Break

Breast cancer is the second leading cause of cancer related death in American women. Cancer cells signal to surrounding tumor stroma to produce cancer associated adipocytes, fibroblasts, and macrophages while secreted factors from stromal cells in the tumor microenvironment induce drug resistant and metastasis in cancer cells. Co-culture and conditioned media-based experiments have demonstrated that paracrine factors induce drug resistance. While multiple co-culture techniques exist, no of them are capable of dynamic sampling of extracellular media during an experiment to investigate cell-to-cell communications. To address this, a novel microfluidic device was designed, fabricated, and tested to study intercellular communications during co-culture. The device consists of three parallel channels connected by an array of 30 µm wide by 100 µm long microchannels spaced 75 µm apart to facilitate the orthogonal diffusion of biomolecules. COMSOL Multiphysics was used to model the mass transfer in the device to optimize channel length and width. The optimized device consisted of three parallel 1500 µm long with the two outermost channels for static cell culture and the center channel connected to a peristaltic pump to allow for continual perfusion and circulating of medium. The operating length of the three channels was 20 mm to allow for sufficient cell numbers for sampling of paracrine factors. Circulating media flowrates of 5, 10, and 15 µL/min were tested, with 5 µL/min providing sufficient mass transfer across the three channels with minimal shear effects on the culture cells A test device was fabricated using a combination of soft lithography and polydimethyl siloxane (PDMS) replication. The device utilized a peristaltic pump with clear, flexible tubing for constant flow of media and a three-way junction incorporating into the tubing outside of the device for dynamic sampling. Pipette-tip reservoirs were positioned at the inlets and outlets of the culture channels for media replacement. Peristaltic pump performance was characterized by running water through the tubing and timing the water collection at different pump speeds. The pipette-tip reservoirs were observed during testing to ensure media replacement without the introduction of bubbles during sampling. A careful sampling technique, which included clipping the opposite junction during sampling, was also developed to address this issue. This novel microfluidic device will be used to analyze the cell-to-cell communications between MDA-MB-231 breast cancer cells, a triple negative cell line, and adipose-derived stem cells (ASCs) to study how stromal cells enhance the proliferation and drug resistance of cancer cells.