Advances in Single-Cell Isolation

The ability to isolate single cells is important for studying rare cell types, for example, and for examining heterogeneity in cell populations, which is key in cancer research. Though recent years have seen advances in single-cell isolation, further improvements are making single-cell investigations more efficient and precise. “There are some key factors scientists could use for optimal single-cell interrogations: flexibility, speed, and throughput,” says Paul Steinberg, Chief Commercial Officer at Lightcast. And as single-cell innovations have become accessible to more researchers, the types of applications are also expanding. “While the focus for single-cell science has largely been on transcriptomic readouts, we are just beginning to see the expansion of this technology into functional analysis, where it is sorely needed,” he says. Here is a look at single-cell isolation methods, both tried-and-true and new, and advances that are expanding this technology.

Manual cell picking & laser capture microdissection

An inexpensive method that uses common lab equipment, manual cell picking works especially for cell-line development. However, it requires a lot of hands-on time, a longer overall workflow, and has a lower efficiency at obtaining single cells compared to other methods.

In laser capture microdissection, the user guides a focused laser beam under a microscope to dissociate and remove individual cells, or groups of cells, from surrounding tissue. This is especially good for isolating rare cells from primary source tissue. However, sample sizes and throughput are relatively low, the process can be time-consuming, and it requires special equipment and training.

Fluorescence- and magnetic-activated cell sorting

Flow cytometry, on which fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are based, is a common method of isolating cells. “One of its greatest strengths is it can analyze individual cells and obtain phenotypic data at high speeds (cells/second),” says Jason McKinney, Senior Director of Application Support at LevitasBio^®. “Cell sorting by flow also allows researchers to design high-parameter panels, enabling the isolation of many different cell types with high accuracy.”

However, flow cytometry can be expensive to invest in, so many labs take advantage of their institution’s core facilities. Other potential disadvantages include requirements of skilled operators, potential shear forces inducing stress on cells, and altered viability. “Even the act of labeling cells for flow sorting can alter their native state, potentially complicating single-cell RNA sequencing or other downstream analyses,” notes McKinney.

FACS is a quantitative flow cytometry technique for sorting cells in suspension according to their fluorescent markers and light scattering properties, and the equipment is available at many research institutions. However, FACS platforms are also complex systems that typically require users to be skilled in their operation.

Cytena provides high-precision lab automation instruments, including single-cell dispensers, and live-cell imaging and cell-line development tools. Zambrano notes that FACS-based single-cell dispensers “offer many of the same benefits as traditional FACS systems, such as multi-fluorescence channels. However, these single-cell dispensers have the added advantages of reduced size, lower maintenance, and simplified cleaning routines.” These systems can be more user-friendly compared to traditional FACS platforms, but both types can impart shear stress while dispensing cells, which can potentially affect cell viability downstream.

Although it uses similar principles, MACS is less complex than FACS, enriching for cells labeled with antibodies attached to magnetic beads. “Various components, such as enzymes, streptavidins, and lectins, can be conjugated to magnetic particles, offering a wide range of separation affinities,” says Adrian Zambrano, Customer Success Manager at Cytena. “Cells expressing the target antigen or other surface markers can be enriched even when present in rare amounts, demonstrating high specificity.” MACS can process large numbers of cells and is amenable to high-throughput methods. However, MACS often functions more as an enrichment than a single-cell isolation technique, and labeling is confined to cell surface markers only.

Microfluidics

Today there are many ways to use microfluidic chips to isolate cells. Microfluidics systems offer extremely low reaction volumes by flowing cells through tiny channels in small chips, and are particularly useful for cell-based assays and single-cell genomics. Some chromatography-based systems use channels containing antibodies that retain selected cells. Other systems use droplet-based methods. “Emerging techniques, such as microfluidic/droplet-based methods and optofluidics, are becoming increasingly intriguing for their ability to manipulate single cells to create intentional interactions, such as between T cells and cancer cells, allowing researchers to capture complex cellular dynamics in action,” says McKinney.

A limitation is that most microfluidic chips or cartridges are not adjustable. “[Many microfluidics systems are custom-made for specific experiments], making it impossible to tweak an experiment on the fly or query cells in a slightly different way,” says Steinberg. Lightcast has a different take to solve this problem: microfluidic chips with transparent sides that allow researchers to use beams of light to control the movement of individual droplets throughout the microfluidic cartridge. Each droplet can be loaded with desired content, such as a cell, a reporter bead, or other compounds/regents. Droplets are then merged, pairwise, to build functional assays and study cell-cell interactions at scale. And by enabling multiple merges, multistep assays become possible. Cells of interest can then be transferred to well plates for additional experiments.

Lightcast’s platform offers the ability to assay functional behavior of individual cells directly, rather than inferring it based on studies of cell populations. Furthermore, the cells can be sorted into subpopulations and assayed again as needed, all using light beams and chips. “The ability to precisely control cell-cell interactions, measure an output, and then export based on user-defined criteria, all at scale— this is really the value our platform offers,” he says. “I believe this will be a game-changer for accelerating single-cell science.”

Imaging-based droplets

Other systems use water-in-oil droplets to encapsulate single cells within individual drops. Imaging-based droplet methods generate picoliter-sized droplets of cell suspension, with the aim of seeding multi-well plates with droplets containing single cells. “Imaging-based proof of single-cell dispensing increases confidence in monoclonality even before well plate imaging,” says Zambrano. “Such proof is sufficient for applications in single-cell genomics and proteomics, where it ensures that only wells with a single cell are sequenced or screened, and adds significant value in ensuring clonal colonies during cell-line development.” Though not common, these systems are valuable in cell-line development, especially those with integrated plate imaging for assessing monoclonality.

Levitation

LevitasBio uses levitation to enrich and isolate cells. Their Levitation Technology™ and LeviCell^® platform take advantage of the unique physical properties of different cell types, which levitate in predictable ways in a magnetic density field, for label-free cell enrichment and isolation. Because the method works without mechanical forces, it exerts no physical stress on cells. “This gentler approach is especially beneficial for fragile cells such as neutrophils, microglia, and dendritic cells, which often do not survive traditional methods,” says McKinney.

Unlike other methods, Levitation Technology can be applied to low-quality or debris-heavy samples. “Samples with less than 5% viability, or those heavily contaminated with debris, can be successfully processed with levitation,” says McKinney. “This technology can also remove residual red blood cells from dissociated tissue samples and myelin debris from neural samples, enhancing the quality of the cell population for further analysis.” It can also be used to pre-enrich samples prior to flow cytometry for better overall results, and/or to clean up samples afterward.

New advances in single-cell isolation and dispensing are prompting improvements in cell sorting, cell-line development, and a myriad of applications in immunology and cancer therapeutics. “Single-cell dispensing technologies have advanced by integrating more sophisticated microscopy techniques, providing the ultimate proof of clonality and enabling direct cell measurement and detection,” says Zambrano. “Fields such as stem cell-based therapies are reshaping how technologies provide stringent data monitoring to support the regulatory requirements for cell therapy development.”