The in vitro angiogenic potential of all materials was assessed using the scratch assay [40 (link)]. Human umbilical vein endothelial cells (HUVECs, C2517A, Lonza, UK) were expanded in specific medium (EGM™-2, Lonza, Ireland). When they reached 85–90% confluence, they were seeded in 48-wellplates and incubated at 37 °C and 5% CO2 until confluent (2 days). Using a sterile pipette tip, 1 mm wide gap was created at the cell monolayer. The cells were then washed three times with PBS to remove cellular debris and treated with medium conditioned with each material. Conditioned media was created by incubating supplemented with 2% FBS and 1% P/S DMEM with each of the materials at 20 mg/mL overnight at 37 °C under continuous agitation in an incubated orbital shaker (MaxQ 4000, Thermo Fisher, Ireland) at 150 rpm. These mixtures were then sterile filtered and poured on the cell monolayer with the gap. DMEM with 2% FBS and 1% P/S and endothelial growth medium (EGM™-2, Lonza, Ireland) were used as negative and positive controls, respectively. Images were taken at 4, 8, 12, and 24 h and the area fold change in the monolayer moving into the scratch zone with respect to the area at time 0 was calculated for each material.
Egm 2
Manufactured by Lonza
Sourced in Switzerland, United States, United Kingdom, Belgium, Germany, Italy, Japan, Australia, Singapore, Ireland, Israel, Austria
The EGM-2 is a laboratory instrument designed for the measurement of oxygen concentration in cell culture media. It provides accurate and reliable data on the oxygen levels within the culture environment.
Automatically generated - may contain errors
Lab products found in correlation
Huvecs, by Lonza (144 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (83 mentions)
Ebm 2, by Lonza (81 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (46 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (44 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (37 mentions)
Huvecs, by American Type Culture Collection (24 mentions)
Egm 2mv, by Lonza (22 mentions)
Fetal bovine serum (fbs), by Lonza (20 mentions)
Penicillin, by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
Matrigel, by BD (17 mentions)
L glutamine, by Thermo Fisher Scientific (16 mentions)
Fgm 2, by Lonza (15 mentions)
Matrigel, by Corning (13 mentions)
Trypsin edta, by Thermo Fisher Scientific (13 mentions)
Rpmi 1640, by Thermo Fisher Scientific (12 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (12 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (11 mentions)
Mda mb 231, by American Type Culture Collection (11 mentions)
912 protocols using egm 2
1
In vitro Angiogenic Potential Evaluation
2
Culturing Diverse Cell Lines for Research
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All cell cultures were maintained in a 5% CO2 atmosphere at 37 °C. Cell cultures with 70–80% confluency were used for all listed experiments.
HSVECs and HSVECs 1 were derived from patient samples (patients undergoing varicose vein surgery). The derived cells expressed endothelial markers (assessed by flow cytometry) and fulfilled the requirements of the endothelial functional assays—tube formation assay and LDL assay. HUVECs and HUVECs 1 (both from Thermo Fisher Scientific, Waltham, MA, USA) and HSVECs, HSVECs 1 were cultured in EGM2 (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. hDFs (National Tissue Center Inc. Brno, Czech Republic), hDFs 1 (ThermoFischer Scientific, Waltham, MA, USA) were cultured in DMEM supplemented with 10% FBS, 100 µM non-essential amino acids, 1% penicillin/streptomycin, 2 mM l-glutamine and 0.1 mM mercaptoethanol (all components purchased from Life Technologies, Carlsbad, CA, USA). hiPSCs were cultured in colonies in mTeSRTM1 (Thermo Fisher Scientific) on MatrigelMT-coated dishes (Stemcell Technologies, Vancouver, Canada). All EC-lines differentiated from hiPSCs were cultivated in EGM2 (Lonza) supplemented with 50 ng/mL VEGF (PeproTech, Rocky Hill, NJ, USA). All cell cultures were routinely passaged at 80% confluency.
HSVECs and HSVECs 1 were derived from patient samples (patients undergoing varicose vein surgery). The derived cells expressed endothelial markers (assessed by flow cytometry) and fulfilled the requirements of the endothelial functional assays—tube formation assay and LDL assay. HUVECs and HUVECs 1 (both from Thermo Fisher Scientific, Waltham, MA, USA) and HSVECs, HSVECs 1 were cultured in EGM2 (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. hDFs (National Tissue Center Inc. Brno, Czech Republic), hDFs 1 (ThermoFischer Scientific, Waltham, MA, USA) were cultured in DMEM supplemented with 10% FBS, 100 µM non-essential amino acids, 1% penicillin/streptomycin, 2 mM l-glutamine and 0.1 mM mercaptoethanol (all components purchased from Life Technologies, Carlsbad, CA, USA). hiPSCs were cultured in colonies in mTeSRTM1 (Thermo Fisher Scientific) on MatrigelMT-coated dishes (Stemcell Technologies, Vancouver, Canada). All EC-lines differentiated from hiPSCs were cultivated in EGM2 (Lonza) supplemented with 50 ng/mL VEGF (PeproTech, Rocky Hill, NJ, USA). All cell cultures were routinely passaged at 80% confluency.
