RESUMO
Mammalian synthetic biology and cell biology would greatly benefit from improved methods for highly parallel transfection, culturing, and interrogation of mammalian cells. Transfection is routinely performed on high-throughput microarrays, but this setup requires manual cell culturing and precludes precise control over the cell environment. As an alternative, microfluidic transfection devices streamline cell loading and culturing. Up to 280 transfections can be implemented on the chip at high efficiency. The culturing environment is tightly regulated and chambers physically separate the transfection reactions, preventing cross-contamination. Unlike typical biological assays that rely on end-point measurements, the microfluidic chip can be integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time.
Assuntos
Proteínas/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , Humanos , Mamíferos , Técnicas Analíticas Microfluídicas , Microfluídica , Proteínas/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Imagem Óptica/métodos , Plasmídeos/metabolismo , Técnicas de Cultura de Células/instrumentação , Regulação da Expressão Gênica , Células HEK293 , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Microfluídica/instrumentação , Imagem Óptica/instrumentação , Plasmídeos/química , Transdução de Sinais , TransfecçãoRESUMO
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 developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.