Methods for Single-Cell Isolation

Every cell is unique and it is only by studying cell-to-cell variations that researchers can understand the significance of rare cell events and discrete cellular changes. This article provides a recap of established methods for isolating single cells and highlights some newer single-cell isolation technologies that are helping to advance scientific research.

Applicability to multiple fields of research

Historically, cell-based assays have measured the average response from a population of cells—whether that be a heterogeneous mixture of cell types derived from excised tissue or a more uniform cell population isolated on the basis of marker expression. However, it is now known that analyzing the genome, transcriptome, or proteome of individual cells can reveal critical insights into the biology underlying normal physiology and disease. This has led to the development of novel technologies for single-cell isolation and analysis with utility across multiple fields of research, including the study of cancer.

“It is well documented that tumors are made up of different populations of cells that may respond to treatment in different ways,” comments Arvind Kothandaraman, Managing Director for Specialty Diagnostics at Revvity. “Targeting and treating tumors with a single drug therefore allows for the possibility that a population of cells that is resistant to that drug continues to thrive. But, in today’s era of precision medicine, technologies exist to analyze tumors at the single-cell level, promising personalized treatment strategies that will, ideally, result in better health outcomes.”

Beyond the oncology field, single-cell isolation and analysis technologies have seen uptake for immunobiology, neurobiology, and stem cell research, where they are used to garner information about cell lineage and function, and for microbiology, where they support investigation of rare microorganisms. They are also increasingly used for cell-line development and engineering. “Being able to isolate and evaluate single cells in this setting is important to select for desired characteristics such as high expression of target proteins or high antibody titers,” reports Jessica Zhou, COO of Namocell.

Established options for single-cell isolation

Established methods for isolating dissociated (suspension) cells include magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), which are used for extracting defined populations from starting material following antibody binding to cellular markers. “A drawback of these techniques is that they require large sample volumes,” notes Zhou. “Moreover, some cell sorting methods have been linked to sorter induced cellular stress, a term used to describe undesirable cellular changes resulting from the sorting process.” Various microfluidics-based technologies are also used, offering the advantages of more precise fluid control (equating to gentler sorting) and lower sample consumption.

When it comes to isolating single cells from fixed tissue samples, researchers have historically relied on either laser microdissection (LMD) or laser capture microdissection (LCM). “The main difference between these two methods is that LCM is gentler and more controllable,” explains Troy Stearns, Product Manager for Arcturus Laser Capture Microdissection at Thermo Fisher Scientific. “During LCM, an infrared radiation laser activates a thermoplastic film that adheres to the cells of interest, which can then be gently lifted away from the sample. LMD instead uses a UV laser to extract the cells for transfer to a tube or microplate, an approach that is both damaging to nucleic acids and restricts custody over the sample.”

Isolating single cells from living tissue has traditionally involved manual cell picking using a micromanipulator, although it can also be performed using a patch clamp system. Both techniques have limited throughput and require highly skilled operatives.

Considerations for technology selection

While the various strategies just described are still widely used, single-cell isolation technologies have evolved to meet growing demand. When deciding on a suitable method, there are several key factors to consider. “Critical attributes for single-cell isolation include the throughput—meaning the number of target cells that can be extracted in a session—and the purity of the resultant prep,” remarks Stearns. “Recovery is equally important; namely, the number of targeted single cells obtained as compared to the total available in the sample.” Zhou adds that researchers should also consider the nature of the starting material, how easy the technology is to use, and the quality of the cells post-isolation. “Many cell types, such as induced pluripotent stem cells, neurons, and protoplasts are especially fragile,” she says. “Methods used for single-cell isolation must not only safeguard cellular viability and integrity but should also avoid inducing stress response genes that could compromise the results of single-cell genomics studies.”

Modern approaches

Modern single-cell isolation technologies are designed to meet different needs. Included among these, Namocell’s Hana™ and Pala™ Single Cell Dispensers uniquely combine flow cytometry, microfluidics, and liquid dispensing to rapidly sort and dispense cells in one step. “Both systems provide fast, gentle sorting to deliver viable single cells directly into 96- or 384-well plates and both employ a disposable cell cartridge to eliminate the risks of sample cross-over or contamination,” explains Zhou. “A further benefit of our technology is that researchers no longer need to perform bead calibration or drop delay adjustment.” With sample input ranging from 100 cells/mL to 150M cells/mL, the Hana and Pala can accommodate either limited sample availability or support the enrichment of rare cell types such as circulating tumor cells.

Revvity's latest product offering, the HIVE™ scRNAseq Solution, was developed in collaboration with Honeycomb Biotechnologies to enable preservation of single cells at the point of collection, prior to single-cell RNA sequencing. “The HIVE™ Collector forms the core of the workflow,” reports Kothandaraman. “It comprises a portable, single-use device with over 65,000 nanowells containing barcoded mRNA capture beads. Once a cell suspension has been obtained and pipetted into the HIVE Collector’s isolated chambers, each cell settles into an individual well, where it is preserved to stabilize its molecular signals. This allows the full diversity of different cell types and their contents to be maintained, ready for analysis at a later date, and can greatly reduce variability across time course studies or multi-center collaborations.”

For increased purity, recovery, and throughput for single-cell harvesting from fixed tissue samples, Thermo Fisher Scientific’s Arcturus™ LCM platform is fitted with both infrared and UV lasers that can be used in combination. “The Arcturus LCM platform scans a heterogeneous tissue sample for targeted single cells, which it marks for polymer-based extraction using the LCM software interface,” explains Stearns. “The pure cell population is then harvested from the tissue and prepared for downstream analysis. Alternatively, a laser microdissection mode can be employed, using the UV laser to physically cut individual cells out of the sample tissue and the infrared laser for polymer-based cell collection. This increases the range of options available to researchers, allowing them to perform live-cell capture, frozen sample staining, immunofluorescence staining, or formalin-fixed paraffin-embedded staining, depending on the nature of their workflow.”