The study of cells has been instrumental in understanding biological processes. Research on genes and proteins expressed in specific types of cells has typically been performed on samples containing many cells, with analysis reflecting the data averaged across them. But recent studies have indicated differences in cells of the same type or within the same tumor. Consequently, researchers are conducting single-cell analysis to study the unique qualities of individual cells.

Although individual cells have been analyzed microscopically for quite some time to study cell morphology and behavior, more recent interest in genomics and proteomics has generated demand for analysis of genes and proteins within single cells. Development of tools for single-cell genomic studies has progressed, with the ability to amplify both DNA and RNA of individual cells, facilitating targeted DNA sequencing, miRNA analysis and gene-expression studies. Advances in flow cytometry, which uses fluorescent probes to detect expression of specific proteins in cells, have extended the capacities of the technique to better resolve expression of gene products in single cells. Such advancements in technology have enabled progression of single-cell research and grown interest among lab instrument and product companies in the market.

Fluidigm is a firm focused on single-cell genomics. Its single cell–specific instrument, the C1 Single-Cell Auto Prep System, prepares cells for sequencing or for the BioMark HD System, its real-time PCR instrument that can perform single-cell gene-expression and miRNA analysis. Fluidigm’s instrument revenues grew 41% in 2013, and its installed base at the end of 2013 was 920 systems, with 300 of them combined C1 and BioMark systems. Single-cell analysis applications accounted for approximately 50% of product revenues in 2013.

The main technology Fluidigm has used to advance single-cell genomic research is microfluidics. Howard High, fellow, Corporate Communication and Press Relations at Fluidigm, told IBO that the C1’s microfluidic chip–based, automated technique allows 96 individual cells to be isolated for analysis at once, thus taking a step toward overcoming certain challenges of single-cell genomics, such as the substantial time investment and high cost of isolating and analyzing individual cells. To ensure statistical significance of results, single-cell analysis often requires testing a high number of cells. Whereas other methods of cell isolation can be a tedious and time-consuming process in which cells face a high risk of damage, Mr. High stated that, due to its enzyme solution that dissolves connective tissue, C1 allows single cells to be obtained more rapidly from samples. In addition, the system’s low reagent volume reduces costs of analysis. Mr. High told IBO that the most popular application for the C1 is gene expression.

According to Mr. High, Fluidigm is working to develop new technologies to advance single-cell genomics that will allow scaling up of cell preparation in a cost-effective and fast manner to an even higher volume of thousands to hundreds of thousands of single cells. He stated that developing a sustainable environment for keeping cells alive for longer periods to allow repeated analysis, such as over several time points in experiments investigating cell development or drug dosing, and developing additional protocols to more thoroughly address areas of customers’ research were also of interest to the company. Fluidigm would also like to develop a combined workflow for genomic and proteomic analysis on the same cell, according to Mr. High, although that capability is further in the future.

Fluidigm gained single-cell proteomics expertise with its acquisition of DVS Sciences earlier this year (see IBO 1/31/14). DVS produces the $630,000 CyTOF 2 system (see IBO 5/31/13), which had an installed base of 70 systems at the end of 2013 and 41% sales growth. Mr. High told IBO that Fluidigm expects the CyTOF 2 to appeal to the same customer base on the institutional or company level as Fluidigm’s single-cell genomic instruments, as genomic and proteomic scientists collaborate in their research.

CyTOF technology enhances the protein-analysis capabilities of flow cytometry, using ICP-TOF MS to quantify levels of protein expression. This allows more information to be gathered on a single cell compared with flow cytometry, which is limited by spectral overlap of fluorophores to analyzing fewer than 20 proteins. CyTOF uses metal-conjugated antibodies to bind to proteins and can analyze up to 34 proteins. Fluidigm plans to develop the instrument’s technology to increase this number.

A company that has more recently entered the market for single-cell analysis technologies is Affymetrix. Last fall, the company’s eBioscience business unit introduced the QuantiGene FlowRNA Assay. The Assay allows simultaneous detection of protein and RNA expression in a single cell. EBioscience, a supplier of flow cytometry reagents for immunology and oncology, was acquired by Affymetrix in 2012 (see IBO 11/30/11). Last year, eBioscience sales grew 5.2% organically.

