A new toolbox for assessing single cells

Abstract

Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments.

Keywords: cell-cell interaction; genomics; microenvironment; proteomics; single-cell analysis; soluble factors; transcriptomics.

Figure 1

An overview of approaches for the analysis and perturbation of single cells. Both conventional and novel methods to perform single-cell intracellular analysis at the genomic, transcriptomic, and proteomic level are provided, along with methods to perturb and analyze single cells at the level of secretory responses, microenvironments, and cell-cell interactions. Abbreviations: ESI MS, electrospray ionization mass spectrometry; FISH, fluorescence in situ hybridization; LOC, lab-on-a-chip; MALBAC, multiple annealing and looping-based amplification cycle; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MDA, multiple displacement amplification; MSI, mass spectrometry imaging; SLB, supported lipid bilayer; STRT, single-cell tagged reverse transcription; WGA, whole-genome amplification.

Figure 2

Two methods for whole-genome amplification. (a) Multiple displacement amplification is a commercially available and broadly used method for single-cell whole-genome amplification. The method amplifies the genomic starting material exponentially, potentially introducing amplification biases. (b) Multiple annealing and looping-based amplification cycle uses a quasilinear preamplification step to reduce biases followed by polymerase chain reaction (PCR). This method has the potential to transform single-cell next-generation sequencing (NGS) but requires further validation and benchmarking.

Figure 3

Overview of two major single-cell RNA-seq library preparation techniques. (a) Single-cell tagged reverse transcription (STRT), a polymerase chain reaction (PCR)-based method to obtain indexed libraries for next-generation sequencing. The STRT kit is commercially available through Clontech, enabling straightforward implementation of this method. STRT also reduces bias during library preparation by exclusively using full-length transcripts. (b) Cel-seq provides indexing capabilities through in vitro transcription (IVT) linear amplification. This method provides higher sensitivity than STRT at lower cost but requires larger efforts to initially implement.

Figure 4

(a) Schematic of a process (microengraving) using a microwell-based system yielding multidimensional data from single cells that provide information about protein secretion, surface-expressed markers, viability, and gene expression. (b) Characterization of polyfunctional T cell responses with single-cell temporal resolution. Cells were isolated in a microwell array, and the dynamic patterns of cytokine secretion were investigated by following operations similar to those described in a. The viability (calcein label) and lineage of the cells were characterized by image cytometry, and cytokine secretion was probed at different time points by microengraving (left). The data collected from thousands of single cells were clustered based on the pattern of cytokine secretion and displayed in a heat map, in which each row represents time-resolved data on secretions from a single cell. The color code represents different combinations of cytokine secretion. Abbreviations: IFN, interferon; PDMS, polydimethylsiloxane; TNF, tumor necrosis factor. Adapted from Reference with permission from Proceedings of the National Academy of Sciences.

Figure 5

Methods to perturb the single-cell microenvironment. Cell-patterning techniques can be used for targeted drug delivery by embedding the drug in spots of polymer. Seeding density on a flat substrate can be precisely modulated while avoiding initial cell-cell contact by loading a single-occupancy microwell chip with cells and inverting onto the substrate. The influence of cell morphology can be studied using patterned adhesive proteins organized in different geometries, distinguishing the area of adhesive contact area per se from cell spreading. Substrate stiffness can be modulated by altering the concentration of polyethylene glycol (PEG) precursor when forming a PEG hydrogel. The effect of 2D versus 3D microenvironments can be determined by comparing growth in microwells to growth on flat substrates. Cells may be exposed to tethered protein coatings on prespecified surfaces of microwells: Subtractive microcontact printing can limit proteins to the inner surface of the well. Alternatively, proteins may be tethered to the basal surface of the well by spotting proteins onto the pins of the microwell mold, with the added benefit of allowing different protein concentrations and combinations to be tethered in different wells.

Figure 6

Single-cell analysis of cellular responses using microwell-based systems. (a) An artificial microenvironment inside microwells was created to activate specific receptors on the surface of T helper (Th) cells by using supported lipid bilayers presenting ligands to mimic antigen-presenting cells. (b) The study of cell-cell interactions can be achieved by spatially isolating the cells to track behavior by microscopy and functional responses (i.e., cytokine secretion) by microengraving. In these examples, cytotoxic killing of target cells by natural killer cells was followed by labeling of dead cells with a fluorescent probe that binds to DNA when the plasma membrane has been compromised by apoptosis. Adapted from References and with permission from the Royal Society of Chemistry.