Multiplexed Microfluidic Chip for Cell Co-Culture
Authors: Craig Watson, Chao Liu, Ali Ansari, Helen C. Miranda, Rodrigo A. Somoza, and Samuel E. Senyo
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. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1h. Finally, we validate the use of the chip 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.
Fig. Glucose diffusion and consumption model. (A) Schematic of media supply by diffusion. Medium is flowed into the reservoir adjacent to the culture chamber. The valve between the chambers is then opened to allow diffusion with no shear stress on the cells. This is repeated approximately every 30 minutes, depending on cell types. (B) Glucose concentration in chamber vs time. Concentration over x axis rapidly becomes homogeneous due to the small distance between channels, therefore results are averaged over the x axis for better visualization. Media changes are modeled by resetting the concentration in the reservoir and channels to 4.5 g/L. (C) Impact of glucose consumption rate on the concentration at end of the chamber farthest from the media reservoir. Media supply period is set to 30 min. (D) Impact of media supply period T on glucose concentration at far end of chamber, with consumption rate set to 0.2 pmol/h/cell.
Fig. Schematic of the experimental groups in the NF-κB demonstration experiments. HEK293 were seeded in chamber B of all units; RAW were seeded in chamber C of units 1-16. The next day, RAW cells in 1-8 were briefly treated with LPS. Then, the valve between chambers B and C in odd-numbered units (top half of chip) was opened to allow exchange of paracrine signals. Normal medium was delivered to all units, except four which received LPS-supplemented medium (top right); and four which received TNFα-supplemented medium (bottom right). This yielded 8 experimental groups with 4 technical replicates each.
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