Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis

Naveen Ramalingam, Brian Fowler, Lukasz Szpankowski, Anne A. Leyrat, Kyle Hukari, Myo Thu Maung, Wiganda Yorza, Michael Norris, Chris Cesar, Joe Shuga, Michael L. Gonzales, Chad D. Sanada, Xiaohui Wang, Rudy Yeung, Win Hwang, Justin Axsom, Naga Sai Gopi Krishna Devaraju, Ninez Delos Angeles, Cassandra Greene, Ming-Fang Zhou, Eng-Seng Ong, Chang-Chee Poh, Marcos Lam, Henry Choi, Zaw Htoo, Leo Lee, Chee-Sing Chin, Zhong-Wei Shen, Chong T. Lu, Ilona Holcomb, Aik Ooi, Craig Stolarczyk, Tony Shuga, Kenneth J. Livak, Cate Larsen, Marc Unger, Jay A. A. West

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.