Figure 5
Culturing, treating and evaluating anti-cancer activity in spheroids through limited-perfusion microfluidic flow. (A) Photographic image and (B) schematic diagram of the limited-perfusion microfluidic setup using a single syringe pump. A fluid filled syringe is placed into the pump which is connected to an adjustable 3-way flow valve. The valves are connected to microfluidic devices and through to universal tubes for the collection of supernatants. During fluid flow the 3-way valve is open. Once complete perfusion through microfluidic devices has occurred the pump is stopped, and the 3-way valve is closed to create a vacuum and statically hold fluids in the microfluidic devices. HT29 cells in suspension were seeded and cultured under static conditions in the microfluidic devices flow chip for 2 days to form 3D spheroids. 3D spheroids were then treated with 5-Fluorouracil (5-FU, 500 µM) under fluid flow for 25 min at 20 µL/min and then incubated for 24 h under static conditions. (C) The supernatant was then collected through fluid flow and plated into 96-well plates and the Lactate dehydrogenase (LDH) assay was performed. Data are shown relative to control treated cultures 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 = 200 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).
