For the isolation of EpiCs, we followed the method referred by Zhou and Pu (33 (link)). Briefly, the heart was dissected from 6 to 8-week-old C57BL/6 and rinsed in D-Hanks' buffer and digested with sterilized D-Hanks' buffer supplemented with 0.08% collagenase IV (Worthington, Lakewood, NJ, USA) and 0.05% trypsin (Gibco, Carlsbad, CA, USA) at 37°C for 8 min under gentle rotation (60 rpm/min) and repeated for 8 times. Subsequently, cells were centrifuged at 1,000 rpm for 5 min and seeded onto 0.1% gelatin-coated 24-well plates with culture medium containing low glucose DMEM (Invitrogen, Carlsbad, CA, USA) and Medium 199 (M199) (Invitrogen, Carlsbad, CA, USA) in a 1:1 ratio, supplemented with 10% heat-inactivated (56°C for 25 min) FBS (Gibco, Carlsbad, CA, USA) and 1% P/S (Invitrogen, Carlsbad, CA, USA). Primary EpiCs at passage 3 were used for subsequent experiments.
Medium 199 m199
Manufactured by Thermo Fisher Scientific
Sourced in United States
Medium 199 (M199) is a widely used cell culture medium formulated to support the growth and maintenance of a variety of cell types. It provides a balanced salt solution, amino acids, vitamins, and other nutrients essential for cell proliferation and survival. M199 is commonly used in basic cell biology research, tissue engineering, and cell-based assays.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Bovine α thrombin, by Enzyme Research (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Low glucose dmem, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Methacrylic anhydride, by Merck Group (2 mentions)
Porcine skin gelatin solution, by Merck Group (2 mentions)
Collagen 1 rat tail solution, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Elastase, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Heparin, by Thermo Fisher Scientific (1 mentions)
23 protocols using medium 199 m199
1
Isolation and Culture of Epicardial Cells
2
Endothelial Cell Isolation and Culture
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Dulbecco's modified Eagle's medium (DMEM), medium 199 (M199), collagenase type-III, elastase, soybean trypsin inhibitor, fetal bovine serum (FBS), penicillin, streptomycin, heparin, endothelial cell growth factor (ECGF), and the Lipofectamine 2000 reagent were from Invitrogen (NY, USA). 5-bromo-2'-deoxyuridine (BrdU), an anti-BrdU antibody, and AngII were from Sigma-Aldrich (MO, USA). Rapamycin, 3-MA, and SAR405 were from Selleck Chemical (TX, USA).
3
Isolation and Culture of Human Epicardial Progenitor Cells
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Cultures of hEPDCs were prepared from anonymous human adult right atrial appendages excised during cardiac surgery, as previously described (22 (link), 23 (link)). The layer of epicardium was stripped from the auricle, after which the tissue was placed in a gelatin coated culture disk and capped with a round coverslip to prevent the tissue from floating. Cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) and Medium 199 (M199) (Invitrogen) 0.5% penicillin (Invitrogen), 0.5% streptomycin (Invitrogen), and 10% heat inactivated fetal calf serum (Invitrogen). Seven days after culturing, when outgrowths of epithelium-like cells were visible the coverslips and remaining tissue were removed. When outgrowth was confluent, the cells were passaged (1:3) to induce spontaneous EMT to create spindle-shaped EPDCs and medium was refreshed every 3 days. Phenotypical profiling of cultured hEPDCs was performed previously to support their epicardial origin (22 (link)–24 (link)). Biophysical and biochemical stimulation experiments were performed with spindle-shaped hEPDCs from P 4–8 of five different patients. Purity of the hEPDC culture was determined with staining for Wilms tumor-1 (WT1) (Table 1 ).
4
Signaling Pathways in VEGF-Induced Angiogenesis
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Recombinant VEGF-A was from PeproTech (Rocky Hill, NJ, USA). DMEM, fetal bovine serum (FBS), TrypLE™, Medium 199 (M199), and all cell culture reagents were from Invitrogen (Carlsbad, CA, USA). Antibodies against VEGFR-2, anti-phospho-VEGFR-2 (Y1175), Akt and anti-phospho-Akt (S473), ERK1/2 and anti-phospho-ERK1/2 (T202/Y204), FAK, anti-phospho-FAK (Y397), Src and anti-phospho-Src phosphorylated (Y416), were from Cell Signaling (Danvers, MA, USA). Antibody against α-tubulin and anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase antibodies were from GeneTex Inc (Irvine, CA, USA). All materials for immunoblotting were from Bio-Rad (Hercules, CA, USA). BD MatrigelTM basement membrane matrix was from Becton Dickinson (Mountain View, CA, USA). The immobilon Western chemiluminescence HRP substrate was from Millipore (Billerica, MA, USA). Cell Proliferation ELISA, BrdU assay kit was from Roche (Indianapolis, IN, USA). Toluidine blue O, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA).
