SKOV3 human ovarian cancer cells were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin and 100 µg/mL streptomycin (P/S, Thermo Fisher, Waltham, MA, USA). Panc1 human pancreatic cancer cells and MDA-MB-231 human breast cancer cells were also obtained from ATCC and maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and P/S. Primary human bone marrow stromal cells (BMSCs) derived from whole human bone marrow aspirates (Lonza, Basel, Switzerland) were cultured in the MesenCult Proliferation Kit (Stem Cell Technologies, Vancouver, Canada). Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and maintained in the endothelial cell growth medium-2 (EGM-2) BulletKit (Lonza, Basel, Switzerland). HUVECs expressing a red fluorescence protein (RFP) reporter gene were obtained from Angio-Proteomie. Cells from 75–90% confluent monolayers were passaged using 1% trypsin in phosphate-buffered saline (PBS, Thermo Fisher, Waltham, MA, USA).
Egm 2 endothelial cell growth medium 2 bulletkit
Manufactured by Lonza
Sourced in Switzerland, United States, Japan
EGM-2 Endothelial Cell Growth Medium-2 BulletKit is a cell culture medium formulation designed for the growth and maintenance of human endothelial cells. It provides the necessary nutrients and growth factors required for the optimal proliferation and survival of endothelial cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Huvecs, by Lonza (5 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Kh2po4, by Fujifilm (2 mentions)
Dulbecco s phosphate buffered saline, by Fujifilm (2 mentions)
Na2hpo4, by Fujifilm (2 mentions)
Polystyrene round bottom tube with cell strainer cap, by Corning (2 mentions)
Trypan blue stain solution, by Corning (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Huvecs, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ebm 2 basal medium, by Lonza (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
67 protocols using egm 2 endothelial cell growth medium 2 bulletkit
1
Cell Culture Protocols for Cancer Research
2
Human Cardiomyocyte and Endothelial Cell Culture Protocols
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The human ventricular cardiomyocyte cell line AC10 (8 (link)) was cultured in Dulbecco's Modified Eagle's Medium-F-12 (DMEM-F12, Gibco-Invitrogen®) supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen®) and 1% penicillin/streptomycin (P/S, Millipore). Primary cultures of human umbilical cord vein endothelial cells (HUVEC) were obtained from Lonza and were grown in Endothelial Cell Growth Medium-2 (EGM-2) BulletKit™ (Lonza). Human coronary microvasculature (HCAEC; ATCC) endothelial cells were grown in Vascular Cell Basal Medium supplemented with the Endothelial Cell Growth Kit-VEGF (ATCC). Fibroblasts were obtained from skin biopsies after informed consent and were cultured in high-glucose DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S. Cell were maintained under control conditions (Nx) in a humidified atmosphere at 37°C containing 5% CO2. AC10 cells cultured in Hx were incubated at 2% O2 for 48 h with glucose deprivation during the first 4 h and DMEM/F12 with depleted FBS during the next 44 h at 2% O2. Depleted FBS was generated by ultracentrifugation of an FBS and DMEM/F12 (1:1) mix at 100,000 g for 16 h. The induction of Hx in cell cultures was monitored by stabilization of hypoxia inducible factor-1 alpha (HIF-1α) expression and cell viability reduction (Figure S1 ).
3
Cultivation of NSCLC and HUVEC Cells
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The human NSCLC cell line, H1299, obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Nichirei Biosciences Inc., Tokyo, Japan) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. H1299-IR cells were previously exposed to 10 Gy of X-ray irradiation [8 (link)]. Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza group Ltd. (Basel, Switzerland) and maintained in an Endothelial Cell Growth Medium-2 (EGM-2) Bullet Kit (Lonza Group Ltd., Basel, Switzerland) at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
4
Isolation, culture and characterization of hASC and HUVEC
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The isolation, culture, and quality control of hASC and HUVEC were performed as described previously [11 (link),48 (link)]. hASC were cultured in DMEM/F12 supplemented with 10% human serum (HS, Lonza Group Ltd., Basel, Switzerland) and 1% L-glutamine (Gibco, Carlsbad, CA, USA). hASC used in the study were heterogenous cell populations obtained by isolating the stromal vascular fraction cells of human adipose tissue. HUVEC were expanded in a Endothelial Cell Growth Medium-2 Bullet Kit (EGM-2, Lonza). In the co-culture, hASC was at passage 2, and HUVEC at passage 4. Both cell types were free of mycoplasma, as tested with MycoAlert® Mycoplasma Detection Kit (Lonza). hASC were characterized for markers CD73, CD90, and CD105 (BD biosciences, Franklin Lakes, NJ, USA) with flow cytometer FACSCAnto II (BD biosciences), as previously published [11 (link)], before experimental use. In the co-culture, hASC was at passage 2, and HUVEC at passage 4.
5
Cultivation and Characterization of HUVEC
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Primary human umbilical vein endothelial cells (HUVEC) and Endothelial Cell Growth Medium-2 BulletKit (EGM-2) were obtained from Lonza (Basel, Switzerland), and Endothelial Cell Growth Medium (ECGM) purchased from Promocell (Heidelberg, Germany). 10 × Dulbecco's-phosphate buffered saline (PBS) (−) (2 g/L KCl, 80 g/L NaCl, 2 g/L KH2PO4, 11.5 g/L Na2HPO4) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 5 mL polystyrene round-bottom tube with cell-strainer cap (12 × 77 mm) and 0.5% Trypan blue stain solution were obtained from Corning Falcon (Corning, NY, USA) and Nacalai Tesque, Inc. (Kyoto, Japan), respectively.
