Development of an in vitro 3D BM niche model. (A) Confocal images of calcein-labeled ND-MSCs, passage 2, (calcein/live cells, green; silk scaffold, red) at 2 and 37 days of culture in osteogenic media. The scale bar represents 100 μm. (B) Confocal images of RFP⁺HUVECs (red) ± GFP⁺MM1S cells (green) on scaffolds (blue) (days 3 and 9; scale bar = 100 μm). Representative image of 3 experiments is shown here, cultured in endothelial growth media. (C) Confocal images at day 30 of culture of GFP⁺MM1S alone (left, green), DiD-labeled MSCs alone (middle, red), and cocultures (right) in 50-50 medium with bortezomib (top, 5 nM) or without bortezomib (bottom) on autofluorescent scaffolds (blue). (Scale bar = 100 μm.) (D) Confocal images of primary patient CD138⁺ MM cells (green with DiI) at day 7 seeded onto ND-MSCs (red with DiD). Channels show the myeloma cell (arrow) as alive (calcein⁺, blue), DiI⁺ (green), and DiD^– (red). Overlay of green and blue appears cyan and demonstrates colocalization of calcein and DiI staining. Samples cultured in 50-50 media (n = 3); the scale bar represents 20 μm.