Spotlight on scientists: overcoming the most challenging cell sorting applications

Dr. Daisuke Doi works as Assistant Professor at the Center for iPS Cell Research and Application (CiRA) in Kyoto, Japan, and is strongly involved in the clinical trial program headed by Dr. Jun Takahashi. The lab is developing a new cell therapy based on iPSC-derived dopaminergic progenitors to treat Parkinson’s disease. After establishing an allogenic iPSC cell line (from an HLA homozygous donor) and neural induction, they have to sort Corin-positive neural progenitors in order to end up with the highly pure target cell population needed for further cultivation. During their pre-clinical work, they sorted these cells using conventional droplet sorters. Corin-positive neural progenitors are differentiated into dopaminergic progenitors and are ultimately transplanted into Parkinson’s disease patients. The researchers achieved good purities after cell sorting, but subsequent stages of the cell manufacturing process were hampered by poor cell viabilities, which resulted in low yields of the final cellular product. In addition, the cell sorting process required a large amount of hands-on-time. “When using a conventional droplet-based system, we were manually exchanging sorting vials every 10 minutes for 16 hours straight; that’s just not practicable for a viable treatment,” explains Dr. Doi. For this reason, they started to search for alternative cell sorting solutions. 

The MACSQuant Tyto Cell Sorter, with its gentle microchip-based sorting principle, closed and sterile cartridge, and GMP-grade consumables, appeared to be the perfect solution. Indeed, Doi and his colleagues were surprised to see that sorting on the MACSQuant Tyto Cell Sorter led to a much better post-sort survival rate of cells, while sorting performance in regards to purity and yield was similar to cells sorted on conventional droplet sorters. This observation was further strengthened by sphere size and neurite extension assays which are performed to get an indication of the cellular quality and of their graft survival capacity. Along with better cell survival rates,  the overall yield of the final cell product was much higher with the Tyto.  “Using the MACS GMP Tyto Cartridge with the MACSQuant Tyto Cell Sorter, we’re able to cut cell production time by a third and increase yields.”  In a nutshell, with the MACSQuant Tyto Cell Sorter and its GMP-compliant consumables, including an extensive documentation facilitating the approval of clinical trials by regulatory authorities, Doi and his colleagues are able to produce more cellular product in a shorter time, allowing treatment to be available at the patient’s bedside more quickly.

Read more about this exciting study and have a look at the data:

Enabling the first human iPSC trial of Parkinson’s disease with the MACSQuant® Tyto® Cell Sorter

Ali Mohamed works at Immatics (USA), a clinical stage biopharmaceutical company focusing on T cell cancer immunotherapies. One of their current programs is the ACTolog® multi-target pilot study. ACTolog® is an adoptive cell therapy (ACT) using a patient’s own (autologous) T cells. This study is very exciting, since for the first time not only one but multiple tumor types are being targeted. But how can Immatics provide such a specific treatment for multiple cancer types? Initially, tumor antigen expression (which is specific for each tumor) is tested from fresh tumor biopsies before the personalized cell therapy is produced. During this biomarker profiling tumor targets are selected. After this, target-specific T cells are isolated from the patient using the MACSQuant Tyto. In a subsequent T cell priming and expansion phase, tumor-specific T cells expand to the kinds of numbers needed for cellular therapy. Now, the T cells are ready to be infused back into the patient and specifically combat malignant cells. 

Ali Mohamed appreciates the ease-of-use, performance, and the closed and sterile cartridge system for the GMP manufacturing of patients’ antigen-specific T cells used in Immatics’ clinical trials. “The gentle, microchip-based sorting mechanism preserves high viabilities amongst patients’ precious cells before they are moved to culture vessels for expansion,” says Mohamed. This study demonstrates that the MACSQuant Tyto can support even complex personalized therapies in a GMP environment facilitated by the GMP-compliant consumables. Importantly, the cell functionality of the sorted cells is preserved which is crucial in efficiently eliminating tumor cells in patients. 

Marissa Fahlberg is the Assistant Director of the Flow Cytometry Core at the Tulane National Primate Research Center (TNPRC), USA. The TNPRC dedicates its research to human and animal health with a focus on developing treatments, vaccines, and diagnostic tools for infectious diseases. The Flow Cytometry Core Facility at the TNPRC contributes to the institute’s mission and is highly frequented. TNPRC core scientists, as well as many affiliate scientists, are supported here, and thus the demand for flexibility and experience with various cell types and applications is high. The Flow Core Facility is continuously adapting to these high demands, not only by providing a great service but also by watching out for state-of-the-art technologies. The MACSQuant Tyto is a recent addition to their equipment, and earns its place with its high level of flexibility, ranging from traditional sorts for cell types such as NK cells, B cells, and T cells, to more rare or advanced sorts (e.g. follicular helper T cells and mucosal stem cells).

