Penicillin streptomycin fungizone

Poly(Ɛ-caprolactone) (PCL, M_n = 80,000), glutaraldehyde, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), Hank’s balanced salt solution (HBSS), antibiotics (penicillin–streptomycin–fungizone), and paraformaldehyde were ordered from Sigma-Aldrich (St. Louis, MO, USA). Hexafluoro-2-propanol (HFIP) was acquired from Oakwood Inc. (West Columbia, SC, USA). Thiolated a₅b₁ integrin-binding peptides, CellPhilic™ xeno-free and serum-free culture medium for human epithelial cells (SKU: ESC-10×50 mL), vinylsulfone-functionalized polyvinyl alcohol (PVA-VS), and 4-arm polyethylene glycol acrylate (PEGTA) were ordered from Biomaterials USA LLC (Richmond, VA, USA). LIVE/DEAD™ Reduced Biohazard Cell Viability Kit, AlexaFluor 488/546 Phalloidin, and 4′-6-diamidino-2-phenylindole (DAPI) were purchased from ThermoFisher (Carlsbad, CA, USA). Alamar Blue™ reagent was obtained from Bio-Rad Laboratories (Hercules, CA, USA). The adult human retinal pigmented epithelium-19 (ARPE-19, ATCC-CRL-2302™) cell line was purchased from American Type Culture Collection (Manassas, VA, USA).

DMEM with and without phenol
red and fetal calf serum (FCS) were purchased from Thermo Fisher Scientific.
Penicillin/streptomycin/fungizone (PSF; 2%), trypsin, and poly-d-lysine were purchased from Sigma, while ITS+ (Insulin-transferrin-sodium
selerite with bovine serum albumin and linoleic acid; 1:100) was from
BD Bioscience. Bacterial chondroitinase ABC (Sigma) was used in a
buffer containing 50 mM Tris base and 50 mM sodium acetate (pH 8.0;
Sigma). Cell counts were conducted using a Countess automated cell
counter (Thermo Fisher Scientific).

Cell cultures: The human oral keratinocyte cell line OKF6/TERT-1 was grown in keratinocyte serum free medium (KSFM) supplemented with L-glutamine and penicillin-streptomycin-fungizone (Sigma-Aldrich) in the presence of 0.03 M calcium chloride and bovine pituitary extract as described previously (19 (link)). Three-dimensional cultures of normal human gingival epithelial cells were obtained from MatTek. These represent confluent, terminally differentiated, multi-layer primary cultures of ginigival epithelium (EpiGingiva), obtained in 24-well transwell plates obtained from the producer (Supplementary Figure S1). After delivery, the transwells are transferred to 6-well plates into the medium provided, and the cultures are maintained in an air-liquid interface for 1–2 days prior to their use in the experiment.

Eye primordia were dissected from stage 24 X. laevis embryos and plated on 50 mm glass-bottom dishes (MatTek) coated with 10 mg ml⁻¹ poly-l-lysine and 10 mg ml⁻¹ laminin (both from Sigma). Explants were cultured at 20°C in 60% Leibowitz's L15 medium (Gibco) containing 5% penicillin/streptomycin/fungizone (Sigma cat. no. P4458) and 50 µg ml⁻¹ gentamycin (GE Healthcare) for 16–18 h.

Cell lines were isolated as previously described [16 (link)]. Hras1, H340T, and H245T tumor cell lines were established from thyroid tumors of Hras^G12V/Pten^−/−/TPO-Cre mice of a pure 129/svJ genetic background. Thyroid tumors and wild-type thyroid glands were dissected and minced, followed by digestion in a solution of 1 mg/mL collagenase Type I (Sigma, St. Louis, MO, USA) and 1 mg/mL dispase (Gibco, Waltham, MA, USA) in Hank’s Balanced Salt Solution at 37 °C with gentle shaking for 1.5 h. Following digestion, samples were centrifuged at 1200 rpm for 3 min and resuspended in Ham’s F12 medium (Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-Glutamine (Gibco), and Penicillin/Streptomycin/Fungizone (Sigma). The samples were then plated into tissue culture flasks and maintained at 37° Celsius in 5% CO₂. To ensure removal of contaminating stromal cells and outgrowth of the pure tumor cell lines, all cell lines were passaged at least 5 times after plating and then genotyped using primers specific for Pten and Pten recombination [21 (link)]. Cell lines were authenticated using Short Tandem Repeat (STR) DNA profiling (DDC Medical) according to ANSI guidelines (ASN-0002). Ten mouse STR loci were analyzed for each sample. Loci and STR profiling results are listed in Table S2.

Bone marrow cells were isolated from 6‐week‐old male C57BL/6J mice (Vrije Universiteit Amsterdam animal facility, Amsterdam, the Netherlands). Femur and tibia from both legs were removed, followed by grinding in a mortar and cells were suspended in 10 ml culture medium. Culture medium was α‐MEM (Gibco; Thermo Fisher Scientific, Paisley, Scotland) supplemented with 5% Fetal Clone I Serum (HyClone; GE Healthcare Life Sciences, Logan, UT) and 1% penicillin‐streptomycin‐fungizone (Sigma–Aldrich, St. Louis, MO). The cell suspension was aspirated through a 21 gauge needle and filtered through 40 µm filter. A total of 4 × 10⁷ cells per mouse were collected. Animal experiments were approved by the Animal Welfare Committee of the Vrije Universiteit Amsterdam.