Protecting Stem Cells and Maximizing Success During Single-Cell Cloning

Single-cell cloning of stem cells has the capability to impact applications across all fields of biomedical research and clinical treatments. “Clonal lines offer the opportunity to study and expand pure populations to enable development of myriad applications ranging from basic science, disease modeling, all the way to novel cell therapies,” says Felix Alonso-Valenteen, Field Application Scientist at Namocell.

A homogenous cell population is crucial to identify pathogenic mutations within a population of cells, such as a tumor, with greater precision, adds Rebecca Kreipke, Field Application Specialist Manager at Molecular Devices. “This in turn, drives discovery programs, giving scientists potential therapeutic opportunities to target,” Kreipke explains. Additionally, clonal lines are necessary during the therapy production stage to ensure consistency, quality, and to assure patient safety and therapeutic efficacy.

“While it is a critical step for producing gene therapies, single-cell isolation remains an extremely tedious, low-throughput, and labor-intensive process,” continues Kreipke. Generating single-cell clones is also tremendously stressful for cells, especially fragile and exacting stem cells. “The biggest obstacles to single-cell cloning of stem cells are isolating and successfully seeding individual cells while maintaining good cell health and phenotype, such as pluripotent potential,” says Daryl Cole, Application Specialist at Sartorius.

Methods to protect these cells from damage during each step, including isolation, culture, and cryopreservation, are a prerequisite to bring newer discoveries to the clinic. The good news is recent advances, including cocktails of molecules designed to enhance cell survival, and newer automated systems of separating and dispensing cells, mean clonal lines are poised to revolutionize the way we understand and treat diseases, such as cancer and neurodegenerative processes. There has been “an explosion of innovation in this area,” according to Kreipke “These advances go a long way to alleviate the burden on researchers to manage their single-cell isolation programs, both by decreasing the burden of conducting these screens on individual researchers, as well as decreasing user error and streamlining data management.”

Here we will discuss how to overcome the obstacles common to generating clonal stem cell lines, so you can focus your time on the bigger picture.

Keep (paracrine) communication open

“Like most cells, stem cells prefer to grow in association with other ‘like’ cells and supporting cells. Paracrine signaling informs development of any cell type and determines lineage paths,” explains Alonso-Valenteen. To keep cells isolated while still allowing for cell-to-cell communication, consider products such as, or similar to, Sartorius’s CellCelector plates, which boast thousands of individual nanowells per standard well or chamber, depending on the plate. Media, including nutrients and signaling molecules secreted by cultured cells, is shared between the nanowells, better mimicking in vivo contexts. Cole says this results in more highly viable and healthy cells.

Finding the right platform for cell isolation and sorting

When it comes to isolating cells, options include manual selection, fluorescence-activated cell sorting (FACS), and microfluidics. “All of these methods have benefits and drawbacks, which will guide researchers in making their decisions in what is right for them,” says Kreipke.

Manual selection

Lab groups newer to single-cell isolation might consider starting with manual selection, which requires little upfront capital investment, and is gentle on cells, advises Kreipke. The main drawbacks are that it can be lengthy, inefficient, and difficult to prove clonality. Researchers should be aware that aside from being labor intensive, reproducibility and throughput issues may eventually motivate labs to seek automated processes, according to Alonso-Valenteen.

Cell sorting

Fluorescence-activated cell sorting (FACS) is a popular automated process, especially as many institutions have core facilities with this capability, and it can significantly increase throughput. However, be aware that some cell sorting methods may not be the best choice for especially fragile cell populations, like stem cells, says Kreipke. She points out that while cell sorting can ensure that a single cell ends up in each well of a microtiter plate, the shear forces that cells undergo during the sorting process can be detrimental and have a substantial impact on cell viability. Adds Alonso-Valenteen, “We have observed via RNA-sequencing that cells undergoing high pressure sorting express higher levels of stress response genes, most notably in mitochondrial gene subsets.”

However, this may depend on the system. Cole points out that the Sartorius CellCelector Flex is a sound choice for stem cell single-cell cloning workflows and allows for manipulation at both the single cell and small colony levels. “The main advantage [is] the highly accurate targeting and picking systems and the very gentle aspiration and dispensing process,” says Cole. This results in specific cell selection along with high viability of picked and seeded cells with minimal phenotype disruption. Further, the CellCelector can work with a wide range of standard tissue culture plates, both adherent and non-adherent cell types, and cells embedded in matrices.

Microfluidics

“Most labs who have approached us [at Namocell] have either tried FACS and had disappointing results or continue to use manual methods,” says Alonso-Valenteen. With microfluidic technology cells travel a shorter distance through a fluidic chip compared to long fluidic lines in traditional sorters, explains Alonso-Valenteen. “Hence, we do not need as high of a pressure, have shorter sorting times and less sample loss.” Most often, microfluidics is done using a 3D printed disposable chip, which also reduces cross-contamination. He stresses that these devices are not meant to replace cell sorting; however, labs might consider microfluidics especially if using sensitive cells such as stem or primary cells, or if their core facility is overtaxed.

Ensuring monoclonality

“Another obstacle is tracking single cell clones from start to finish to ensure that the clones you have at the end of your workflow are indeed monoclonal,” says Cole. The CellCelector Flex has highly accurate targeting and picking systems and can image and track clones over time, all the way through late-stage analysis of cultured clones, with fluorescence-based assays. In this way, you “can determine monoclonality from the inception of your studies with a full record of development of each clone over time.”

Molecular Devices also offers a variety of technology solutions for clonal line development. Kreipke says they offer a low pressure cell dispenser along with CloneSelect Imager, which quickly collects high-resolution images of plates to demonstrate proof of monoclonality, for groups who may screen up to hundreds of clones. “By pairing two forms of proof of monoclonality from the cell dispenser and the imager, one can be confident in meeting the FDA threshold for proof of monoclonality during a single round of cloning.”

For those requiring higher throughput solutions, the company offers ClonePix with monoclonality verification. “This solution eliminates the need to use a single-cell dispenser by immobilizing hundreds of cells in semisolid media in 6 well plates and imaging their precise locations on day 0.” As cells form colonies, images from day 0 can be coupled with images 10–21 days later to select clones that meet requirements of the screen. “This allows researchers to be confident that the colonies they are picking are monoclonal when moving them downstream in their workflow.”

Some closing advice

There are many options available for efficiently generating robust monoclonal stem cell lines, but don’t forget the basics, like thoroughly researching protocols and good cell culture practice. “Testing multiple media conditions is very important and depending on how your cells are modified, published reagents may still need tweaking to best suit your cells,” says Alonso-Valenteen. “While on the cutting edge of human knowledge, you can hardly ever follow a cookbook approach to research.”