Fahlberg also points out that “the microfluidic chip confers exceptionally easy setup, while also eliminating the worry that a fluidic issue or clog will cause a long delay or spraying like a traditional cell sorter”.  The MACSQuant Tyto sorts in a “walk-away” fashion, relieving the busy environment and fully booked calendars of the core facility staff. The Flow Cytometry Core Facility at the TNPRC also has a BSL3 laboratory for sorting biohazardous samples. Cell sorting of biohazardous material, such as infected samples, poses a challenge, and precautions like placing conventional droplet sorters in a space-consuming safety cabinet, as well as time-consuming decontamination protocols, are mandatory. Notably, cell sorting with the MACSQuant Tyto happens only in the fully closed and sterile cartridge, thereby reducing the hazards of aerosolization of infectious particles for the operators. The flexibility, ease-of-use, closed, sterile, and aerosol-free sorting process of the MACSQuant Tyto Cell Sorter represents a meaningful addition to the Flow Core Facility equipment, and opens doors for sorting applications where aerosols have been a challenge before.

Patrick Wilson is Professor of Medicine and the Principal Investigator of the antibody biology laboratory at the University of Chicago. His group dedicates its research to the basic understanding of the underlying B cell response, as well as determining specificity and activity of antibodies. The knowledge gained thereby can be used in translational settings, such as direct therapies or vaccine design. In one of their recent studies, Patrick Wilson and his group teamed up with an international research team in order to investigate B cell responses in convalescent COVID-19 patients by using a high-throughput B cell sorting and sequencing platform. The corresponding findings provide a comprehensive tool for studying B cell responses to SARS-CoV2 or vaccination.

In order to sort this rare population of antigen-specific B cells, Wilson’s group took advantage of the MACSQuant Tyto. They utilized a PE-coupled antigenic probe derived from SARS-CoV-2 to target specific B cells. Notably, antigen-specific B cells had a frequency of only 0.26% (Wilson et al., 2020). Thanks to the aerosol-free sorting mechanism of the MACSQuant Tyto, as well as its closed and sterile cartridge system, they were able to sort their BSL2+ materials at the campus’  flow cytometry facility without further precautions. “The low pressure and gentle microchip sorting mechanism maintained high cell viability, which enabled us to easily perform 10x Genomics™ on very rare populations of cells,” declares Wilson. Next generation sequencing techniques are very sensitive, and require high quality samples to minimize sequencing artifacts. The gentle sorting mechanism of the MACSQuant Tyto fulfills these requirements by sorting cells with high viabilities.

If you are curious about this study, read the article here: 
https://www.researchsquare.com/article/rs-80476/v1

Mariane Schleimann is Project Manager and Postdoc at the Aarhus University Hospital in Denmark. The strength of the research in University Hospitals lies in the closeness to the patients and the cooperation of medical doctors and scientists. Previously mainly focusing on HIV-related research, Mariane and her colleagues started to work on a novel therapeutic approach for COVID-19 utilizing neutralizing antibodies. Currently, they use the MACSQuant Tyto predominantly to sort B cells from COVID-19 patients. Subsequently, they extract DNA from these B cells and amplify antibody gene sequences via PCR. These sequences are used to produce the antibodies of interest, which are eventually tested for their ability to neutralize the virus. 

To sort the virus-specific B cells, they use its spike protein coupled to fluorophores. “Being able to rely on a closed, sterile containment is incredibly useful to our work: the sorting itself is what created a safety issue, and was the most critical part,” explains Schleimann, and continues: “With the Tyto, everything’s far more practical: there is no longer a critical step as such.” For conventional droplet sorters, a space-consuming safety cabinet is needed, and sorting should take place in BSL3 rooms because of aerosol production. In contrast, the group installed and uses the MACSQuant Tyto Cell Sorter in a BSL1 lab. Schleimann also points out that no in-depth training is needed to use the MACSQuant Tyto. “Once it’s all set up, a lab manager can be trained to use it easily: they don’t need all the flow cytometry background knowledge,” says Schleimann. Taken together, the research project at Aahrus highlights the benefits of sorting in a closed and sterile system, thereby providing safety not only for the samples, but also for the laboratory staff. 

If you want to know more, please read the interview with Mariane Schleimann.

After Quy Nguyen completed his PhD thesis at the University of California, Irvine (UCI), in Dr. Kai Kessenbrock’s laboratory, he recently transitioned to the genomics core facility at UCI, where he supports scientists with the design and execution of their genomics workflows. During his PhD thesis, his work focused on profiling the cellular diversity of the mouse mammary epithelium on the single cell level, in order to identify changes that happen during initiation and progression of breast cancer. Sorting of epithelial cells is fundamental for this workflow because the mammary gland has a very complex cellular composition, comprising a lot of different cell types. After preparing a single cell suspension from mammary glands, epithelial cells were sorted, either by using a conventional droplet sorter, or by using the MACSQuant Tyto Cell Sorter.

In order to study cellular heterogeneity, Nguyen then was using the 10x Genomics platform. Nguyen observed that the cell types were the same between cells sorted via a conventional droplet sorter and cells sorted with the MACSQuant Tyto. However, in the subsequent transcriptome analysis more gene reads were captured from cells sorted on the MACSQuant Tyto, indicating a better sample quality. In addition, sorting with a conventional droplet sorter led to an upregulation of stress response genes in contrast to the gentle sorting with the MACSQuant Tyto. Finally, Nguyen could also observe higher cellular growth rates in cells sorted with the MACSQuant Tyto when performing mammosphere formation assays to test the functionality of the cells. In summary, Quy Nguyen’s work impressively shows that cell sorting with the MACSQuant Tyto dramatically improves the quality of  samples for genomics, but also functional analyses.

For more information, dive into the results: 

Learn how to get optimal sequencing results with the help of gentle cell sorting