Single-cell analysis enables the acquisition of detailed cell-by-cell data, which was not possible with conventional bulk analysis. This article introduces the benefits and principles of single-cell analysis, the equipment used, and application examples.
Single-cell Analysis Is a Cell-by-Cell Analytical Technique
To understand the concepts and benefits of single-cell analysis, it is easiest to compare it with bulk analysis, which has been the main approach.
Bulk analysis uses samples prepared by homogenizing multiple cells together to obtain overall average data. In contrast, single-cell analysis is a technique that obtains and analyzes independent data for each cell. It has recently been shown that even cells classified as the same species or tissue differ in their expression of proteins and messenger RNAs (mRNAs), and that these differences play an important role in their functional expression. However, bulk analysis cannot obtain such cell-specific difference data.
Single-cell analysis allows us to measure mRNA expression levels on a cell-by-cell basis. It is also possible to analyze cell-to-cell differences in gene sequences of proteins such as immunoglobulins, which have great intra-individual diversity.
Benefits of Single-Cell Analysis and How it Attracted Attention
Traditionally, it has been common to obtain only average data of the cell populations that make up a tissue. However, recent advances in technology have made it possible to analyze trace amounts of material in a short time and to collect samples with detailed manipulation, thereby obtaining information on individual cells.
As a result, it is becoming clear that cell types are more diverse than previously predicted and that even cells of the same species are unique. As the diversity of individual cell types has become clearer, the importance of single-cell analysis has been understood, and it is rapidly becoming a useful research technique.
Single-cell analysis has the benefit of obtaining data on a cell-by-cell basis, and is attracting particular attention for its use in real clinical practice.
For example, in cancer therapy, cancer cells are heterogeneous compared to normal cells, which may contribute to therapeutic resistance. Single-cell genetic analysis of individual cancer cells will help identify genes of therapeutic resistance that only certain cells carry and assist in the selection of effective approaches.
As for COVID-19, it has also been used as a method to study mutations in viral genes such as the Omicron variant and changes in immune response after vaccination.
Furthermore, the technology has been utilized to induce differentiation of induced pluripotent stem cells (iPS cells), and its practicality is expected to increase in the future.
Principle of Single-Cell Analysis
Single-cell analysis can be broadly divided into three steps: cell isolation, measurement, and data analysis. Although some devices have a series of platforms from cell isolation (sampling) to measurement, this section describes the three steps from cell isolation to data analysis, excluding the prerequisite cell culture.
Cell Isolation (sampling)
Single-cell analysis starts with culturing cells and sampling single cells from the cell population in culture. A variety of devices and techniques are used to sample single cells, including flow cytometers, microfluidics, and capillary picking. Some sampling devices are designed as a series of platforms that include sample preparation for subsequent measurements.
Measurement
Samples are prepared for each single cell collected and analyzed in various ways. One typical single-cell analysis approach is gene expression analysis called single-cell RNA sequencing using a next-generation sequencer (NGS). First, complementary DNA (cDNA) is produced by a reverse transcription reaction using all the mRNA in a single cell as templates. The cDNA is then amplified by polymerase chain reaction (PCR) while fragmented and tagged. Finally, the amplified product is subjected to a next-generation sequencer (NGS) to obtain cDNA sequence data.
Data Analysis
Single-cell RNA sequencing enables comprehensive analysis of each gene expression level (transcriptome analysis) from the acquired sequence data. Individual cells can be clustered (grouped by feature) from dimensionality-reduced data by principal component analysis or other means. It can be seen that even cells that look the same have different features.
Evolution of Single-Cell Analysis
From Genetic Analysis to Protein and Metabolite Analysis
Single-cell analysis currently focuses primarily on genetic analysis of mRNA and DNA. In contrast, single-cell analysis of proteins, which play critical roles in biological phenomena, has lagged far behind. This is because, whereas genetic analysis can quantitatively amplify the analysis targets and has thus encouraged development of technologies one after another to stably measure even ultra-micro samples of single cells, proteins cannot be amplified and are therefore difficult to detect. In recent years, however, with the increased sensitivity of mass spectrometers and the development of new sample preparation techniques, analyses other than genetic analysis, such as protein and metabolite analysis, have also been developing.
