3D Culture of Dendritic Cells in Collagen Gels

Collagen gels were prepared as previously described (Imle et al, 2019 (link)). Briefly, concentrated rat tail collagen I (Corning) was mixed with bicarbonate‐buffered MEM on ice (15 μl 10× MEM, 17 μl 7.5% NaHCO₃ (both Gibco), and 120 μl rat collagen I). To culture iDCs in 3D collagen, 2 × 10⁶ cells/ml were mixed 1:1 with collagen, and 100 μl cell‐collagen per‐well mix was transferred in 96‐well F‐bottom plates and let polymerize for 20 min at 37°C. For suspension cultures, 2 × 10⁶ iDCs/ml were mixed at 1:1 ratio with RPMI, and 100 μl per‐well of cell‐media mix was transferred in 96‐well U‐bottom plates. For 2D collagen cultures, 100 μl of collagen mix, polymerized in 96‐well F‐bottom plates, were overlaid with 2 × 10⁶ iDCs/ml supplemented with 20 ng/ml IL‐4 and 20 ng/ml GM‐CSF. 2D suspension and 3D collagen cultures were overlaid with 100 μl RPMI supplemented with 20 ng/ml IL‐4 and 20 ng/ml GM‐CSF per well.