High Throughput Single Cell Analysis
Individual cell heterogeneity within a population has invalidated historic classification methods based on macroscopic considerations and given rise to new evaluation techniques based on single cell transcriptional signatures.
In this context, the use of high throughput screening capacities, easy fluid handling, and reduced costs related to device miniaturization, microfluidics has emerged as a powerful tool for single cell analysis and manipulation.
Introduction to Single Cell Analysis
Most cellular research from fundamental cell biology and microbiology to applications in biotechnology is performed using cell populations with high cell numbers. This is convenient, as a huge variety of experimental techniques exist to analyze cell behavior. However, cell population data only covers information of an imaginary average cell (i.e. the average of the microbial culture), and not the mechanistic information of one cell. (1)
Substantial evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their chance of survival. Methods that use average responses from a population often mask the difference from individual cells. To fully understand cell-to-cell variability, a complete analysis of an individual cell, from its live state to cell lysates, is essential (1,2).
Properties of single cells within a population can be quantified by flow cytometry, deciphering cell-to-cell differences and thereby elucidating the heterogeneity of the population. This is a snapshot analysis, reflecting the state of a cell at a certain moment in time. Neither the history nor the temporal development of a cell is traceable. Next to flow cytometry, developments are rapid and aim at the ultimate goal: spatiotemporal high-throughput single cell analysis to mechanistically elucidate cellular functions. The enthusiasm of the field is reflected by the exponential increase of single cell studies and conferences in the last years. The development is pushed by the technical advances in microfluidic chip manufacturing and analytical methods, and pulled by the increasing demand from biologists (3).
Highly sensitive detection of multiple components and high throughput analysis of a large number of individual cells remain the key challenges to understand this aim. In this context, microfluidics and lab-on-a-chip technology have emerged as the most promising avenue to address these challenges.
Droplet Microfluidics applied to Single Cell Analysis
Recent analysis of healthy and diseased tissue homogeneous at the macroscopic scale revealed striking heterogeneities at cellular levels. This variability is particularly well illustrated in polyclonal tumors which constantly undergo mutations. In this respect, single cell analysis is necessary to fully capture the complexity of such tissue. However, working at cellular scale equally exposes many variations in gene expression: from specific biomarkers to insignificant delays in gene expression. High throughput analysis is then needed to multiply the number of profiled cells and discriminate relevant biomarkers from intrinsic population noise.
Droplet microfluidics is particularly well suited and extensively used for high throughput single cell analysis: individual cells are isolated and confined at high speed in pico-volumes to analyze biological processes at the cellular level, streamlining multiple procedures on a single chip, with scope for parallelization.
Recently developed droplet microfluidics has also emerged as a new forerunner for single-cell encapsulation and analysis with massive parallelization. High throughput screening of rare cells to a drug library has been achieved, providing additional information on cell heterogeneity response. The use of microdroplet confinement has enabled new insights into the nature of quorum sensing, suggesting it is a “cell-autonomous mechanism for diffusion or efficiency sensing”.
Benefits
High monodispersity: unique liquid-handling capabilities of microfluidic systems
Reduced costs: volume down scaling from µL to pL compared to pipetting robots
Time saving: molecular diffusion length reduced in small volumes
Higher sensitivity: smaller molecular quantity (1.106-fold less) required to reach minimum detectable concentration
High throughput: up to 1.106 cells compartmentalized per second.
Applications
Single cell genomic or transcriptomic analysis: Single-cell genome or transcriptomic sequencing aims to increase our understanding of complex microbial ecosystems and disease in multicellular organisms by isolating the contributions of distinct cellular populations.
Tissue analysis: Single cell analysis makes it possible to create an atlas of genes expressed in all cell types in the human body, and therefore of the genes expressed in each human tissue. It allows cell-to-cell heterogeneity to be investigated, providing numerous advances in polyclonal tumor research.
Lineage tracing: Single-cell lineage analysis reveals genetic and epigenetic interaction of drug resistance in blastoms, as it enables genomic and biochemical analysis. It is more flexible than classical FACS sorting. (Stem cell lineage selection for stem cell therapy)
Cell sorting with microfluidics: Single cell sorter microfluidic platforms provide numerous advantages over conventional methods by reducing the size of necessary equipment, eliminating potentially biohazardous aerosols, and simplifying the complex protocols commonly associated with cell sorting.
Personalized medicine: single cell analysis of a tumor’s heterogeneity associated with selection and amplification of specific corresponding T cells for personalized cancer immunotherapy.
Related Products
Selected publications from our customers:
Yin, H. and Marshall, D. (2012) “Microfluidics for single cell analysis,” Current Opinion in Biotechnology, 23(1), pp. 110–119. Available at: https://doi.org/10.1016/j.copbio.2011.11.002.
Andersson, H. and van den Berg, A. (2004) “Microtechnologies and nanotechnologies for single-cell analysis,” Current Opinion in Biotechnology, 15(1), pp. 44–49. Available at: https://doi.org/10.1016/j.copbio.2004.01.004.
Yin, H. and Marshall, D. (2012) “Microfluidics for single cell analysis,” Current Opinion in Biotechnology, 23(1), pp. 110–119. Available at: https://doi.org/10.1016/j.copbio.2011.11.002.