The establishment of a stromal network is necessary to reproduce stromal-dependent B-NHL cell growth. (A) Effect of Matrigel (Mg) and TSC on spheroid growth. Left panel, representative images of spheroids obtained from B cells encapsulated alone (3D) or with Mg (3D+Mg) or with Mg+TSC (3D+Mg+TSC) at D7 (HLY1), D6 (SUDHL4), or D10 (DOHH2) of culture. Scale bar, 200 µm. Right panel, the B cell number was evaluated over time in the different conditions (n = 3). (B) TSC network needs Mg to be established. Left, example of GFP-TSC encapsulated without Mg and B-cells. Right, example of TSC encapsulated with Mg but without B-cells. TSC were visualized in green by stable expression of GFP, and nuclei were stained in blue with Hoechst 33252. Capsules were imaged 3 days after encapsulation. Images were maximum intensity projection from z-stacks. Scale bar, 100 µm. (C) TSC network is necessary to induce DOHH2 spheroid growth. DOHH2 cells were coencapsulated with TSC-GFP with or without Mg for 10 days. TSC were visualized in green by stable expression of GFP. (D) TSC/Mg interaction. GFP-TSC were encapsulated alone in the presence of Mg. After 3 days in culture, capsules were fixed and immunofluorescence was performed. Mg and TSC were visualized by staining with anti-human pan laminin (in red) and anti-GFP (in green), respectively. Nuclei were counterstained in blue with Hoechst 33258. Images were maximum intensity projection from z-stacks. Scale bar, 50 µm. Yellow square is a crop showing the anchoring of TSC on Mg coating. Scale bar, 10 µm.