Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells 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 and synthetic gene circuits can be optimized on chip. 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. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies.

We designed a microfluidic module that generates complex and dynamic concentration profiles of multiple molecules over a large concentration range using pulse-width modulation (PWM). Our PWM module can combine up to six different inputs and select among three downstream mixing channels, as required by the application. The module can produce concentrations with a dynamic range of three decades. We created complex, temporal concentration profiles of two molecules, with each concentration independently controllable, and show that the PWM module can execute rapid concentration changes as well as long-time scale pharmacokinetic profiles. Concentration profiles were generated for molecules with molecular weights ranging from 560 Da to 150 kDa. Our PWM module produces robust and precise concentration profiles under a variety of operating conditions, making it ideal for integration with existing microfluidic devices for advanced cell and pharmacokinetic studies.

High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We have developed a simple, yet powerful method that uses contact spotting to generate highdensity nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries. High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We have developed a simple, yet powerful method that uses contact spotting to generate highdensity nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.Cell-based assays and the tools used to perform them are constantly undergoing improvements toward higher experimental throughput, reduced reagent consumption, and advanced control of the cell microenvironment 1,2 . There are currently two main approaches for conducting highcontent cell culture experiments. In the first, cells are cultured in microtiter plates and robotic equipment is used to perform all necessary fluidic operations 3 . Although effective, this method remains unavailable to many laboratories due to the requirement for expensive robotics. A second approach that can be used for high-throughput cell studies is reverse transfection [4][5][6] . Here, cells are seeded onto an array of DNA, RNA, or small molecules. Large gene expression and silencing studies can be conducted in this manner, but complex cell libraries cannot be investigated since each array is limited to a single cell type.As an alternative to these approaches, contact spotting offers an affordable yet high-throughput platform. This technique is extensively used to generate high-density DNA and protein arrays and has been adapted to a variety of other purposes. For example, Hart et al. created a high-content immunoassay by spotting fixed mammalian cells that were cultured under different conditions 7 . To date, contact spotting has not been used to array live mammalian cells due to rapid spot evaporation and consequent cell death. Therefore, arraying of live mammalian cells has been limited to inkjet printing, which lacks the ability to handle a large number of different samples 8,9 .Large collections of mammalian cell lines have recently become available, including primary tumor and cancer cell lines 10 , stably transfected expression cell lines, and GFP-fusion libraries 11 . Novel approaches are required to efficiently assemble complex arrays of hundreds to thousands of genetically diverse cells. To address this arising need, we developed a simple, fast, and scalable method that uses standard microarray printing tools to generate high-density nanowell arrays. A minimal sample requirement of 500 cells enables the interrogation of cells that are availab...