Microfluidic platforms for high-throughput mammalian cell printing, transfection, and dosage-dependent studies

Abstract

With the advent of high-throughput and genome-wide screening initiatives, there is a need for improved methods for cell-based assays. Current approaches require expensive equipment, rely on large-scale culturing formats not suited for small or rare sample types, or involve tedious manual handling. Microfluidic systems could provide a solution to these limitations, since these assays are accessible, miniaturized, and automated. When coupled with high-content analysis, microfluidics has the potential to drastically increase throughput in cell biology and drug discovery. In light of these benefits, we developed 3 microfluidic approaches for mammalian cell-based assays: (1) printing of live mammalian cells into nanowell arrays, (2) a high-throughput transfection device, and (3) a module that generates complex, continuous concentration profiles. Our first technique generates high-density nanowell arrays of live mammalian cells (LMCAs) using a standard contact microarrayer. Both commonly used cell lines and primary cells cultured on the arrays are highly viable and maintain their signature phenotype, making the platform suitable for long-term stem cell differentiation studies. Our 675-well array is 2.6x more dense than a 1,536-well microtiter plate and can be frozen and thawed, facilitating the handling, storage, and screening of large libraries of cells. LMCAs are also compatible with transfection, a technique that could enable analysis of the entire proteome in the natural cellular context. Transfection is routinely conducted on high-throughput arrays, but this setup requires manual cell culturing and precludes precise control over the cell environment. To this end, we created a microfluidic chip that streamlines cell loading and culturing and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration. This platform is well-suited for optimizing synthetic gene circuits; we co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. To complement the biological assays that could be performed on our transfection chip, we lastly generated an accurate and automated method to manipulate molecular concentrations on chip. Our pulse-width modulation (PWM)-based microfluidic module combines up to 6 different inputs and produces arbitrary concentrations with a dynamic range of 3-5 decades. We created complex concentration profiles of 2 molecules, with each concentration independently controllable. The PWM module can execute rapid concentration changes as well as long-timescale pharmacokinetic profiles under a variety of operating conditions, making it ideal for integration with existing devices for advanced cell and pharmacokinetic studies. Taken together, the 3 microtechnologies developed in this work integrate and automate mammalian cell handling, culturing, transfection, imaging, and solution preparation. These features have far-reaching implications in fields such as synthetic biology, stem cell research, and drug development.