Microtissues 3d petri dish micro molds

SF A549 spheroids were generated using MicroTissues 3D Petri Dish^® micro-molds (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, the micro-molds were sterilized with anhydrous ethanol and allowed to dry under UV light for 30 min. Then, they were filled with sterile 2% (w/v) agarose solution, prepared in Milli-Q water. After solidification, the gelled agarose molds were released from the flexible 3D Petri Dish^® micro-molds and transferred to 6-well plates (3 agarose molds per well). To equilibrate the agarose gels, each well was filled with 1 mL of growth medium. After equilibration, the plates were placed in the incubator and incubated overnight.
Before seeding the cells, the medium was removed from both the culture plate and the molds. Then, 1.3 mL of fresh growth medium was added to each well and aliquots of 150 μL of growth medium with 40,000 cells were gently added into the molds. The medium was changed every other day. The size and morphology of the grown spheroids were examined by bright field microscopy using AxioVert.A1 microscope (Zeiss, Oberkochen, Germany) equipped with a Plan 10×/0.25 phase-contrast objective. On day 7, the spheroids were harvested for the following experiments.

The chondrospheres were formed using MicroTissues 3D Petri dish micro-molds (Sigma-Aldrich, catalog no. Z764019-6EA, 81 circular wells 800 μm × 800 μm) as previously described (28 ). The concentration of human chondrocytes was 3.4 × 10⁶ per milliliter, which resulted in a final concentration of 8000 cells per well/spheroid. The chondrospheres were incubated at 37°C in a humidified atmosphere with 5% CO₂ for 2 days. On the second day of fabrication, the diameters and roundness of chondrospheres were estimated using bright-field microscopy (Nikon Eclipse Ti-E inverted microscope, Japan) and measured using ImageJ 1.48v software [National Institutes of Health (NIH), Bethesda, MD, USA] as previously described (28 ).

Organoids were fixed for 20 min in 4% (v/v) Roti Histofix (Carl Roth) followed by embedding into MicroTissues 3D Petri Dish micromolds (Sigma–Aldrich) using 2% (w/v) Agarose LE (Sigma) in PBS supplemented with 0.5 mM DTT. Thereafter, organoids were subjected to dehydration steps and embedding in paraffin. Formalin-fixed agarose/paraffin-embedded sections (3–5 µm) were manually cut from blocks with a microtome (Leica RM 2145) and transferred to glass slides (Superfrost, Thermo Fisher Scientific) before H&E staining using automated staining devices.