Co-culture of HSC-3 cells and Mfs induce invasion in an organotypic 3D tissue model.

<p>(A) HSC-3 cells were treated with Mf-CM and/or 10 μM BAY 11-7082 (n = 6) and adhesion to fibronectin-coated wells was analysed after 2 hours with Crystal violet-staining. Stained wells were analysed by measuring absorbance at 540 nm. (B) Labeled HSC-3 cells were cultured on top of Matrigel and Mfs, HSC-3 or THP-1 cells were cultured in the lower chambers (n = 6). Invaded cells were analyzed from the membrane by measuring fluorescence. HSC-3 cells and Mfs were co-cultured on top of human myoma tissue (C,D, n = 3) for 10 days where after tissues were fixed and processed for immunohistochemistry. Pan-cytokeratin stained sections (in C, scale bars: 500 μm) were photographed and invasion areas and invasion depths were analysed with the Leica Qwin3 software. Conditioned medium was collected at day 4 and 8 from myomas and medium containing 0.5 (Mf CMs) or 15 μg (HSC-3) protein were subjected to gelatin zymography (E, day 4). Samples were done in triplicate, representative picture of samples is shown in E. Recombinant MMP-2 and -9 were used as positive controls. Relative intensity of MMP-9 bands were analyzed with the ImageJ-software (F).