Insulin

For the generation of multicellular spheroids with adipocytes, ASCs were cultured in adipogenic differentiation medium consisting of growth medium with insulin (final concentration 1.7 µM; PromoCell, Heidelberg, Germany), dexamethasone (1 µM; Sigma-Aldrich), 3-isobutyl-1-methylxanthine (IBMX, 500 µM; Serva-Electrophoresis, Heidelberg, Germany), and indomethacin (200 µM; Sigma-Aldrich). Cells were differentiated in conventional 2D culture for 14 days prior to detachment and subsequent spheroid formation. Successful differentiation was verified by qRT-PCR of adipogenic marker genes and determination of intracellular triglyceride content. Adipocyte-containing mono- and co-spheroids were generated as described above for ASCs.

Lung samples were collected from IPAH patients and individuals without PAH (mentioned as Donors) according to the protocol approved by the ethics committee at Faculty of Human Medicine of the University Hospital Giessen (Giessen, Germany) according to European IPS registry (AZ 111/08) and DZL Biobank (58/15) and in accordance with national law and with Good Clinical Practice/International Conference on Harmonization Guidelines. Tissues were obtained during lung transplantation. Human tissue donation was approved by the ethics committee of the University Hospital Giessen in agreement to the principles stated in the Declaration of Helsinki. Patients with IPAH had a mean age 35.38 ± 10.85 (years ± SD). Control individuals had a mean age 43.70 ± 10.81 (years ± SD) [24 (link)].
Human PASMCs were cultured in smooth muscle cell (SMC) growth medium-2 (SmGM-2) with inclusion of the supplement mix, containing 5% fetal bovine serum, basic fibroblast growth factor (2 ng/mL), epidermal growth factor (0.5 ng/mL), and insulin (5 μg/mL) (PromoCell GmbH).

Patient-derived cells were dissociated from solid biopsies through mechanic grinding followed by enzymatic digestion using a 0.1% collagenase mixture for 1−2 h at 37 °C under gentle agitation. The cell mixture was then passed through a 100 μm mesh, and the collagenase solution was replaced by fresh culturing medium.
Most sarcoma cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12) supplemented with 15% fetal bovine serum (FBS), non-essential amino acids, penicillin and streptomycin. Angiosarcoma cells were cultured in endothelial cell media containing vascular endothelial growth factor (VEGF), insulin growth factor (IGF) and basic fibroblast growth factor (FGF). Muscle cells were grown in skeletal muscle cell media containing 5% fetal calf serum, basic FGF, insulin, and dexamethasone (Promocell, Germany).
Ewing’s sarcoma (ES), alveolar rhabdomyosarcoma (aRMS) and angiosarcoma samples were obtained by fine needle aspiration (FNA) from palpable tumours. The content of tumour cells in the FNA biopsy was assessed on site by microscopic evaluation of Grunewald−Giemsa-stained FNA smears prior to in vitro cultivation.
Logarithmic growing patient-derived cells (PDC) were established within a range from 2 days to 18 weeks depending on the biopsy size, cell viability and histological subtype.

Human uterine smooth muscle cells (also known as HUt-SMC [PromoCell, Heidelberg, Germany]),will be identified as commercial primary myometrial cells, and abbreviated as cpMYO) were derived from human myometrium and were grown in smooth muscle cell growth medium 2 and supplemented with 5 % fetal calf serum (PromoCell), 0.5 % epidermal growth factor (PromoCell), basic fibroblast growth factor (PromoCell), and insulin (PromoCell). Human uterine leiomyoma cells (GM10964, will be identified as commercial primary leiomyoma cells and abbreviated as cpLYO) were purchased from Coriell Institute for Medical Research (Camden, NJ, USA), and were cultured in Medium 199 (1x) (Gibco, Grand Island, NY, USA) and 15 % fetal bovine serum (FBS) (Gibco), heparin (Sigma-Aldrich, St. Louis, MO) and endothelial cell growth supplement (ECGS) (PromoCell). All cell cultures were incubated in 5 % CO₂at 37 °C. Upon reaching 70–80 % confluence, the cells were passaged using 0.05 % trypsin-EDTA (Gibco).

Human nasal epithelial cells (HNEpC; PromoCell) were cultured in Airway Epithelial Cell Growth Medium (basal medium supplemented with BPE, EGF, insulin, hydrocortisone, epinephrine, triiodo-L-thyronine, transferrin and retinoic acid; PromoCell) at 37 °C in a 5% CO₂ atmosphere. HNEpC cells at passage 5 were used in the experiments. The cells were plated in 48-well plates (BD Falcon) coated with type I collagen (Corning) at the initial density of 1 × 10⁴ cells/cm², 24 h prior to the experiments. The growth medium was then changed to the experimental cell culture medium containing nanoparticles or vehicle control (water). The experimental medium consisted of phenol red-free DMEM, prepared from powder using water or a mixture of water and 214 mg L⁻¹ TA-AgNP suspension in water and supplemented with 1 gL⁻¹ glucose, NaHCO₃ (Gibco), sodium pyruvate (Lonza), 1% bovine serum albumin (BSA), L-glutamine and P/S (50 units/mL of penicillin and 50 µg/mL of streptomycin). Water (used as vehicle control) was a component of the cell culture medium, not an additive to the medium, and as such, the osmolality of the experimental medium was not compromised. A 48 h incubation in the experimental medium had no adverse effects on the viability of the cells compared to the PromoCell culture medium. Cell culture reagents were from Sigma unless otherwise specified.