HCASMC were purchased from Life technologies and cultured according to manufactures recommendation in Medium 231 supplemented with smooth muscle growth supplement (GIBCO). As per manufacturer information, cells were isolated by the enzyme digestion method as previously described by Ray et al.60 (link). Expression of αSMA, a specific adult SMC marker, and absence of the specific endothelial cell protein CD31 was confirmed in HCASMC by immunoblotting using anti human SMA IgG (abcam) and anti human CD31 IgG (RD systems). As control cell line we used Human Aortic Endothelial cells, which abundantly express CD31 and was a kind gift by Dr.Yun Fang, Section of pulmonary and critical care, Department of Medicine, University of Chicago (Supplementary Fig. 15 ). Three different batches of HCASMC (from Life technologies) were propagated and passage 2–7 were used for experiments. With the exception for the microarray experiment, which was performed on HCASMC from only one donor, all other experiments utilized three different batches of HCASMC and each cell line was tested in triplicates.
Hcasmc
Manufactured by Thermo Fisher Scientific
Sourced in United States
HCASMCs are primary human coronary artery smooth muscle cells. They are used for in vitro research and studies related to the cardiovascular system.
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Lab products found in correlation
Medium 231, by Thermo Fisher Scientific (3 mentions)
Smooth muscle growth supplement, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Harvard Bioscience (2 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti human cd31 igg, by R&D Systems (1 mentions)
Sh sy5y cell, by American Type Culture Collection (1 mentions)
Sh sy5y cells, by Thermo Fisher Scientific (1 mentions)
Ang 2, by Merck Group (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Smooth muscle cell growth media, by Thermo Fisher Scientific (1 mentions)
Smooth muscle cell growth supplement, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Beyotime (1 mentions)
Streptomycin, by Beyotime (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Beyotime (1 mentions)
11 protocols using hcasmc
1
HCASMC Culture and Characterization
2
Carbon-Nanotube Reinforced Vascular Conduits
3
HCASMC Cell Characterization Experiments
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All experiments were performed in triplicates and repeated two times. With the exception for the microarray experiment, which was performed on HCASMC from only one donor, all other experiments utilized three different batches of HCASMC (from Life technologies) and each cell line was tested in triplicates. Results are presented as mean ± standard deviation. Statistical differences were analyzed using independent sample T-test and one-way analysis of variance were used for mean comparison between two or multiple groups, respectively. Two-tailed probability values of p less than 0.05 were considered statistically significant for each test to ensure an overall study significance level of P < 0.05.
4
SH-SY5Y and HCASMC Cell Culture and AngII Exposure
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Human neuroblastoma SH-SY5Y cells were obtained from ATCC (Manassas, VA) and cultured in T75 flasks in a humidified incubator at 37°C with 5% CO2. SH-SY5Y cells were grown in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, L-glutamine (2 mM), non-essential amino acids (1 mM), and sodium pyruvate (1 mM). HCASMC were obtained from Gibco and were cultured in a humidified incubator as SH-SY5Y cells (at 37°C with 5% CO2). HCASMC were grown in Medium 231 supplemented with Smooth Muscle Growth Supplement and 1% penicillin–streptomycin. In MTT experiments, the cells were exposed to 1, 10 and 100 nM AngII (Sigma, A925) for 6, 24 and 48 hours. For Western blotting experiments, the cells were exposed to 10 nM AngII for 6 hours. The entire experiment was performed under a laminar flow hood.
5
Overexpression of key signaling proteins in human coronary artery smooth muscle cells
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Smooth muscle cells from the human coronary artery (hCASMC, lot 1130140, Gibco, Life Technologies) were cultured in 6 well plates (Nunclon, Thermo Scientific) containing Medium 231 (Life Technologies) with 5% growth supplement (SMGS, Life Technologies) and 50 U/50 μg/ml penicillin/streptomycin (Biochrom, A 2212) in a water-jacketed incubator with an atmosphere of 5% CO2 in air (37 °C). Cells were passaged by trypsin treatment and used between passage 3 and 8. Full length h-MAML2, the intracellular domain of NOTCH2 (nucleotides 5107–7425 of Notch2 cDNA sequence AF308601), a C-terminal fragment of h-FRYL and full length JAG1, all under control of the CMV promoter, were overexpressed using custom-made human adenoviruses Type 5 (dE1/E3) from Vector Biolabs. Ad-CMV-null at the same multiplicity of infection (MOI) was used as control for all transductions. Virus was added 24 h after seeding and maintained for 96 h. For harvest, cells were washed with ice-cold PBS and lysed in Qiazol (RNA) or Laemmli sample buffer (protein).
6
Cell Culture of HUVEC and HCASMC
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Human umbilical vein endothelial cells (HUVEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). First, HUVEC was cultured in an endothelium medium (EGM-2 BulletKit, Lonza, Benicia, CA, USA) with 1% (v/v) penicillin/streptomycin (P/S, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 atmosphere, and HCASMC was cultured in a human vascular smooth muscle cell basal medium (Medium 231, Gibco) with smooth muscle growth supplement (SMGS, Gibco) under the same conditions as the HUVEC culture.
7
Cultivation of Human Coronary Artery Smooth Muscle Cells
8
Proliferation and Migration Modulation of HCASMCs by SBP
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HCASMCs were obtained from Sigma. We cultured the HCASMCs with a humidified incubator at 37°C provided with 5% CO2 growing inappropriate volume of DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin (100 μg/ml) and penicillin (100 U/ml) (Beyotime, Shanghai, China) bought from Yiyuan Biotechnologies (Guangzhou, China) to create an unusual lipid environment and contribute to proliferation and migration models. The SBP-treated group was cultured with extra 0, 1, 2.5, 5, and 7.5 mg/L SBP for 0, 12, 24, 36, and 48 h to explore the effect of SBP on the proliferation and migration of HCASMCs.
9
Fabrication of Tissue-Engineered Blood Vessels
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Fabrication of 3D tissue-engineered intimal layers (TEILs), media layers (TEMLs), and the complete tissue-engineered blood vessel was achieved using human umbilical vein endothelial cells (HUVECs) and human cardiac artery smooth muscle cells (HCASMCs), both obtained from GIBCO, Life Technologies. Cells were cultured with medium 200 and 231, respectively, also obtained from GIBCO, Life Technologies, and used between passage numbers 2 and 5. The construction of the TEIL, TEML, and TEBV constructs was performed using our previously described methodology [18 (link)], as such these protocols are outlined briefly below.
10
Human Coronary Artery Smooth Muscle Cell Culture
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Primary human coronary artery smooth muscle cells (HCASMCs) from two different donors (Lot: 1130140 and 1689414) were purchased from Gibco Life Technologies (#C-017-5C) and maintained in Medium 231 (Life Technologies, #M231500) supplemented with 5% smooth muscle growth supplement (Life Technologies, #S-007–25) and 1% penicillin/streptomycin (Biochrom, #A2212). Cells were cultured at 37°C in 5% CO2 and used until passage 8. Media was changed every other day.
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