Abstract
The hanging drop (HD) cell culture method is widely used for the formation of in vitro three-dimensional (3D) cell aggregates, avoiding the cellular distortion typically seen in the two-dimensional cell culture. The conventional HD technique consists of placing small drops of cell suspension solution on a plastic substrate, inverting the substrate, and incubating for the desired or permitted length of time. However, the size of the HD is restrained by gravity, and hence only a limited number of cells can be sustained without cumbersome periodic replenishment. In this study, we presented a microfluidic HD (HD) platform allowing for the automated HD formation, cell loading, medium exchange and retrieval of cells without pipette-based manual labor. The platform consisted a microfluidic channel incorporating openings at its bottom, which was fabricated using photolithography followed by soft lithographic and membrane transferring techniques. When the microchannel was filled with liquid, pedant drops formed automatically and hanged from openings at the bottom. Cells were loaded into the microchannel and docked in the HD. Untrapped cells could be rinsed off. Different extracellular solutions were introduced into the HD through the microchannel with the precise temporal and spatial control. We demonstrated the automatic formation of an array of HDs of various sizes and the formation of cell aggregates from multiple types of cells, including human lung cancer cells (A549), mouse embryonic fibroblasts (M-EF001, Bioresource Collection and Research Center, Taiwan) and stem cells (mES) cells (ES-D3). This HD platform provides an array of 3D cell culture space, which allows easy and low-cost fabrication, operation, and automation, and should find applications in reproductive medicine, cell biology, tissue engineering, stem cell biotechnology, and high-throughput pharmaceutical testing, among others