) supplemented with 5% fetal calf serum and 1% antibiotics (10000 U Penicillin/ml and 10000 U Streptomycin/ml, Lonza). This medium was developed by PromoCell for culturing human ECs from either microvascular or macrovascular origin. HUVEC cells were purchased from Lonza (# cc-2817) and cultured in EGM2-MV medium (Lonza). Cells were passaged at confluence after treatment with trypsin-EDTA buffer (170000U trypsin/l, Lonza). Confluence is mimicking the native in vivo state of ECs. All cells were used at passages 0–2. TGFbeta concentration in the medium, provided by the serum, was measured with the ELISA assay (Ready-Set-Go, human/mouse TGFbeta, 2nd generation, eBioscience). Phase contrast images of the isolated cells in culture were acquired with a Nikon Eclipse TE 2000-E inverted microscope with a 10x air objective.
10
Cigarette Smoke Impact on Bladder Endothelial Cells
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Human bladder microvascular endothelial cells (HBMEC) were grown in EGM‐2MV medium (Lonza, Walkersville, MD) and maintained at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. Cells were treated with cigarette smoke extract (CSE, 20 μg/mL) for indicated times as previously described (Sharma et al. 2012 ). CSE was obtained from Murty Pharmaceuticals (Lexington, KY).
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