5
In Vitro Culture of Porcine Embryos
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All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise. The base medium for the IVG culture for SAFCOCs was Minimum Essential Medium alpha medium (a-MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 0.1% (w/v) polyvinyl alcohol (PVA), 0.4 mM pyruvate, and 75 μg/mL kanamycin. For the IVM of immature porcine oocytes, medium-199 (M-199) (Invitrogen, Grand Island, NY, USA) was used. This IVM medium was supplemented further with 10% (v/v) porcine follicular fluid, 0.4 mM pyruvate, 0.6 mM cysteine, 10 ng/mL epidermal growth factor (EGF), 1 μg/mL insulin, and 75 μg/mL kanamycin. The medium for the in vitro culture (IVC) of PA embryos was porcine zygote medium (PZM)-3, which contained 0.3% (w/v) bovine serum albumin (BSA). The IVC medium was modified in this study by adding 0.34 mM tri-sodium citrate, 2.77 mM myo-inositol, and 10 μM β-mercaptoethanol [28 (link)].
6
Zebrafish and Fathead Minnow Cell Cultures for Virus Propagation
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Zebrafish liver (ZFL) cells were obtained from a pool of 10 adult zebrafish livers, whose sex were not recorded (American Type Culture Collection, ATCC, www.atcc.com ). ZFL cells were cultured at 28°C in 5% CO2 in Ham’s F12 nutrient mixture medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Epithelioma papulosum cyprini (EPC) cells were derived from fathead minnow (Pimephales promelas) without sex record [42 (link)], which is belongs to Cyprinidae and is also phylogenetically closely related to zebrafish. EPC cells were obtained from China Center for Type Culture Collection (CCTCC) and were maintained at 28°C in 5% CO2 in medium 199 (M199) (Invitrogen) supplemented with 10% FBS. SVCV, a negative ssRNA virus, was propagated in EPC cells until a CPE was observed, and then cell culture fluid containing SVCV was harvested and centrifuged at 4 × 103g for 20 min to remove the cell debris, and the supernatant was stored at -80°C until used. CaHV was provided by Prof. Q. Y. Zhang (Institute of Hydrobiology, Chinese Academy of Sciences) and propagated by intraperitoneal injection into healthy gibel carp. The isolation method of CaHV was used as previously described [43 (link)].
7
GelMA-Collagen Hydrogel Fabrication
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GelMA macromer was prepared by dissolving 8 ml of methacrylic anhydride (Sigma) in 100 ml of a 10% (w/v) porcine skin gelatin solution (Sigma) at 60 °C. The mixture was then diluted with 100 ml DPBS and dialyzed at 50 °C for 1 week before being lyophilized. 20% (w/v) GelMA pre-polymer solution with 0.25% (w/v) photo-initiator Irgacure 2959 (Specialty Chemicals) was prepared to be mixed with collagen. The GelMA-collagen prepolymer solution blend was prepared by first mixing 0.9 μl 1N NaOH with 7.2 μl of 10× Medium 199 (M199, Gibco). Next, 36 μl of 3 mg/ml collagen I rat tail solution (Gibco) was gently added to the NaOH/M199 solution and mixed thoroughly until the collagen was neutralized. Once the collagen was neutralized, 25.2 μl of 20% GelMA was added to the collagen mixture to generate the GelMA-collagen blend.
8
Culture of Human Mesothelial Cells
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Human mesothelial cells (MeT-5A) were obtained from American Type Culture Collection (ATCC® CRL-9444TM, ATCC, Manassas, VA, USA). They were cultured in Medium 199 (M199) (Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Cell cultures were maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere.
Cell cultures were maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere.
9
Isolation and Culture of Endothelial and Mesothelial Cells
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Human endothelial umbilical vein cells (HUVEC), human cardiac microvascular endothelial cells (HCMEC) and the immortalized mesothelial cell line (MeT-5A) were purchased from established vendors (HUVEC and HCMEC from Promocell, Heidelberg, Germany; MeT-5A (ATCC® CRL-9444™) from LGC Standards, Wesel, Germany). Endothelial cells were grown in endothelial cell growth medium (Promocell, Heidelberg, Germany) with supplements and antibiotics according to the manufacturer’s instructions. MeT-5A were cultured in Medium 199 (M199, 31150022, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) foetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% (v/v) penicillin/streptomycin (P/S, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Human peritoneal mesothelial cells (HPMC) were isolated from four non-uremic patients and cultured as previously described [11 (link)], as approved by the Ethics Committee of the Medical Faculty, Heidelberg University (S-501/2018). Informed written consent was signed by the patients. The cells were grown in M199 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 0.5 μg/mL insulin, 0.5 μg/mL transferrin, 0.4 μg/mL hydrocortisone and 2 mM L-glutamine (all from Merck, Darmstadt, Germany).
10
Angiogenesis Assay with Arnebin-1 and VEGF
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Arnebin-1 was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and recombinant human VEGF was from PeproTech Inc. (Rocky Hill, NJ, USA). Growth factor-reduced Matrigel basement membrane matrix was obtained from BD Biosciences (Bedford, MA, USA). Medium 199 (M199) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LY294002, a PI3K inhibitor, was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents utilized were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
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