The ability to isolate single cells empowers numerous applications. These include cell-line development, monoclonal antibody production, single-cell multiomics, and the characterization of rare cell types such as circulating tumor cells. But for such applications to deliver on their potential, single-cell isolation must be fast, gentle, and efficient. Here, we discuss some methods used to isolate single cells and share tips for producing viable single-cell preparations.

Challenges of single-cell isolation

Researchers wishing to isolate single cells must overcome a number of challenges. Not only must they attain a pure population of the target cell type, but they must also ensure that any cellular characteristics remain unchanged. A good isolation method should maintain cellular viability, provide a sufficient number of cells for the intended purpose, and be scalable to future needs. It should also prevent aggregation and be relatively easy to perform without incurring tremendous expense. Elisa Lam, Ph.D., application scientist at On-chip Biotechnologies, comments that throughput (the number of cells isolated in a certain time) and recovery (the number of isolated target cells compared to the number of target cells in the original sample) are other key factors to consider when selecting a suitable technique.

Pitfalls of conventional FACS

Fluorescence-activated cell sorting (FACS) is one of the most commonly used methods for single-cell isolation due to its speed and throughput. However, these advantages are offset by several inherent shortcomings. “The combination of high pressure, electric charge, and the frequency at which high-speed collisions occur during conventional FACS can cause a phenomenon known as sorter induced cellular stress (SICS),” explains Lam. “This limits the utility of the sorted cells for downstream research and is especially problematic for researchers working with large or sensitive cell types such as spheroids or neurons.”

According to Rachel Agoglia, application scientist at Namocell, other disadvantages of FACS are its large sample input requirement and operational complexities. “FACS typically requires at least 100,000 cells, so it cannot be used to sort samples that are available in only short supply—such as many clinical samples,” she says. “It is also vulnerable to cross-sample contamination and system clogging. In addition, FACS instruments have a large footprint and require specially trained experts for operation, so are often inconveniently housed in core facilities. For these reasons, many researchers are switching to alternative methods for single-cell isolation.”

Enabling technologies improve single-cell isolation

Microfluidic cell sorters are widely recognized to improve on conventional FACS, not least because they reduce the risk of SICS by using a lower sorting pressure. They also benefit from lower input volumes, particularly where samples are loaded into a disposable microfluidic chip or cartridge for sorting. “The ability to handle microliter volumes is just one advantage of using a microfluidic chip-based cell sorter,” reports Lam. “These systems also eliminate the risk of sample-to-sample contamination and can enhance the quality of sorted cells where they allow the sheath fluid to be tailored to the cell type.”

In recent years, microfluidic technology has been integrated with other key disciplines to further enhance single-cell isolation. Namocell’s co-founder and COO, Jessica Zhou, explains that combining microfluidics with flow cytometry and liquid dispensing allows researchers to both sort and isolate cells directly into microplates in one step. “Our cartridge design provides the ability to process millions of cells per minute, enabling rapid isolation of rare cells, or the best clones for antibody development, which is something neither FACS nor conventional microfluidic sorters can do,” she says. “It also eliminates the complex operational steps of these sorters, such as bead calibration and drop delay adjustment. And because there is no dead volume, single-cell isolation can be achieved with as few as 100 cells for sample input—an important consideration for precious clinical samples. At the same time, low sorting pressure helps deliver good clonal outgrowth for cell-line development/engineering and enhances single-cell genomics studies by preventing the induction of stress genes.”

Aside from microfluidic cell sorters and cartridges, other technologies advancing the study of individual cells include systems that combine cell sorting with gene-expression profiling. Hye-Won Song, Ph.D., staff scientist at BD Life Sciences–Biosciences, explains that without first sorting cells, it can be extremely time-consuming to study gene expression on a single-cell level. “Although the single-cell RNA sequencing method, sci-RNA-seq, can be used to interrogate cellular gene expression without the need for single-cell isolation (it instead uses combinatorial cellular indexing), it is labor-intensive,” she says. “The BD Rhapsody™ Single-Cell Analysis System was designed to overcome this issue and improves experimental efficiency by both isolating single cells and performing transcriptome analysis in one platform.”

Top tips for single-cell isolation

The following tips combine the insights from all of the contributors to this editorial.
  • Minimize the time spent preparing a single-cell suspension to help preserve cell viability
  • Consider using a cell strainer to filter out clumps or doublets
  • Pay close attention to buffer selection, including both the sorting and collection solutions
  • When sorting cells into 384-well plates, choose plates with straight (non-tapered) square wells to prevent cells sticking to the walls
  • If you intend to culture cells after sorting, use cells that are in log-phase growth—dissociate adherent cells at 80–90% confluency and split suspension cells 1:2 the day before sorting—and identify optimal culture conditions for the outgrowth of single cells
  • When sorting transfected cells for clonal outgrowth, it is often best to wait 72 hours post-transfection to improve the viability of the post-sort cell population
  • If isolating a rare cell type with a fluorescent antibody, always spin down the antibody prior to staining to remove any fluorescent particles that could be mistaken for the target cells
  • For single-cell genomics applications, be sure to spin the plates after sorting to ensure the cells reach the bottom of the wells
  • Speak with suppliers to identify a technology that best suits your needs
  • Choose a robust analysis method for evaluating the quantity and quality of sorted cells