Microtissues 3d petri dish micro mold spheroids

To form hASC spheroids, commercial MicroTissues® 3D Petri Dish® micro-mold spheroids (Sigma-Aldrich) were used as molds in hydrogel-replicated microwells. The autoclaved molds were prepared and transferred into a hood. Sterilized 1.5% w/v agarose (UltraPure™ Agarose; Invitrogen) in phosphate buffered saline (PBS) was transferred into the hood and stirred with a magnetic bar on a hotplate. The agarose solution was pipetted into the molds and allowed to solidify at room temperature for 5 min. The agarose molds were transferred into individual wells of 24-well plates. The molds were then immersed in PBS and stored at 4 °C until use. The prepared hASCs were seeded on the mold and cultured with cell culture medium at a density of 2.5 × 10⁵ cells/mold in 24-well plates. After 72 h, 35 spheroids about 200 μm in diameter were formed from a mold (Fig. 1).Fig. 1

Formation of hASC-spheroids from agarose mold. A prepared agarose mold consisting of 35 cylindrical wells was placed in the wells of 24-well plates. Aliquots of 2.5 × 10⁵ hASCs were seeded and cultured in the molds. A Aliquots of 2.5 × 10⁵ hASCs were separated into 35 cylindrical wells of the agarose molds (scale bar: 200 μm), B and each cylindrical well-formed spheroids about 200 μm in diameter after 72 h of culture (scale bar: 200 μm)

To evaluate the 3D cell proliferation, a spheroid formation assay was performed in micro-molds containing 81 pores. Approximately 700 µL of the 2% agarose solution in saline was added to the micro-mold (MicroTissues^® 3D Petri Dish^® micro-mold spheroids; Sigma, St. Louis, MO, USA). After 30 min, the micro-molded agarose was misinformed from the micro-mold in a 24-well plate. After the agarose was completely dried, approximately 20 min, DMEM containing 2.5% SFB was added to micro-molded agarose and was incubated for 15 min in an incubator at 37 °C and 5% CO₂. After removing the medium, the cell suspension (approximately 1 × 10⁶ cells) was resuspended in 200 µL of medium and added to the micro-molded agarose. After spheroid formation (24 h later), cells were treated or not with 20 and 40 μM BH4 or 2 and 4 mM DAPH for 5 days, and sphere growth was monitored for 5 days, and the tumorsphere area was measured.

The information below on spheroid preparation and growth conditions is based on MISpheroID recommendations (21 (link)). 3D spheroids were made in micro-molds according to the manufacturer’s instructions (MicroTissues 3D Petri Dish micro-mold spheroids, Sigma-Aldrich) with 2.5 × 10⁵ cells in one mold (monocultures; 7000 cells/spheroid) or 5.0 × 10⁵ cells in one mold (cocultures; 14 000 cells/spheroid). In cocultures the cell ratio was 1:1 (cancer cells and fibroblasts, respectively). The spheroids were grown in serum-free DMEM medium for 5 days at 37°C in an incubator environment of 20% O₂ and 5% CO₂. Ascorbic acid (50 µg/ml) was added daily. The spheroids were washed out from the molds with hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 10 µg/ml DNase I (DN25, Sigma-Aldrich)) and collected into Eppendorf tubes. The spheroids were then washed twice with hypotonic lysis buffer and centrifuged between washes 2 min/1000 x g. After that, the spheroids were incubated with hypotonic lysis buffer o/n in +4 °C in a rotator. The next day, spheroids were washed twice with hypotonic lysis buffer as described above. The pellets were frozen in -20 °C for further mass spectrometric analysis.