96 well suspension culture plate

PaCaDD or MIA-PaCa2 cells were seeded in DMEM cell culture medium containing 2% heat-inactivated FCS and 0.25% methylcellulose (PureCol, Biomaterials, Fremont, USA) in a 96-well suspension culture plate (Greiner Bio-One, Frickenhausen, Germany) with a density of 10,000 cells/well for spheroid formation. The cells were used for co-incubation experiments with infected MSCs 72 h later. MSCs were stained with the PKH26 red fluorescent cell linker kit for general cell membrane labeling (Sigma-Aldrich, Munich, Germany), and 5 × 10⁴ MSCs/well were seeded in a 24-well plate in DMEM cell culture medium supplemented with 2% FCS. The MSCs were infected 24 h later with a virus titer of 2000 TCID₅₀. Two hours after infection, the MSCs were overlaid with a gel layer of methylcellulose, Matrigel and collagen (PureCol, Biomaterials, Fremont, USA) at a ratio of 1:1:1 in DMEM supplemented with 2% FCS. PaCaDD183 or MIA-PaCa2 spheroids were seeded on top of the gel layer and co-incubated with MSCs for 16 h. Next, the spheroids were transferred with a cut tip of a 10 μl pipette into new 96-well suspension culture plates. Forty-two hours after infection, images of non-fixed spheroids were acquired under a BIOREVO BZ-9000 microscope (Keyence, Neu-Isenburg, Germany).

For CLL 3D co‐culturing conditions, the protocol previously adapted to CLL in our lab has been followed.²⁵ Briefly: Gelatine disks of 4 mm of diameter and 3 mm height were cut from Spongostan™ gelatine sheets (Ethicon) using a sterile biopsy punch and then pre‐seeded with BM derived stromal cells HS5 (200.000/scaffold) for 4 h in 96‐well suspension culture plate (Greiner Bio‐one). Scaffolds were then transferred to 10 mL High Aspect Ratio Vessels (HARV) in 600 μL DMEM culture medium supplemented with 10% v/v FBS and cultured overnight in the RCCS™ bioreactor. The day after, CLL cells were added to the vessels, following the optimal CLL cells:stromal cells ratio, set in previous experiments.²⁵ After 5 h, vessels were filled with growth medium (RPMI 1640 culture medium supplemented with 20%). After 48 h of 3D dynamic culture in the bioreactor, supernatants were withdrawn from the vessels and centrifuged at 1500 rpm for 5 min. Recovered cells were counted. Clarified supernatants were put again in the vessels with or 4 μM defactinib (Verastem Oncology). Cultures were stopped after 4 h of treatment and cells in the supernatants and in the scaffolds were recovered and submitted to Trypan Blue exclusion test for viability and cell count.