3
Cardiac Fibroblast and Endothelial Cell Isolation
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Normal human cardiac fibroblasts (NHCFs; passage 6) and human cardiac microvascular endothelial cells (HMVECs; passage 6–7) obtained from Lonza (Allendale, NJ, USA) were used in the experiments. After culture in fibroblast growth medium (FGM-3, Lonza), NHCFs were dissociated with 0.1% trypsin (FUJIFILM Wako Pure Chemical) and centrifuged for 5 min at 160 × g. After the supernatant was removed, the cells were suspended in phosphate-buffered saline (PBS) (Nacalai Tesque) and used in the layer-by-layer approach described below. HMVECs were cultured in endothelial growth medium (EGM-2, Lonza) and then dissociated with 0.25% trypsin-EDTA and centrifuged for 5 min at 160 × g. After the supernatant was removed, the cells were suspended in EGM-2 and used for tissue construction without the layer-by-layer approach.
4
Isolation and Culture of Human Endothelial Cells
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Human umbilical vein endothelial cells were isolated from freshly collected umbilical cords using collagenase (Roche Diagnostics) 0.2% in EBM‐2 media (Lonza). Cells from 3 to 5 cords were pooled. Human adult dermal microvascular endothelial cells (HMVECs) were purchased from Sigma (100‐05a). HUVECs and HMVECs were cultured in EGM‐2 (Lonza) or EGM‐2 10% FBS and no heparin, respectively, and used for experiments up to passage six. Immortalized human mammary normal fibroblasts (iNF) and cancer‐associated fibroblast (iCAF) cell lines (kindly provided by Professor Akira Orimo; Kojima et al, 2010 ) and the melanoma cancer cell lines, B16F1, B16F10, and B16F10‐GFP+ (from the CRUK Beatson Institute), were cultured in DMEM (Gibco, Thermo Fisher Scientific), 10% FBS, 2 mM glutamine. The prostate cancer cell line PC3 was grown in RPMI medium (Gibco, Thermo Fisher Scientific) with 10% FBS, 2 mM glutamine. The E0771 breast cancer cells were purchased from Tebu‐Bio and cultured in RPMI with 10% FBS. The LLC cells were purchased from Sigma‐Aldrich and cultured in DMEM with 10% FBS. All cell lines were harvested with trypsin (0.025% in PBS + EDTA) and grown at 37°C, 5% CO2, 21% O2. Blebbistatin was from TOCRIS. All cell lines are routinely tested for mycoplasma. For primary antibodies, RNAi, and primers, see Appendix Supplementary Methods .
5
Evaluating E-Liquid Impacts on Endothelial Cells
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Commercially available (Lonza Inc, Walkersville, MD) HAECs were cultured from passages 4 to 7 in EGM-2. When cells achieved 80–90% confluence, serum was withdrawn for 4 h, and the cells were exposed to either a JUUL e-liquid, PG:VG or nicotine salt diluted in media (phenol-red-free EGM-2, Lonza) for 90 min at 37°C before measurements of apoptosis, oxidative stress, inflammation, and nitric oxide production as discussed below. Controls were HAECs in the same media conditions without e-liquid exposure. For the heated fraction studies, endothelial cells were exposed to particle fractions collected at stage 6 diluted to 0.001% for 90 minutes (stage with the highest mass concentration; 0.56 μm cutoff) as described above and stimulated NO production was evaluated.
6
KSHV Virus Stock Production and Cell Maintenance
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iSLK cells that harbor wild-type KSHV BAC-16 [86 (link),87 (link)] were used to make KSHV virus stocks and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, 11965–092) with 10% FBS (GIBCO, 10437028), 50 μg/mL gentamicin sulfate (Caisson Labs, ABL05), and selection antibiotics 1 μg/ml puromycin (Thermo Fisher Scientific, A1113803), 250 μg/ml G418 (Thermo Fisher Scientific, 10131035) and 1.2 mg/ml hygromycin B (Corning, 30-240-CR). In addition, the iSLK line were cultured in DMEM with 1 μg/ml puromycin and 250 μg/ml G418. The iSLK BAC16 were cultured in DMEM with selection antibiotics 1 μg/ml puromycin, 250 μg/ml G418, and 1,2000 μg/ml hygromycin B. The WT1 siRNA and control siRNA were obtained from Thermo Fisher. HuARLT-1 cells were cultured in endothelial cell growth medium EGM-2 (Lonza, CC-3162) with 2μg/ml of doxycycline (Sigma-Aldrich, D9891-1G). Human umbilical vein endothelial cells (HUVEC) (Lonza, C2517A) were cultured in EGM-2. HUVEC ORFE4, a kind gift from the lab of Shahin Rafii at Weill Cornell Medicine were cultured in EGM-2 and did not require selection. All primary cell cultures and cell lines have undergone testing for mycoplasma and were confirmed negative.
