Figure 6

Time course monitoring of chemosensitivity in spheroids through continuous microfluidic flow. (A) Photographic image and (B) schematic diagram of the continuous perfusion microfluidic setup using peristaltic pump. Fluid filled syringes were connected to the peristaltic pump that was connected to the microfluidic devices and through to universal tubes for the collection of supernatants. HT29 cells in suspension were seeded and cultured under static conditions in the microfluidic devices flow chip for 2 days to form 3D spheroids. Spheroids were then treated with 5-FU (200 μM) through continuous perfusion at 20 µL/min for 0, 1, 2, 3 and 5 days. The independent channel controllable pump allowed flow through devices to be independently controlled permitting flow to stop through one device without influencing flow through other devices. (C) The supernatant was collected at the different time-points through fluid flow and plated into 96-well plates and the Lactate dehydrogenase (LDH) assay was performed. Data are shown relative to cell media control and represent means with standard deviation of 3 independent experiments (3 replicates per experiment). (D) Spheroids were also stained with Hoechst 33342 (Blue) and propidium iodide (Red) fluorescent dyes through flow and fluorescently imaged. Scalebar = 100 μm. Images are representative of at least 3 independent experiments. (E) Hoechst 33342 and propidium iodide fluorescent intensity from 3D HT29 spheroids was digitally quantified using ImageJ. Data represents means with standard deviation of 3 independent experiments (3 spheroids per experiment).