Cell Sorting in Stem Cell Research

Stem cells offer vast potential for new medical treatments

With the unique abilities to self-renew and rebuild functional tissues, stem cells have long been of interest to researchers as potential tools for regenerative medicine. 2006 saw a major turning point for stem cell research, when Shinya Yamanaka and Kazutoshi Takahashi of Kyoto University succeeded in generating pluripotent stem cells from fibroblast cultures¹. This breakthrough paved the way for rapid growth within the field and there are now more than 7,000 trials involving the use of stem cells listed in the WHO International Clinical Trials Registry². Yet stem cell research continues to face many challenges, not least how best to isolate the cells of interest. We spoke with Bio-Techne and Miltenyi to learn about some of the innovative cell sorting solutions available to stem cell researchers.

Stem Cell Hierarchy

Stem cells can be broadly classified into three main groups. The first of these, the totipotent stem cells, exists from fertilization until the developing embryo reaches 4-8 cells in size and has the potential to differentiate into any cell type. The cells subsequently become pluripotent, with the ability to form distinct embryonic germ layers: ectoderm, mesoderm or endoderm. These so-called embryonic stem cells (ESCs) then undergo consecutive divisions to become adult stem cells (ASCs). ASCs are multipotent, meaning they have a more limited capacity for differentiation and can form only the specialized cell types of the tissue or organ in which they reside. Another category of stem cells, known as induced pluripotent stem cells (iPSCs), is generated in vitro by reprogramming adult somatic cells into an ESC-like state, such as described by Yamanaka and Takahashi¹. In addition, cancer stem cells (CSCs) represent a small sub-population of cancer cells that can fuel tumor growth.

Challenges for Stem Cell Research

Stem cells present multiple challenges for scientific research, several of which relate to their source. For example, the use of ESCs was once hampered by ethical concerns, since the derivation of these cells historically involved destroying human embryos. However, with the creation of the first ESC line in 1998³, and advances in iPSC technology, this issue is being addressed. In contrast, the use of ASCs poses difficulties for isolation due to low cellular abundance and the fact that surgery is often required to remove the relevant host tissue. In addition, ASCs can harbor abnormalities, such as toxin-induced damage or errors acquired during replication, which risk causing tumorigenicity upon transplantation.

Other challenges for stem cell research center on cellular identification, which is partly due to marker redundancy. For example, as a result of their shared lineage, hematopoietic stem cells and endothelial cell precursors share the markers CD31, CD45, and Tie-2⁴. As such, correctly identifying different stem cells (and removing contaminating cell types) often requires simultaneously detecting large numbers of targets. Minimizing cellular stress throughout the stem cell workflow can also be problematic. Cryopreservation, routine long-term passaging, single-cell cloning, and gene editing can all contribute to extensive cell death if not performed carefully, while using incorrect growth conditions can lead to unintentional differentiation.

Cell Sorting Advances

Cell sorting is the process of isolating distinct cell types from a heterogeneous population. Fluorescence-activated cell sorting (FACS), a well-established form of the technology, uses fluorophore-labeled antibodies for detecting specific cellular markers. After labeling, the sample is introduced into the cell sorter, where a stream of fluid directs the cells one-by-one past an interrogation point. The cells are then encapsulated in liquid droplets, each of which is given an electric charge based on the detected signals to allow for deflection into collection tubes or micro-titer plates.

In recent years, various studies have focused on understanding the impact of shear forces, osmotic forces, laser excitation, and electrical charges on cell health during traditional FACS⁵. While the sensitivity of different cell types to conditions resulting from FACS is not yet fully understood, newer technologies have evolved to address potential pain points. One such technology is Miltenyi Biotec’s MACSQuant® Tyto® Cell Sorter, a cartridge-based system that offers several advantages for stem cell research.

“The MACSQuant Tyto Cartridge consists of three compartments – an input chamber, a positive collection chamber, and a negative collection chamber – located on top of a microchip,” explains Frank Thiel, Ph.D., Global Marketing Manager at Miltenyi Biotec. “The fluorescently-labeled cells are loaded into the input chamber and driven into the microchip by filtered air at low pressure (<3 psi), where they are interrogated by lasers. When a target cell is identified, a magnetic field is applied to the microchip, triggering a sorting valve to open and directing the cell into the positive collection chamber.”

A main advantage of the MACSQuant Tyto is that it does not expose the cells to charge, decompression, or high pressure and shear force. And, because the entire sorting process takes place in a disposable cartridge, the samples remain sterile, with no risk of sample-to-sample carryover or the generation of biohazardous aerosols – something that is of paramount importance when working with patient-derived material. “The MACSQuant Tyto also benefits from being easy to use,” reports Thiel. “You simply load your sample into the cartridge under a sterile hood and insert it into the instrument. There is no need for specialized technical expertise during daily operation as flow control, laser alignment, speed detection and valve timing are all automated.”

Bio-Techne cell sorter and single cell dispensers also utilize a cartridge-based cell sorting technology that overcomes known challenges for cell-based research. The Hana™ and Pala™ Single Cell Dispensers provide sorting and single-cell dispensing in one step, using low sorting pressure (<2 psi) to preserve cell viability and integrity. Ryan McComb, Ph.D., Product Manager at Bio-Techne, notes that key features of the Hana and Pala systems include their speed (single cells can be dispensed into a 96-well plate in 1 minute or a 384-well plate in 6 minutes) and throughput (a sample input of up to 150 million cells can be processed at a rate of 3 million cells/minute).

“The Bio-Techne single cell dispensers have three sorting modes,” says McComb. “The single cell sorting mode dispenses single cells at a density of 1 cell/well, while the enrichment mode can process millions of cells to isolate rare cells based on a fluorescent label. It is also possible to perform bulk sorting. By combining the enrichment mode with single cell sorting, researchers can easily isolate rare cell types, such as adult stem cells, using up to 11 fluorescence channels for detection. The microfluidic technology means there is no risk of system clogging, which can be catastrophic when working with limited sample material, as well as reduces the amount of sheath fluid required by as much as 300-fold compared with traditional methods.”

Bio-Techne also offers a cell culture supplement, the CEPT Cocktail Kit, for improving stem cell viability and fitness.

Therapeutic Applications of Stem Cell Research

Stem cell-based therapies are being developed for a broad range of conditions. These include Parkinson’s disease, for which iPSC-derived dopaminergic neurons have been shown to improve behavioral deficits in rats, and multiple sclerosis, where autologous mesenchymal stem cell transplantation has demonstrated therapeutic benefit in a phase II clinical trial^6,7. Stem cells are also being investigated for their utility to treat spinal cord injuries, macular degeneration, and diabetes, as well as for dentistry applications such as the restoration of dental pulp, periodontal tissue, and mandibular bone⁸. In addition, stem cell transplants are administered to people with hematopoietic cancers including leukemia, lymphoma, and multiple myeloma to replace cells lost during chemotherapy or radiotherapy treatments⁹.