In order to reduce the number of passages and to use the cells at the same passage when preparing microvessels, primary HUVEC were thawed upon reception, amplified for two passages in EGM-2 and frozen again. When fabricating microvessels, cells were thawed in EGM-2, seeded in 21 cm2 culture dishes, cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air, and used at 70–80% confluence. Cells were harvested by rinsing once with PBS, incubating with 0.25% trypsin-EDTA solution for 3 min at 37 °C in 5% CO2/95% air and collected in ECGM. Cells were passed through a 35 μm cell strainer to dissociate cell aggregates; stained with Trypan Blue and live cells were counted using a hemocytometer.
In order to reduce the number of passages and to use the cells at the same passage when preparing microvessels, primary HUVEC were thawed upon reception, amplified for two passages in EGM-2 and frozen again. When fabricating microvessels, cells were thawed in EGM-2, seeded in 21 cm2 culture dishes, cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air, and used at 70–80% confluence. Cells were harvested by rinsing once with PBS, incubating with 0.25% trypsin-EDTA solution for 3 min at 37 °C in 5% CO2/95% air and collected in ECGM. Cells were passed through a 35 μm cell strainer to dissociate cell aggregates; stained with Trypan Blue and live cells were counted using a hemocytometer.
6
HUVEC Isolation and Culture
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Studies were performed with Primary human umbilical vein endothelial cells (HUVEC; Catalog #C2519A, Lot #0000699241; Lonza, Basel, Switzerland) that were cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2; Lonza). They were frozen in liquid nitrogen at passage 4 to 5, thawed and cultured for three days in culture dishes, and then used to load MV chips.
7
Microvascular Model with GFP-HUVECs
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Pooled human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein (GFP) were commercially obtained from Cellworks. GFP-HUVECs were cultured in the Endothelial Cell Growth Medium-2 Bullet Kit (EGM-2; Lonza). Instead of the fetal bovine serum supplied with the kit, 2% HS (Biowest or Serana) was used. In the microvascular network model, GFP-HUVECs were used in passages 4–6.
HUVECs isolated from human umbilical cord vein tissue samples were received from two donors. Tissue samples were obtained in planned cesarean sections at the Tampere University Hospital with the patient’s written informed consent together with the favorable opinion of the Regional Ethics Committee of the Expert Responsibility area of Tampere University Hospital, Tampere, Finland (R13019). Cells were isolated using the enzymatic procedure as previously described [19 (link),20 (link)] apart from using the collagenase type II (1.0 mg/mL; Merck Millipore, Burlington, MA, ÙSA) enzyme in cell isolation. HUVECs were cultured in EGM-2 medium, where fetal bovine serum was replaced with 2% HS (Biowest). The cells were used in a microvascular network model in passage 4 and multilineage spheroids and chip experiments in passages 2–6.
HUVECs isolated from human umbilical cord vein tissue samples were received from two donors. Tissue samples were obtained in planned cesarean sections at the Tampere University Hospital with the patient’s written informed consent together with the favorable opinion of the Regional Ethics Committee of the Expert Responsibility area of Tampere University Hospital, Tampere, Finland (R13019). Cells were isolated using the enzymatic procedure as previously described [19 (link),20 (link)] apart from using the collagenase type II (1.0 mg/mL; Merck Millipore, Burlington, MA, ÙSA) enzyme in cell isolation. HUVECs were cultured in EGM-2 medium, where fetal bovine serum was replaced with 2% HS (Biowest). The cells were used in a microvascular network model in passage 4 and multilineage spheroids and chip experiments in passages 2–6.
8
Breast Cancer Cell Lines and Immortalized Microvascular Cells
9
Differentiation of Endothelial Cells from Induced Pluripotent Stem Cells
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Differentiation of ECs from piPSCs was progressive. First, piPSCs were passaged on Matrigel (1:150) (Corning, Corning, NY, USA) to preclude the presence of mouse embryonic fibroblasts. Then, piPSCs were digested by 0.5 μM EDTA (Thermo Scientific, Waltham, MA, USA) and planted on Matrigel at a dilution of 1:10 with 10 μM ROCK inhibitor Y27632 (EMD4 Biosciences, Darmstadt, Germany). After 2 days, culture medium was switched to EC differentiation medium (FGF2 and LIF were removed from MX medium) with 6 μM CHIR99021 (Stemgent, Boston, MA, USA) for 2 days. Next, the cells were cultured in EC differentiation medium supplemented with 25 ng/mL BMP4, 10 ng/mL FGF2 and 50 ng/mL VEGF (R&D Systems, Minneapolis, MN, USA) for 2 days. At last, EC differentiation medium was changed to Endothelial Cell Growth Medium-2 BulletKit (EGM-2, Lonza, Guangzhou, China) with the addition of 50 ng/mL VEGF. The medium was changed every day [28 (link)].
10
HUVEC Culture and Characterization
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Human umbilical vein endothelial cells (HUVECs, Catalog #C2519A, Lot #0000699241; Lonza, Basel, Switzerland) were cultured in an Endothelial Cell Growth Medium-2 BulletKit (EGM-2; Lonza, Basel, Switzerland). The cells between passages 4 and 7 were used in this study.
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