Development of a Microfluidic Platform for Cell-Cell Communication
Authors: Craig Watson
Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. This dissertation details the development of a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. Several versions of the chip were developed, culminating in a chip featuring 128 individually-addressable chambers arranged in 32 sets of 4 chambers; 16 fluidic inputs; and 2 outputs. The chip enables a wide range of cell culture studies including co-culture and can support assays with optical readouts such as immunocytochemistry, as well as cell or media recovery. We demonstrate the ability of the chip to support culture of a variety of cells including cell lines and primary cells, and validate its use for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened. In parallel, a pneumatic controller was developed to operate microfluidic chips, offering manual and automated control via a PC interface of the hardware which features pressure sources, high-precision digital pressure regulators and arrays of up to 32 solenoid valves.
Fig. Schematic comparison of Transwell inserts, the widely accepted standard for co-culture, and microfluidic chambers. In the dish, cells are cultured in much larger volumes of media (~100x more), which dilutes cell-secreted signals; in addition, imaging is complicated by the vertical stacking of cells. In microfluidic chips, chamber height and media supply can be adjusted to prevent signal dilution.
Fig. Setup used for cell culture studies in the chip. An environmental chamber around the microscope and a stage-top enclosure provide temperature and CO2 control, respectively.
Keywords: Microfluidics; cell culture; co-culture; coculture; paracrine signaling; multiplexing; PDMS; soft lithography; photolithography; computational modeling; open source; open hardware; instrumentation; automation; Qt; Arduino; bubble trap