Cell culture insert

HCC cell lines [400,000 cells/well (HepG2), 200,000 cells/well (Hep3B, HuH-7 and PLC/PRF/5)] were seeded in 6-well culture plates in DMEM supplemented with 0.5% dialyzed FBS and 1% supplements as previously described. TWNT-1 (50,000 cells/well) were seeded into the Cell Culture Insert™ of 1.0-µm pore size (353102; Corning) in the same medium as used for HCC cells, inserted into the 6-well plates where HCC cells were seeded, and incubated for 3 days. After this, cells were seeded and cultured in this manner in case of indirect co-culture unless otherwise specified.

Cell proliferation ability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HCC cell lines were seeded at 40,000 cells/well in 500 µl of DMEM supplemented with 1% dialyzed FBS in 24-well culture plates (353047; Becton-Dickinson, Franklin Lakes, NJ, USA). TWNT-1 (30,000 cells/well) were seeded into the Cell Culture Insert™ of 1.0-µm pore size (353104; Corning) in the same medium as used for HCC cells, and plated into the 24-well plates where the HCC cells were seeded. After 48 h of quiescence, 50 µl of MTT (5 mg/ml in PBS) was added to each well, and the wells were incubated for an additional 3.5 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 500 µl of dimethylsulfoxide (Sigma-Aldrich) and applied in 100 µl volumes in a 96-well culture plate in the fourth replicate. The activity of mitochondria, used as a measure of cellular growth and viability, was evaluated by measuring optical density at 570 nm with a microplate reader (Model 680 microplate reader; Bio-Rad).

MC38 cells were cocultured with MPE macrophages using a cell culture insert with 0.4 μm pores (Corning, NY, USA). MPE macrophages isolated from WT and IL-10^−/− mice were seeded within the upper chamber, while MC38 cells (2 × 10⁵ cells/mL) were plated on the lower chamber. Subsequently, the upper chamber was placed onto the plates. After coculture for 24 hr, MC38 cells were collected, and the apoptotic cells were analyzed using a fluorescein isothiocyanate Annexin V Apoptosis Detection Kit (BD Biosciences).

First molar tooth germs were exenterated from the mandibles of ED14.5 mice. To determine the crown-root direction, the dental lamina, which is a transparent band and gives rise to the primordia of the enamel organs of the teeth, is used as a reference point. Each isolated tooth germ was ligated using a fine thread (8–0 nylon surgical suture; Natsume, Tokyo, Japan) by manipulating the thread around the mesiodistal centre of the tooth germ and slowly tightening the thread until the two regions were constricted into a bottleneck shape. To determine the ligation force value, we strictly defined an inner diameter of 50 μm with an 8–0 nylon thread. The ligated tooth germs were incubated for 5 min at 37 °C, placed on a cell culture insert (0.4 μm pore diameter; Corning, NY, USA), and then further incubated at 37 °C for 6 days in in vitro organ culture, as described previously26 (link)41 (link).

THP-1 derived macrophages and lung cancer cells were co-cultured using a cell culture insert (Corning, NY, USA) with a porous membrane to separate the upper and lower chambers. The macrophages (7.5×10⁵ cells/well) were seeded into the upper chamber of the transwell apparatus, and the A549 and H1299 cells were placed in the lower chamber (3×10⁵ cells/well). The chambers with the THP-1-derived macrophages were then placed directly on top of six-well plates containing the A549 and H1299 cells, and the resulting co-culture systems were incubated for 24 h or 48 h in serum-free RPMI 1640. Lung cancer cells and THP-1-derived macrophages were cultured for 24 h in serum-free RPMI 1640 as controls. To study the role of TGF-β, anti-TGF-β antibody (R&D Systems, Minneapolis, MN, USA) was added to the co-culture system.

MSCs (PαS cells) were cultured in 10-cm dishes using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% HEPES (Gibco, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco), 20% fetal bovine serum (FBS), and basic fibroblast growth factor (5 ng/mL; Pepro Tech, Cranbury, NJ, USA). The medium was replaced every 3–4 days. Before MSCs became confluent, they were passaged and cryopreserved. MSCs were cultured and used for further experiments at passage 3–4. For co-culturing HSPCs with MSCs, 1.0 × 10⁴ PαS cells/24-wells were seeded 2 days before seeding 1.0 × 10⁴ HSPCs on a cell culture insert (Corning, Durham, NC, USA, 0.4 μm pore size). For collecting conditioned medium, the culture medium was replaced with S-clone (Iwai North America, San Carlos, CA), and cells were further cultured for another 24 h before collecting the conditioned medium. The MSC conditioned medium was collected and centrifuged at 300 × g for 3 min to remove debris. HSPCs were cultured with MSC conditioned medium or control medium (S-clone) for 6 days. For analysis of the differentiation ability of HSPC-derived cells, the percentages of myeloid lineage (CD11b⁺/Gr-1⁺) cells, lymphoid lineage (CD3e⁺/CD45R⁺) cells, and erythroblasts (Ter119⁺ cells) were measured via flow cytometry (FACSAria II, BD Biosciences).

To analyze the interaction between organoids and NFs, we employed a non‐contact co‐culture method. NFs were pre‐seeded at the bottom of 24‐well plate (Corning). Upon reaching confluence, organoids were suspended in Matrigel, and 10 μL of this suspension was placed in a cell culture insert (0.4 μm; Corning) containing the medium used for the establishment of each type of organoid (please see Section 2.1). The medium was changed every 3–4 days, and three NFs were used in each experiment. On the 10th day of culture, the diameter of organoids was measured: eight organoids were measured in order of size per field of view for a total of 64 organoids. To evaluate the significance of the changes in organoids during co‐culture, adenoma organoids were cultured in an NFs‐conditioned medium. Once the NFs seeded in the dish reached confluence, the medium was replaced with Advanced DMEM/F12 (Thermo Fisher Scientific) containing 1% penicillin–streptomycin (Thermo Fisher Scientific) and collected after 3 days to prepare the complete medium. The medium was replaced every 2–3 days, and the changes were observed under a microscope (CKX41; Olympus Scientific Solutions) on Day 7 of culture.