Cytokine Stimulation of 3D Brain Organoids

For cytokine-stimulation experiments, d150 hCSs were transferred to low-attachment 6-well plates (Corning) that were placed on a gentle cell-culture incubator rocker in order to achieve an optimal flow of cytokines during the stimulation period. hCSs were incubated with 5 ng/mL TNFα (Sigma-Aldrich T0157-10UG; in PBS) or 5 ng/mL IL-1β (Sigma-Aldrich SRP3083-10UG; in PBS) for 4 h, 12 h or 36 h. The cytokine concentrations and stimulation time points were chosen based on our previous studies, studies utilizing 2D cell models [17 (link),18 (link),19 (link),20 (link),21 (link)], and the only study published thus far that dealt with cytokine (TNFα) stimulation of a 3D human brain organoid [22 (link)]. Following stimulation, spheroids were harvested for RNA isolation and the RNA was subjected to RNA-seq analysis as described below. Unstimulated hCSs from the same batch were used as controls. For pro-inflammatory stimulation experiments with HMC3 and HOG cells, the cells were incubated with 100 ng/mL LPS (Sigma-Aldrich, L4391-1MG) (dissolved in PBS) for 24 h (24 h) with subsequent incubation with 5 ng/mL TNFα (in PBS) and 5 ng/mL IL-1β (in PBS). Following stimulation, HMC3 and HOG cells were harvested for RNA isolation and the RNA was subjected to RNA-seq analysis, as described below.