7
Culture of HUVEC and THP-1 Cells
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Human Umbilical Vein Endothelial Cells (HUVECs) were grown in EGM-2 medium supplemented with 2% of Fetal Bovine Serum (FBS), hydrocortisone, Vascular Endothelial Growth Factor (VEGF), R3 Insulin-like Growth Factor 1 (R3 IGF-1), ascorbic acid, human Epidermal Growth Factor (hEGF), Gentamicin sulfate/Amphotericin (GA-1000), and heparin (medium EGM-2, Lonza, Basel, Switzerland). Cells were used from passage number 2 to 10, consistent with manufacturer recommendations to assure the viability and an adequate metabolism of cells.
THP-1 (ATCC® TIB202™, Manassas, VA, USA) is a commercial cell line of monocytes isolated from peripheral blood. THP-1 was grown in suspension in RPMI-1640 media supplemented withl -glutamine, 2% of FBS and ampicillin/streptomycin (RPMI-1640, Gibco, Gaithersburg, MD, USA), and THP-1 were used from passage number 0 to 7.
THP-1 (ATCC® TIB202™, Manassas, VA, USA) is a commercial cell line of monocytes isolated from peripheral blood. THP-1 was grown in suspension in RPMI-1640 media supplemented with
8
HUVEC Angiogenic Response Assessment
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Human Umbilical Vein Endothelial Cells (HUVEC; Lonza) were used to assess the angiogenic response. For cell expansion, HUVEC were seeded at a density of 2500 cells/cm2 and maintained with endothelial growth medium-2 bulletkit (EGM-2) (Lonza; Ref. H3CC-3162), containing VEGF, rhFGF-B, rhEGF, r-IGF-I, hydrocortisone, ascorbic acid, gentamicin sulfate, amphotericin-B and 2% FBS. When cells reached 70–85% confluence, they were passaged using 0.25% Trypsin-EDTA (Gibco). Cells were maintained in standard culture conditions (37°C and 5% CO2). Passages equal or below P5 were used for subsequent assays. Preliminary experiments tested two types of cell culture media supplemented with ions: i) basal medium (Lonza; Ref. CC-3156), supplemented with 2% FBS and amphotericin-B; ii) and EGM-2 medium, previously described.
9
Isolation of Endothelial Progenitor Cells from Cord Blood
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Endothelial progenitor cells were isolated from human umbilical cord blood using the methodology first developed by the Yoder laboratory27 (link),28 (link) and detailed in video by Ravishankar et al.29 (link) Briefly, the mononuclear cell fraction from human cord blood units (40–80 mL) was isolated using a Ficoll density gradient separation. The total viable mononuclear cell (MNC) yield varied from 4 to 6 × 108 MNCs per cord blood unit (approximately 6 × 106 MNCs per mL of blood). The MNC fraction was resuspended at a density of 1 × 107 cells per mL in endothelial growth medium-2 (EGM-2, Lonza)-, and 5 mL of cell suspension was plated into each well of a 6-well dish precoated with collagen. After an overnight incubation, the nonadherent fraction was removed. Endothelial cell colonies were observed in most wells by day 7. The primary colonies were pass at day 14. The initial cell yield at the first passage typically yields 0.5 to 2 × 106 cells. Endothelial cell cultures were maintained in EGM-2 (Lonza) growth medium.
10
Isolation and Culture of Endothelial Cells
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HDMEC were isolated from neonatal foreskin by incubating the obtained cells with magnetic beads coated with an antibody to CD31 (Invitrogen) as described previously [26 (link)]. HLMEC and HUVEC were obtained from Lonza. HDMEC and HLMEC were grown in EGM-2 MV (Lonza) and HUVEC were grown in EGM-2 (Lonza). Cells were used for experiments between passages 5 and 10. For all experimental protocols, cells were seeded at 105 (HLMEC and HDMEC) or 4x104 cells/cm2 (HUVEC) and incubated for 48–72 h prior to any treatment to obtain mature cell-cell junctions.
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