The QuantiGene FlowRNA Assay is based on an oligonucleotide probe designed to facilitate branched transcript amplification, a linear amplification that increases signal, when hybridized in situ to target RNA transcripts in a single cell. Using flow cytometry, up to three RNA transcripts can be detected with these probes. The detection of both proteins and RNA in the same cells using the Assay can provide data on the relationship between transcriptional regulation and protein expression in a cell.

Tony Ward, senior vice president and general manager of eBioscience, told IBO that the early-access release of the Assay was in fall 2013, and the company is now refining and further developing the product based on feedback. “The multiparameter nature of the QuantiGene Flow RNA Assay on the genetic side was relatively small, limited currently to three colors, but we’re looking at how to increase that,” he explained. “Combining the three [colors] now with a more robust antibody-based phenotypic analysis is where we’ve made the first improvements, and [it] will be capable of eight-color experiments, including the three RNA measurements.” The QuantiGene FlowRNA Assay will be released at the end of the summer.

Combining single-cell genomics and protein analysis, said Mr. Ward, is necessary to understanding changes in cell regulation and development and the effects of drug therapies. “What’s interesting when you combine the two is the whole interplay that you have in a systems-biology way. I think the importance is looking at the phenotypic potential using mRNA; it’s looking at the control and regulation using noncoding RNA; it’s looking at transcription factors; it’s looking at what happens downstream,” he explained. “It really allows people to get a more comprehensive understanding of the entire process and modifiers to that process.”

Asked about advancing single-cell analysis, Mr. Ward told IBO that Affymetrix has over 17,000 probes for its Luminex bead–based RNA QuantiGene assays, whose use can be extended to the QuantiGene FlowRNA Assay. He said the company is considering and prioritizing product development based on customer interest. However, noncoding RNA is one area that has a high level of interest. As Mindy Lee-Olsen, vice president of Marketing Services at Affymetrix, told IBO, FlowRNA adds to Affymetrix’s products for noncoding RNA analysis.

Mr. Ward also said that another possibility for development is tools for research on organisms for which few products have been developed but that may be better models for studying disease than conventional models. For single-cell analysis beyond flow cytometry, Mr. Ward said it also would be useful to be able to study cells in tissues rather than in suspension, perform high-content screens and apply single-cell technologies to drug discovery.

The US is one of several countries funding the advancement of single-cell analysis on a national level. The NIH Common Fund Single Cell Analysis Program began in 2012 and will allocate funding totaling $90 million over five years. The Program is organized around three initiatives: transciptional profiling to study cell heterogeneity, innovative tools and technologies for single-cell analysis, and technology integration and translation for studying biological processes.

According to Andrea Beckel-Mitchener, PhD, chief, Functional Neurogenomics Program, the National Institute of Mental Health, and working group member of the Single Cell Analysis Program, the techniques increasing the capabilities of single-cell analysis have involved applying genomic tools to the level of the single cell. “[T]he real challenges were around things that you couldn’t amplify and things that were a little bit tougher to measure, like proteins, for example, and biochemistry, like the pH of a cell, and oxygen, and some of the gases of the cell are a little bit tougher ,” she said. “Things that were impossible before are still extremely difficult, but at least we have people working on them now.”

Dr. Beckel-Mitchener told IBO that opportunities to advance the field lie with technologies to distinguish biological variation among individual cells from measurement error. Developing nondestructive techniques for analysis of cells also will be important. “One of the other challenging areas is to measure multiple things at the same time,” she explained. “So not just the DNA, or not just the RNA, or not just the metabolomics end points, but can we combine all of those and get a really good picture of what the cell is doing overall?”

She also indicated great interest by the NIH and the scientific community in tools that are nondestructive to cells and allow measurement of the same individual cells over time, which is necessary to understand the changes on a cellular level in areas including aging, cell development and disease processes. As she told IBO, there has been little activity to develop techniques enabling repeated measurements on single cells, and the NIH hopes to help stimulate this.