HMEC-1 (generous gift from the Welch lab, University of California, Berkeley) were cultured in MCDB 131 medium (Fisher Scientific; 10372–019) supplemented with 10% FBS (GemBio; 900-108), 10 ng/mL epidermal growth factor (Sigma; E9644), 1 μg/mL hydrocortisone (Sigma; H0888), and 2 mM l-glutamine (Sigma; 56-85-9). HUVEC (Lonza C2517A) were cultured according to the manufacturer’s instructions (EGM Bullet Kit-2, Lonza CC-3162). Passages used were between P4–P8.
Mcdb 131 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, France, Germany, Australia, Italy, Belgium
MCDB 131 is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It is a basal medium that provides the necessary nutrients and components for cell proliferation and survival. The medium is formulated to maintain a consistent pH and osmolarity to create an optimal environment for cell culture.
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Lab products found in correlation
Hydrocortisone, by Merck Group (41 mentions)
L glutamine, by Thermo Fisher Scientific (36 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Glutamax, by Thermo Fisher Scientific (20 mentions)
Penicillin, by Thermo Fisher Scientific (20 mentions)
Streptomycin, by Thermo Fisher Scientific (19 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (18 mentions)
Epidermal growth factor (egf), by Merck Group (15 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (15 mentions)
L glutamine, by Merck Group (13 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Penicillin, by Merck Group (9 mentions)
Hmec 1, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Merck Group (7 mentions)
Ascorbic acid, by Merck Group (7 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (6 mentions)
Hmec 1, by American Type Culture Collection (6 mentions)
Recombinant human epidermal growth factor, by R&D Systems (5 mentions)
Chemically defined lipid concentrate, by Thermo Fisher Scientific (5 mentions)
168 protocols using mcdb 131 medium
1
Culturing HMEC-1 and HUVEC Cell Lines
2
Cultivation of Human Dermal Microvascular Endothelial Cells
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Human dermal microvascular endothelial cells HMEC-1 (generous gift from the Welch lab, University of California, Berkeley previously obtained from Centers for Disease Control, Biological Products Branch) were maintained in MCDB131 medium (Fisher Scientific; 10372-019) supplemented with 10% fetal bovine serum (GemBio, 900108), 10 ng/mL epidermal growth factor (Sigma, E9644), 1 μg/mL hydrocortisone (Sigma, H0888), and 2 mM L-Glutamine (Sigma, 56-85-9) (Reed et al., 2014 (link)). Cells were passaged 1:6 at 90% confluence.
3
Culturing Primary Human Brain Endothelial Cells
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ACBRI376 cells (primary human brain microvascular EC) were cultured in complete Complete Classic Medium (Cell Systems, Kirkland, WA, USA) supplemented with 1% antibiotic-antimycotic (Fisher Scientific, Brussels, Belgium) and 2% CultureBoost (Cell Systems). According to the manufacturer, these cells issued from human brain cortex tissue express after plating Cluster of Differentiation 31 (CD31) and von Willebrand Factor, known as EC markers, as well as Zonula Occludens 1 (ZO-1), a biomarker of tight junctions.
HepaRG (hepatocyte cell line) was maintained in William’s E medium supplemented with 10% fetal bovine serum (FBS), 13% thaw, plate, and general purpose medium supplement, and 1% GlutaMAX (all from Fisher Scientific). N18(H) (neuroblastoma cell line) and 1321N1 (astrocytoma cell line) cells were cultured in DMEM (4.5 g/L glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS and penicillin/streptomycin 1% for N18(H) or 2% for 1321N1 (all from Fisher Scientific). HUVEC (human umbilical vein EC) cells were cultured in MCDB131 medium supplemented with 20% FBS, 1% L-glutamine, 1% antibiotic-antimycotic, and 0.14% heparin 5000 U/mL (all from Fisher Scientific).
Experiments on N18(H) cells were performed after differentiation. Cells were immobilized on the appropriate support, and the differentiation was induced the second day, using medium containing 0.2% FBS for 48 h.
HepaRG (hepatocyte cell line) was maintained in William’s E medium supplemented with 10% fetal bovine serum (FBS), 13% thaw, plate, and general purpose medium supplement, and 1% GlutaMAX (all from Fisher Scientific). N18(H) (neuroblastoma cell line) and 1321N1 (astrocytoma cell line) cells were cultured in DMEM (4.5 g/L glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS and penicillin/streptomycin 1% for N18(H) or 2% for 1321N1 (all from Fisher Scientific). HUVEC (human umbilical vein EC) cells were cultured in MCDB131 medium supplemented with 20% FBS, 1% L-glutamine, 1% antibiotic-antimycotic, and 0.14% heparin 5000 U/mL (all from Fisher Scientific).
Experiments on N18(H) cells were performed after differentiation. Cells were immobilized on the appropriate support, and the differentiation was induced the second day, using medium containing 0.2% FBS for 48 h.
