Static_BKF_10uM_3
Homo sapiens
Protocols: Organoids were extracted from Matrigel using dispase and collagenase in HBSS as described before ('Thyroid follicle extraction and re-plating in membranous carriers'). A single-cell suspension was prepared by incubation with TrypleE Express (Thermo Fisher) for 15 min at 37˚C. Differentiation medium was added to inactive the enzymes. After centrifugation, cells were washed with FACS buffer (PBS containing 2% FBS) and dead cells were stained with DRAQ7 (Biolegend) dye (1:500 in FACS buffer). Samples were filtered through a 30 µm cell strainer. FACS sorting of viable (DRAQ7-) NKX2-1/GFP+ cells was performed using a BD FACS Fusion cell sorter (BD Biosciences) and data was processed using FACSDiva software (BD Biosciences). Human thyroid follicles were generated from hESCs following a 58-day protocol developed by Romitti et al (2022). The HES3 hESC cell line was genetically modified to produce a NKX2-1wt/GFP reporter cell line (Golburn et al. 2011), which was used to generate a tetracycline-inducible NKX2-1-PAX8 hESC line. NKX2-1-PAX8 hESCs were cultured on Matrigel-coated 6-well culture dishes in Stem Flex medium (Thermo Scientific). Highly confluent hESCs were detached and cultured in hanging drops (2000 cells per droplet) for two days to form embryoid bodies (EBs). Then, EBs were collected, embedded in growth factor-reduced Matrigel (Corning) and plated in 50 µl drops (20 EBs per drop). EBs were cultured in differentiation medium: DMEM/F12 + Glutamax (Gibco) supplemented with 20% FBS, 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 100 U/ml Penicillin-Streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 50 µg/ml L-ascorbic acid (Sigma). To induce foregut endoderm, basal differentiation medium was supplemented with 50 ng/ml Activin A for three days. Next, NKX2-1 and PAX8 expression was induced by treatment with 1 µg/ml doxycycline for four days (Sigma). In the following week, cells were cultured in basal differentiation medium to allow expansion of thyroid progenitors. In the next weeks, proliferation and differentiation was induced by incubation with 300 µM 8-br-cAMP (Biolog Inc.), followed by treatment with 1 mU/ml hrTSH (Genzyme), 50 nM dexamethasone (Sigma) and 10 µM SB431542 (R&D systems) to promote folliculogenesis, maturation and TH synthesis. EDC exposure experiments were performed in flow (ThyO chip) and static conditions (organoids). Thyroid follicles were exposed to 10 µM benzo[k]fluoranthene (BkF, Cas number: 207-08-9) or an equal amount of DMSO (0.5%, control) in the culture medium (unexposed control). After seven days of exposure, samples were collected from ThyO chips or culture plates for downstream analysis as described in the respective sections. Between eight to sixteen ThyO chip models were assembled per experiment by stacking vertically a membranous carrier between two fluidic gaskets. The system was closed inside the housing components and compressed by screwing a set of six M3 screws to provide leak-tight conditions. The assembled devices were placed on a white Delrin® manifold and connected upstream to an IPC-N Ismatec peristaltic pump (78000-47, Metrohm, The Netherlands) and downstream to glass reservoirs. All fluidic connections were made via Luer-type connectors and Ø0.8 mm Masterflex™ Puri-Flex™ tubing (11739799, Fisher Scientific). To avoid bubble formation, a positive pressure was established throughout the fluidic system and all equipment, including pump and reservoirs were incubated in an ICO240 incubator (Memmert GmbH + Co. KG, Schwabach, Germany) with active humidity control (set to 70% rh). The flow rate was set to 25 µL/min for seven days. Sorted cells were collected in FACS buffer. Sorted viable NKX2-1/GFP+ cells were lysed using QIAzol Lysis Reagent (Qiagen) and RNA was extracted using the miRNAeasy Micro Kit (Qiagen) according to manufacturer's instructions. RNA-Seq libraries were prepared using 2 ng of RNA as input with the SMART-Seq® mRNA LP (with UMIs) (634762, Takara Bio Europe, France). The libraries were quantified on a Qubit 2.0 Fluorometer (ThermoFisher) and quality control was performed on the BioAnalyzer 2100 expert (Agilent).