Cell culture dish

MDA‐MB‐231 and MCF‐7 cell lines were trypsinized and suspended using trypsin (Gibco, pro. no. 25200‐56), and they were then centrifuged (100g for 5 min at room temperature). The suspended cells were counted using a hemocytometer lam (Marienfeld), and several droplets of 20 µl suspended cells (~1 × 10³ cells) were deposited on the lid of a cell culture dish. The lid was returned to the cell culture dish (SPL Life Sciences), and the droplet hanged upside down and incubated for 2 days in a standard cell culturing incubator (37℃, 5% CO₂, RH ~95%). Following that, the desired spheroids were collected and added to the collagen matrix (~100–150 µm, using an inverted microscope; grown spheroids should have a tight and compact spherical shape; disintegrated spheroids and those with loose aggregations are not suitable) (type I rat tail, Corning). The spheroid and collagen mixtures were incubated for 30 min (37℃, 5% CO₂, RH ~95%). After gel cross‐linking, the cell culture medium was added to the combination.¹⁸, ¹⁹

For 3D cell culture, we encapsulated hMSC in 3D_SF/LD-PEG hydrogel. Hydrogel precursor solutions were prepared with 6 wt% of PEG4NB, dithiol linkers (7 mM, molar ratio was 50:50 of DTT and MMPs) and 1 mM LAP. hMSC were suspended at a cell density of 2 × 10⁶ cells/mL in the hydrogel precursor solution, and 25 μL cell-suspended solution was carefully transferred into a cylindrical mold (diameter: 5 mm) containing the 3D_SF. The cell-suspended 3D_SF/LD-PEG hydrogel was prepared by exposing it to UV light (365 nm, 5 mW/cm²) for 2 min. Finally, HD-PEG hydrogel was deposited on the top of the cell-laden 3D_SF/LD-PEG hydrogel as described previously in section Preparation of 3D SF Construct Embedded Dual Layer (3D_SF/LD/HD) Hydrogel. The cell-laden 3D_SF/LD/HD-PEG hydrogel was cultured in the normal culture medium or differentiation medium for chondrogenesis. For 2D cell culture, hMSC cells were cultured on a cell culture dish (SPL, Korea) at a cell density of 5 × 10³ cells/cm².

The mRNA expression associated with EMT and drug resistance was analyzed by RT-qPCR. To obtain samples for reverse RT-qPCR, cells in 2D and 3D were cultured on a cell culture dish (100 × 20 mm; SPL Life Sciences, Pocheon, Korea). To analyze cell proliferation and drug resistance, cells were cultured in 96-well plates. For the harvesting of cells and RNA extraction, 2D-cultured cells were scraped off the dish bottom using a cell scraper. 3D microtumors were carefully collected using a pipette. Then, 2D- and 3D-cultured samples were homogenized by hand using a tissue grinder on ice. Isol RNA lysis reagent (5 Prime, Hilden, Germany) was used to extract total RNA from cells in accordance with the manufacturer’s instructions. Samples were mixed with this reagent, followed by phase separation using chloroform. After collection of the aqueous phase, RNA was precipitated by treatment with isopropyl alcohol and glycogen for 1 h at −20 °C. Extracted RNA was collected and washed with 70% ethanol three times, followed by dehydration. Dried RNA was dissolved in a suitable amount of diethylpyrocarbonate water. The concentration of total RNA was determined by measuring the absorbance at 260 nm using a spectrophotometer. Only RNA samples with A260/A280 ≥ 1.8 were used. Samples were stored at −20 °C until future use.