Fibroblast growth medium

REF52 cells, stably transfected to express β3-integrin with EGFP tag on its intracellular domain (Dr. Bernhard Wehrle-Haller, University of Geneva, Switzerland) [45] (link), [48] (link)–[50] (link), were cultured in Dulbecco’s modified Eagle’s media (DMEM) (Gibco, Invitrogen) supplemented with 10% fetal calf serum (Gibco, Invitrogen) and 1% penicillin–streptomycin (Gibco, Invitrogen). HFF cells (PromoCell) were cultured in fibroblast growth medium (PromoCell). For experiments, the culture media for HFF cells were changed to the GlutaMAX-1 containing DMEM (Gibco, Invitrogen) supplemented with 10% newborn calf serum (Gibco, Invitrogen) and 1% penicillin–streptomycin (Gibco, Invitrogen). All cells were cultured in culture flask (BD Biosciences) in a humidified incubator at 37°C with 5% CO₂ and 95% air for several passages before experiments.

The inhibitory potencies of monomeric 2C1 and all four 2C1-Fc fusion proteins were determined by stimulating normal human dermal fibroblasts with recombinant IL-17A and TNF, as they act in synergy (26 (link)). Normal human dermal fibroblast cells (ATCC PCS-201-010) were seeded in 96-well plates (TPP) in fibroblast growth medium (Promocell). The cells were allowed to adhere. Supernatant was exchanged with medium containing IL-17A (34 pm, R&D Systems) and TNF (1.2 pm, Pierce), with and without the anti-IL-17A compounds (2C1, 2C1-Fc fusions, and commercially available IL-17R-Fc (R&D Systems)). As negative control, equal volume of PBS was added to the medium. Additionally, cells were treated with TNF only to mimic IL-6 levels after full IL-17A inhibition (“TNF control”). After 24 h, supernatants were collected, filtered (0.45 μm pore size), and used to determine IL-6 concentrations by ELISA according to the manufacturer's instructions (DuoSet, R&D Systems). Data points were measured in triplicate. Data indicate a representative result. The percentages of inhibition were plotted, and IC₅₀ values were calculated using Prism software (version 5). The percentage of IL-17A inhibition was determined with the following formula.

Ang II, ET-1, PD123319, gallein (G_βγ inhibitor), and BQ788 were obtained from Tocris Bioscience (Ellisville, MO, USA). Recombinant human TGF-β1, valsartan, bosentan, ambrisentan, LY2109761, FR180204, and SB203580 were obtained from Sigma Aldrich (Saint Louis, MO, USA). SIS3 (Smad3 inhibitor) and FR900359 (G_αq inhibitor) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Fibroblast growth medium and related cell culture reagents were obtained from Promocell (Heidelberg, Germany).

All cells used in this study were checked for contamination with mycoplasma before and after cell growth. The pancreatic tumor cell lines A818-1, AsPC-1, BxPC-3, CFPAC-1, Colo357, MIA PaCa-2, Paca44, PANC-1, Pt45P1 and SK-PC-1 and six primary PDAC cell lines were used in the analysis (Table 1); the primary cells have been described in detail previously [44 (link)]. Normal human dermal fibroblasts (NHDFs) (PromoCell, Heidelberg, Germany) acted as control. All cell lines but the fibroblasts were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen, Darmstadt, Germany) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The fibroblasts were grown in PromoCell fibroblast growth medium. Cells were cultured to 85-90% confluency; then the medium was removed and the cells were washed three times with phosphate-buffered saline (PBS) followed by two washes with serum-free growth medium. Subsequently, the cells were incubated in serum-free growth medium for 12 h in order to synchronize cell growth. The medium was replaced and cells were incubated for another 48 h. Then, the medium was collected, centrifuged at 3500 g for 10 min, filtered through 0.22 μM nylon filters and stored at -80°C.

Normal human dermal fibroblasts (NHDFs) and Fibroblast Growth Medium containing 2% fetal calf serum (FGM), insulin (5μg/mL) and FGF-2 (1ng/mL) were obtained from PromoCell (http://www.promocell.com/) and cells were cultured as specified by the supplier. Human Primary Mammary Fibroblasts (HPMFs), HPMF growth media (HPMF-GM), the Fibroblast Medium Supplement Kit (FBS, hydrocortisone, L-glutamine, FGF and an antibiotic-anti-mycotic solution) and gelatin coating solution were obtained from Cell Biologics (http://www.cellbiologics.net). HPMF cells were cultured in HPMF-GM with the added supplements for 6–7 passages as specified by the supplier. MDA-MB-231 breast cancer cells were obtained from ATCC and cultured for 12–15 passages in MCF media. MCF medium is DMEM/F12 (Life Technologies, Carlsbad, CA) supplemented with 5% horse serum, human recombinant EGF, insulin, hydrocortisone and cholera toxin, all obtained from Sigma Aldrich (St. Louis, MO), and penicillin-streptomycin from Thermo -Fisher (Grand Island, NY) [21 (link)]. GFP tagged human dermal fibroblasts (NHDFs-GFP) were obtained from Angio-proteomie (www.angioproteomie.com) and cultured according to the supplier’s directions.

Low-adherent well plates were prepared
by coating them with 5 mg/mL poly(2-hydroxyethyl methacrylate) solution
(Sigma) and stored sealed at 4 °C until use. BC and BC-Ppy materials
were sterilized with UV irradiation for 15 min on each side and secured
by polytetrafluoroethylene rings at the bottom of each well.
The scaffolds were immersed in DMEM containing 4.5 g/L glucose, 50%
FBS (Gibco, Invitrogen), and incubated overnight inside a cell culture
incubator at 37 °C with 5% CO₂. Scaffolds were coated
with collagen (100 mg/mL, VitroCol, Advanced Biomatrix) prior to myoblast
seeding.
Thirty thousand cardiomyoblasts/cm² or fifteen
thousand cardiac fibroblasts/cm² were seeded on top of
each scaffold. Culture plastic wells (without scaffolds) were used
as controls. Adult human cardiac fibroblasts (p5-p6, PromoCell) were
cultured in fibroblast growth medium (PromoCell), whereas H9c2 rat
cardiac myoblasts (p2-p9, ATCC) were cultured with DMEM containing
4.5 g/L glucose, 10% FBS, 1% l-glutamine (Gibco, Thermo Fisher),
and 1% penicillin/streptomycin (Gibco, Invitrogen). Cultures were
maintained in a humidified atmosphere with 5% CO₂ at 37
°C, and culture medium was changed every 2 days.