Fig. 8 | Scientific Reports

Fig. 8

From: Comparative analysis of 3D-culture techniques for multicellular colorectal tumour spheroids and development of a novel SW48 3D-model

Fig. 8

Cellular organization of CRC spheroids cultured with colonic fibroblasts. (Top) MCTSs of DLD1 (left) or HCT116 (right) stained with Cell Tracker Green CMFDA (green) co-cultured with CCD-18Co fibroblasts stained with BioTracker 555 Orange Cytoplasmatic Membrane Dye (red). Images were taken after 48 h of culture in MT-Plates with 2.5 mg/mL methylcellulose, using Images were taken in a LLEICA DM1600B fluorescence microscope with 4X objective. (Middle) Stitched images of spheroids collected, decanted, and transferred to bottom-glass 8-well chambered slides (three spheroids per chamber), imaged using an Abberior STEDYCON super-resolution confocal microscope. Despite random orientation after transfer, CCD-18Co fibroblasts (red) consistently localize to the spheroid core. Yellow triangles indicate spheroids selected for Z-stack analysis. (Bottom) Composite Z-stack images of a DLD1/CCD-18Co MCTS (left) and a HCT116/CCD-18Co MCTS (right), with slices separated by 10 μm. CCD-18Co fibroblasts (red) occupy the central region, surrounded by cancer cells (green). Fluorescence signal intensity notably decreases beyond 50 μm depth.

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