Microfluidic Sample Preparation for Single Cell Analysis

Sanjin Hosic, Shashi K. Murthy, and Abigail N Koppes

Single cell analysis is the measurement of transcription, translation, regulatory, and signaling events within individual cells at the molecular level. The goal is to analyze and synthesize information from single cells in order to holistically understand the cell population. This reductionist approach allows researchers to unravel how molecular events within a single cell link to the behavior of tissues, organs, and eventually whole organisms. Single cell analysis has gained significant traction over the past decade, as evidenced by the number of recent reviews.1-3 The field continues to expand exponentially and necessitates a review of developments that have occurred over the past three years. The transition from bulk to single cell analyses has been fueled in part by studies highlighting single cell heterogeneity and stochasticity relative to whole cell populations.4-5 The random variability in these cell populations is likely due to intrinsic noise. Intrinsic noise refers to cell-to-cell variation in transcription and translation products such as ions, mRNA, and proteins. These components are governed by phenomena such as reaction rates and molecular collisions. Given the flexible and dynamic nature of the cell membrane, reactions and molecular collisions will occur stochastically. Thus, it is unreasonable to assume that all cells within a population are equal at any given moment, and only a large number of single cell measurements will reveal this heterogeneity and provide the statistical power to model it. Modeling approaches are necessary for interpreting the massive amount of data generated with single cell analyses such as whole genome sequencing. Furthermore, these models may ultimately guide the optimum operation of a bioprocess such as the production of valuable biotherapeutics via cell culture or deterministic stem cell reprogramming for regenerative medicine.6 Such findings have driven the development of new analytical systems to probe biology at the resolution of a single cell. In order to study single cells accurately and efficiently, systems with high sensitivity and throughput are needed. The small dimensions of microfluidic systems enable single cell and reagent manipulation with minimal dilution,8 resulting in high sensitivity assays. Furthermore, microfluidic systems offer several key advantages toward the study of single cells including facile automation, parallelization, and reagent reduction.8 Early researchers found that sample preparation such as cell manipulation, compartmentalization, and lysis was significantly more difficult to implement at the single cell scale compared to in bulk. However, sample preparation preceding molecular analysis has also been miniaturized, allowing facile sample processing. As such, microfluidic systems have been developed and applied toward the study of single cells extensively.9-10 Given microfluidics’ instrumental role in single cell analysis up to this point, we can expect continued innovations in microfluidics to better enable single cell biology. In this review, novel microfluidic techniques currently used toward sample preparation and subsequent single cell analysis are highlighted. Techniques are discussed in terms of discrete sample preparation steps that may be necessary for characterizing single cells; tissue dissociation into cell suspensions, sorting heterogeneous cell populations into homogenous populations, isolating, and lysing single cells (Figure 1). With each discrete step, conventional approaches are discussed first and then microfluidic based strategies are reviewed. Finally, the future direction for developing microfluidic single cell analysis technology is discussed.

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