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

Abstract

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.

Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex

“…To map physiological response patterns onto cellular diversity, we developed an approach using the microfluidic Polaris device (Ramalingam et al, 2016) to couple the recording of Ca 2+ responses to transcriptomic analysis of the same single cells ( Figure 4A). Cells dissociated from the GZ or CP and SP of mid-gestational cortical tissue were loaded with a Ca 2+ dye, and single cells were captured on microfluidic chips.…”

“…The Polaris system has been described in detail recently (Ramalingam et al, 2016). Polaris Integrated Fluidic Circuit (IFC) chips were operated according to the manual (PN 101-0075 A1).…”

Integrative analysis and machine learning based characterization of single circulating tumor cells

“…This poses a significant challenge in differentiating CTCs from immune cells. We addressed this challenge by integrating two commercially available microfluidic systems namely Biolidics ClearCell FX System 31 and the Fluidigm Polaris™ system 16 (Methods, Fig 4-a) . In the proposed workflow CTCs are enriched in two steps - size-based enrichment by ClearCell, followed by CD45 (leukocyte marker) and CD31 (endothelial cell marker) based negative selection by Polaris 16 .…”

“…The Polaris IFC is first primed using Polaris system (Fluidigm) 16 to fill the control lines on the fluidic circuit, load cell capture beads, and block the inside of PDMS channels to prevent non-specific absorption/adsorption of proteins. To capture and maintain the single cells in the sites, the capture sites (48 sites) are preloaded with beads that are linked on-IFC to fabricate a tightly packed bead column during the IFC prime step.…”

“…Almost all studies that reported molecular profiles of single CTCs resorted to marker based bioinformatic annotation of cell types or applied post-capture staining of CTCs using epithelial/cancer-specific molecular markers 10, 15 . In this study we collated published scRNA-seq datasets of human CTCs and peripheral blood mononuclear cells (PBMCs) to do an integrative analysis and build a machine-learning based classification system that accurately labels CTCs in a marker-agnostic manner and also present the ClearCell ® Polaris™ workflow 11, 16 that involves size-dependant enrichment of CTCs, followed by negative selection based on CD45.…”