PBMCs were cultured and used as described above. Silicone droplets containing media was prepared as follows: 100 μl of silicone liquid (SYLGARD 184, Dow Corning) was added to 3 ml of cell media into a 10-ml syringe barrel and further mixed by pushing and pulling the plunger in the barrel three times. Cells were embedded with silicone microdroplets in a 3D collagen I gel with a final concentration of 1.6 mg/ml. A total of 18,000 cells per condition were gently mixed with 22.5-μl silicone droplets containing media and added to 45 μl of the previously prepared collagen I master mix in a sterile tube on ice. The cell-containing collagen I solution was gently mixed until a homogeneous bright pink color was obtained and subsequently transferred to a 96-well plate. Polymerization of the collagen gel and equilibration of gas conditions within the collagen matrix took place for 20 to 30 min at 37°C and 5% CO2 to obtain a final pH of 7.4 to 7.5 (fig. S10B). The pH values were determined using indicator paper strips. Cells embedded in the silicone droplet containing collagen matrices were maintained for 3 days with medium replacement every 1 to 2 days.

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