Foetal bovine serum fbs

HeLa (cervical adenocarcinoma cells) and SCC-13 (skin squamous carcinoma cells) human cancer cell lines were used for cell cultures. They were grown using DMEM (Dulbecco’s Modified Eagle’s medium) culture media, supplemented with 10% Foetal Bovine Serum (FBS) and 1% antibiotics (G penicillin, 100 U/mL; streptomycin, 100 µg/mL), purchased from Fisher. They were incubated in HERACell incubator (Heraus) at 37 °C with 5% CO₂ concentration and 95% relativity humidity.

Well inserts containing Alvetex^® Polaris polystyrene membranes (Reprocell Europe, Glasgow, UK), which consist of 3 µm and are not commercially available, washed in 70% ethanol overnight, before being rinsed twice with sterile phosphate buffered saline solution (PBS). Inserts were then coated using Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning) to facilitate EB attachment before being placed in 6-well plates with 5 mL of the appropriate culture medium in the well consisting of either Knockout DMEM, 20% Knockout Serum Replacement (Fisher Scientific UK), 1 mM L-glutamine, 1% non-essential amino acids and 0.1 μM β-mercaptoethanol or high glucose DMEM (Fisher Scientific), 10% Foetal Bovine Serum (FBS, Fisher Scientific UK), 2 mM L-glutamine, 55 µM β-mercaptoethanol, 0.1 mM nonessential amino acids and 100 U/mL Penicillin/Streptomycin (Fisher Scientific UK).
Once PSC-derived EBs had formed, they were transferred to the prepared Alvetex^® inserts, with around 50–100 EBs placed on each polystyrene insert and allowed to attach overnight at 37 °C, 5% CO₂ and 95% air. The following day wells were topped up to 10 mL total media. EBs were then maintained with regular media changes for a defined timescale, before fixation in 4% paraformaldehyde (PFA, Fisher Scientific UK) at 4 °C overnight.

The C2C12 myoblast cells were cultured in growth media [Dulbecco’s modified eagle medium (DMEM) with 10% (v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (all from Fisher Scientific, UK)] at 37 °C with 5% CO₂. Subculturing by passaging was carried out using TrypLE recombinant enzyme (Fisher Scientific, UK) to maintain the cells below 60% confluency. The cells younger than passage 20 were used in all assays in this study.

Folate free RPMI-1640 media, Gibco McCoy’s 5A (modified) medium, foetal bovine serum (FBS), penicillin–streptomycin (5000U/mL), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic Acid (DiI), nitric acid, Trypan Blue, isopropanol, NaH₂PO₄ (sodium dihydrogen phosphate), Na₂HPO₄ (disodium hydrogen phosphate), and Trypsin-EDTA were purchased from Fisher Scientific (Loughborough, UK). Soy phosphatidylcholine (PC), cholesterol (Chol), methoxy(polyethyleneglycol)-2000-distearoyl-phosphatidylethanolamine (mPEG-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-folate(polyethylene glycol)-2000 (DSPE-PEG₂₀₀₀-FA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-folate (polyethylene glycol)-5000 (DSPE-PEG₅₀₀₀-FA) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). VECTASHIELD Hard Set Mounting Media with DAPI were obtained from Vector Labs (Peterborough, UK). Phosphate-buffered saline (PBS), ATO, nickel acetate, dimethyl sulfoxide (DMSO), thiazolyl blue tetrazolium bromide powder (MTT), Whatman Anotop 0.1 µm syringe filters, and dialysis tubing were purchased from Sigma (Welwyn Garden City, UK). Ham’s F-10 Nutrient Mix, methanol, and dichloromethane were obtained from Thermofisher (Paisley, UK).

Bombyx mori silk was purchased from Jiangsu, China. Na₂CO₃ (11552), DMSO (dimethyl sulfoxide, A13280), curcumin (B21573.09), and dopamine hydrochloride (A11136) were purchased from Alfa Aesar, Haverhill, MA, USA. Methanol (34860), CaCl₂ (C1016), C₂H₅OH (ethanol, 51975), 2-Methylimidazole (M50850), a zinc assay kit (MAK032-1KT), a dialysis tubing cellulose membrane (D9777-100FT), paraformaldehyde (158127), and hydrochloric acid (7647-01-0) were purchased from Sigma-Aldrich, St. Louis, MO, USA. PI/RNAse Staining Solution, Zinc nitrate hexahydrate (228737), Tris Base (BP152-1), MTT (M6494), DAPI (D1306), DiD (V22887), and foetal bovine serum (FBS) were purchased from Fisher Scientific, Waltham, MA, USA. PBS (BE17-512F), RPMI-1640, DMEM (−)Pyruvate and DMEM (+)Pyruvate were purchased from Lonza, Basel, Switzerland. Human Caucasian breast adenocarcinoma cells (MDA-MB-231) and AD-293 human embryonic kidney cells were purchased from ECACC, Salisbury, UK. Human breast cancer cells (SK-BR-3 and MCF-7) were purchased from ATCC, Manassas, VA, USA.