Towards Analysis that Retains Spatial Information of Cells
In conventional single-cell analysis, cells are detached from the culture vessel as a population, prepared with a cell suspension, and then isolated using flow cytometers or microfluidic techniques. However, technological advances have recently introduced devices that allow sampling only specific cells while maintaining culture conditions without prior cell suspension. Such devices can now retain spatial information of cells for analysis. In addition, devices that can sample with pinpoint accuracy only at specific sites in the cell or where organelles are localized allow for even more detailed analysis of cell functions.
Yokogawa’s Single CellomeTM System SS2000 enables analysis that retains spatial information as described above. Learn more.
Applications of Single-Cell Analysis
Organelle Sampling and Genetic Analysis
The Single Cellome System SS2000 allows precise positioning of glass tips for sample collection while capturing high-resolution 3D images with a confocal microscope, and easy operation with a fully automated system. This technology has enabled sampling of intracellular components at the single-cell level.
Selective sampling and analysis of specific intracellular components contribute to the elucidation of unknown cellular functions and disease mechanisms.
Related article
https://www.yokogawa.com/library/resources/application-notes/lsc-organelle-sampling-and-genetic-analysis/
Single-Cell Sampling and Single-Cell RNA-Seq
Cells in the differentiation process were sampled cell by cell and analyzed for changes in gene expression using a next-generation sequencer. The SS2000 records image information of individual cells, enabling sampling with spatial information such as cell location and morphology. This makes it possible to link changes in cell morphology to changes in gene expression. In addition, only target cells can be sampled while maintaining culture conditions. The ability to repeatedly sample from the same culture vessel at different times reduces variability among culture vessels during analysis.
Related article
https://www.yokogawa.com/library/resources/application-notes/lsc-single-cellome-system-ss2000-single-cell-sampling-and-single-cell-rna-seq/
Sampling and Mass Spectrometry of Drug-Treated Intracellular Components
This is a case in which intracellular components were sampled from drug-treated cultured cells to examine the intracellular localization of the drug. When HepG2 cells were treated with amiodarone, aggregation of lipid droplets was observed. The aggregated lipid droplets from a single cell were collected with a glass tip, and mass spectrometry was performed on the obtained samples. As a result, the peak of amiodarone was detected, indicating the accumulation of amiodarone in the lipid droplets.
Sampling and mass spectrometry of specific intracellular sites allow analysis of the intracellular localization and metabolic levels of administered drugs.
Sampling in Different Cells
The Single Cellome System SS2000 is capable of sampling intracellular components from different cell types, such as from suspension cells or from human iPS cells while maintaining pluripotency. We have confirmed that it can sample whole cells of various sizes, such as HeLa cells larger than 20μm. It also allows selective sampling of target organelles or focused sampling of specific parts such as cell bodies or axons. The system supports complex experimental techniques such as single cell cloning of only cells that exhibit specific behavior under microscopic observation, or continuous sampling from the same culture vessel.
Related article
https://www.yokogawa.com/library/resources/application-notes/single-cellometm-system-ss2000-examples-of-sampling-for-various-cell-types/
Single-Cell Analysis Solution in Collaboration with Novel Intracellular Delivery Technology
The Single Cellome Unit SU10 is an innovative device that delivers target substances directly into cells or nuclei using a nanopipette with a minimum tip outer diameter of several tens of nanometers. We used SU10 to deliver foreign genes into specific cells and observed the changes in the cells with SS2000. In addition, the SS2000 allows selective sampling of only cells that have undergone changes. We provide a series of solutions for manipulation, observation, and sampling of cells at the single-cell level.
Yokogawa's Single Cellome Adds New Value to Single-Cell Analysis
Single-cell analysis is a very important analytical technique that allows us to analyze every single cell now that the diversity of individual cell types has become apparent. Yokogawa’s Single Cellome System SS2000 enables single-cell analysis with spatial information and analysis of specific intracellular components at the single-cell level. For more information on our products, please visit here. If you have any questions or concerns about using this system, please do not hesitate to contact us.
Related Products & Solutions
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Nano-point Delivery / Nano-point Sampling SU10
This system component automates the penetration and injection of single cells using a nanopipette. Its low invasiveness enables the manipulation of live single cells. The system integrates with multiple manufacturers' inverted microscopes.
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Subcellular Sampling System SS2000
The SS2000 is a system that automatically samples specific regions of cells or whole cells at the single-cell level while imaging cells in culture with a confocal microscope. Because cells in culture do not need to be detached, positional and morphological information is maintained.