4
Endothelial Cell Treatment with Antioxidants
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Human dermal microvascular endothelial cells, HMEC-1 (purchased from ATCC, Cat. No. CRL3243™), were cultured in MCDB 131 Medium (Thermo Fisher Scientific) supplemented with FBS (10%, Thermo Fisher Scientific), L-glutamine (2 mM, Thermo Fisher Scientific), hydrocortisone (0.05 mg/ml, Sigma-Aldrich), Epidermal Growth Factor (5 ng/ml), 100 U/ml penicillin, 10 μg/ml streptomycin, and 250 ng/ml amphotericin B (Sigma-Aldrich). HMEC-1 were cultured under standard conditions (37°C, 5% CO2) and passaged two times a week. In all experiments, cells between the second and tenth passages were used only when they reached full postplating confluency. Bardoxolone methyl (CDDO methyl ester, Cayman Chemical), dimethyl fumarate (Sigma-Aldrich), and L-sulforaphane (Sigma-Aldrich) initially diluted in DMSO were added to the culture medium at final concentrations of 100 nM, 300 nM, 500 nM, 1 μM, 3 μM, and 5 μM in triplicates, if not stated otherwise. Control cells were treated with 0.05% DMSO added to the culture medium. To check for acute toxicity, the cells were incubated with tested agents for 3 hours. Standard toxicity assessment time was set to 24 hours, according to the literature [30 (link)].
5
Aortic Ring Angiogenesis Assay
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According to previously described methods,18 the thoracic aortas were removed from 6‐ to 9‐week‐old BALB/c mice after cervical dislocation and cut into 1‐mm‐thick rings. Each ring was placed on a prechilled 96‐well plate coated with 40 µl Matrigel and incubated at 37°C for 20 minutes. Then, additional 40 µl Matrigel was added and incubated at 37°C for 20 minutes. The embedded rings were incubated with 150 μl MCDB 131 medium (ThermoFisher) in the presence or absence of 20 ng/ml VEGF, with replacement of the medium every 2 days. In the cisplatin‐treated rings, cisplatin (5 µM) was added for 2 days, and cisplatin was withdrawn for subsequent incubation. In the VEGF + cisplatin + FIR group, cisplatin (5 µM) was added for 30 minutes, followed by exposure to FIRx2/day for 2 days. Then, the medium without cisplatin was replaced, and the treatment of the rings with FIRx2 was continued for 3 days. On day 5, the angiogenesis was evaluated by the capillary sprouts from aortic rings by using an inverted microscope equipped with a digital camera (SAGE VISION).
6
Directed Differentiation of Definite Endoderm and Primitive Gut
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Cells were directed towards definite endoderm (DE) and primitive gut tube (PG) in MCDB 131 medium (cat# 10372-019, Thermo Fisher Scientific) with 1% 100x GlutaMAX, 1.5 g/L NaHCO3, 0.5% BSA, and a 10 mM final glucose concentration. Differentiation to DE was done in 3 days by daily adding 100 ng/mL Activin A (cat# 120-14, PeproTech) and 0.3 μM CHIR-99021 (reduced to 0 on the last day) (cat# S2924, Selleckchem). Further differentiation to PG was done in 2 days by daily adding 0.25 mM ascorbic acid (cat# A4544, Sigma) and 50 ng/mL FGF7 (cat# 100-19, PeproTech). The cells were analyzed by flow cytometry.
7
Isolation and Culture of BMVECs
8
Efficient Pancreatic Cell Differentiation
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For differentiation, P2 organoids, which highly express PDX1 protein, were considered equivalent to hESC stage 3 (Rezania et al., 2014 (link)), washed with PBS (Mg/Ca free), and changed to MCDB131 medium (Thermo Fisher Scientific) supplemented with compounds as listed in Table S5 for 3 days each for stage 1 (S1)–S3 and 6 days for S4. Medium was changed daily. Samples were taken from each step for RNA analysis. Control organoids were kept in MCDB131 medium supplemented with 1× GlutaMAX.
9
Culturing Various Cell Lines
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HEK293E (ATCC, CRL-10852) and Jurkat cells (ATCC, TIB-152) were cultured in RPMI media (Gibco, #61870-010) supplemented with 10% fetal bovine serum (FBS) (Sigma, #10500-064) and 1% PenStrep (Sigma, #15140-122). HT-29 colorectal cancer cells (ATCC, HTB-38) were grown in McCoys 5 A basal media (Gibco) with 10% FBS and 1% PenStrep. A431 epidermoid carcinoma cells (ATCC, CRL-1555) were grown in DMEM (Gibco, #41966-029) with 10% FBS and 1% PenStrep. MCF7 breast adenocarcinoma cells (ATCC, HTB-22) were grown in DMEM (Gibco) with 10% FBS and 1% PenStrep. CHO Chinese hamster ovary cells (ATCC, CCL-61) were grown in DMEM with 10% FBS and 1% PenStrep. 3T3 and 3T3-EGFR cells lines were cultured in DMEM media with 10% FBS and 1% PenStrep as previously described26 (link). HMEC1-FcRn cells (previously generated in collaboration with Novozymes)18 (link) were cultured in MCDB131 medium (Thermo Fisher Scientific, #10372019) supplemented with 10% FBS, 2 mM L-glutamine, 10 ng ml−1 human EGF (Peprotech), 1 ng ml−1 fibroblast growth factor (Peprotech), 50 µg ml−1 gentamicin (Sigma, #G1397) and 0.25 µg ml−1 Fungizone (FisherScientific, #15290018). All cell lines were cultured in humidified atmosphere at 37°C with 5% CO2.
10
Culturing Human Dermal Microvascular Endothelial Cells
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Human microvascular endothelial cells (HMECs) of the dermal HMEC-1 line (catalogue no. CRL-3243) were purchased from ATCC® (Manassas, VA, USA) and grown in MCDB131 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) GibcoTM fetal calf serum (FCS), 2.5 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 pmol/L hydrocortisone, and 5 ng/mL epidermal growth factor (EGF).
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