Scientists share their stories about how the MACSQuant® Tyto® Cell Sorter has helped them facilitate their cellular investigations. Thanks to the closed and sterile cartridge and the gentle sorting mechanism, the MACSQuant Tyto is a great fit for multiple applications, from basic research through translational, and up to clinical and GMP-compliant workflows.
Renzo Mancuso works at the VIB-Center for Molecular Neurology, University of Antwerp on microglia, neuroinflammation and their role in neurodegenerative disorders like Alzheimer’s disease. In his recent publication together with Prof. Bart de Strooper, they detail the novel, cutting-edge MIGRATE (microglia in-vitro generation refined for advanced transplantation experiments) method. This consists of a combined approach for differentiation of human induced pluripotent stem cells (hiPSCs) into microglia and xenotransplantation of these cells into the mouse brain, together with a quantitative analysis of engraftment. Using this xenotransplantation model, he was able to analyze human specific aspects of microglia biology in Alzheimer’s disease that often lack adequate homology in mice. These findings were published together with Bart de Strooper in Nature Neuroscience.
The MACSQuant® Tyto® Cell Sorter was the perfect fit for such experiments producing superior results in terms of sorting efficiency and cell viability. In addition, due to the closed and hermetically sealed one-use cartridge system, the cells were kept sterile and therefore could be used for transplantations into the mouse brain without any risk of infection or contamination. The gentle microchip-based sorting procedure also provided ideal conditions for downstream applications including transcriptomic studies, minimizing the impact of mRNA profile cell sorting.
If you want to know more, read the full blog article.
Rajeev Gupta works as Associate Professor at the Cancer Institute of the University Colleague London, where researchers study all types of cancer. They dedicate their work to understanding cancer biology and ultimately translating this knowledge into the clinics. Gupta and his team are investigating hematopoietic stem cells (HSCs) and the fate of this cell type during malignant progression.
When COVID-19 changed all our lives in 2020, it also redirected the research focus of Gupta and his team by an intriguing observation. During microscopy analysis of blood samples from patients suffering from severe COVID-19 with lung complications, they observed that neutrophils and monocytes have a characteristic vacuolated appearance which was not found in SARS-CoV-2-positively tested individuals with mild symptoms. These innate immune cells were analyzed by flow cytometry and showed a higher expression of complement receptor 3. This captivating finding motivated them to further examine it in functional studies. To do this, however, they had to isolate these cells from whole blood. Monocytes and neutrophils are among the most delicate cell types, as they easily get activated by any kind of stress. This, of course, makes it complicated to study them and obtain meaningful, unbiased data in functional assays.
Gupta and his team knew they could not sort these cells using a conventional droplet cell sorter. “Our previous experience is that monocytes that have been isolated from healthy volunteers by conventional cell sorters seem to be “activated” by the process of sorting and are “hyper-responsive” to practically any stimulus.”, Gupta explains. Fortunately, Gupta heard about the MACSQuant® Tyto® and its gentle microchip-based cell sorting technique. Working with patient samples infected by SARS-CoV-2, they were compelled to safely sort the cells and ensure operator safety. Gupta stated, “that needed a Tyto: no fluidics whatsoever within the instrument itself, so no risk of carryover contaminations but, more importantly, no aerosol formation, which made it completely safe – even in BSL 1 or 2 conditions”. The MACSQuant Tyto Cartridge´s safe and sterile sample containment allowed Gupta and his team to continue their SARS-CoV-2 project, and the gentle sorting principle enabled functional analysis of fragile monocytes and neutrophils.
Securing certainty
It will come as news to no-one that COVID-19 has dominated 2020 and, although at the time of writing it has yet to be announced, it will be similarly unsurprising if the Word of the Year is not very closely related to virus or pandemic, or some other aspect of the new reality we find ourselves in. Researchers everywhere have trained their sights on the novel coronavirus, even those for whom studying infectious diseases is not par for the course. One such example is Rajeev Gupta, Associate Professor of Hematology at the UCL Cancer Institute and his team. Towards the end of a surreal year, we caught up with him to find out how cancer research lends itself to better understanding the pathology of the current viral threat.
Please give us a brief overview of your organization
I work at the University College London’s Cancer Institute, which is a standalone research institute that is part of UCL Medical School. We study the biology of all cancers, and we have a very strong translational interest, being closely allied to University College London Hospitals.
And what is your research currently aimed towards?
We study the biology of normal human blood stem cells (HSCs), and how they ‘go wrong’ in blood cancers such as leukaemia and lymphoma. Our ultimate aim is to develop novel treatments. A particular interest of late has been understanding how HSCs “decide” when they differentiate from their baseline multipotent state into mature blood cells, and once they make that decision, how they “choose” which type to become. In March 2020, a new area of interest emerged, and it’s a very clinical one that we sort of stumbled into at first. At the start of the pandemic, we noticed some strange features in the blood of the more seriously ill COVID-19 patients. We found that their innate immune cells - monocytes and neutrophils- had a characteristic vacuolated appearance when viewed under the microscope, and when we profiled them by conventional flow cytometry, we found that they expressed high levels of complement receptor 3. We didn’t see these changes in patients who tested positive for the virus but who had remained well; so, there were clear differences in the early innate immune response to SARS-CoV-2 in those patients who suffered lung complications. This was interesting enough for us to want to study it further by comparing the functional properties of innate immune cells from patients with mild and severe COVID-19, but first we needed to isolate them from whole blood.
Enter cell sorting?
Enter cell sorting. But that threw up an immediate challenge: traditional cell sorting is done using droplet sorters, and the way that they work means that cells are exposed to significant pressures – and then rapid decompression, shear forces, and electrical charge. Now, cells vary in their robustness, and the cells of the innate immune system are really extremely delicate, as you’d expect from what are essentially flexible “first responders”. In practice, what this means is that the process of sorting neutrophils and monocytes in a traditional manner is so brutal, that it affects their functional properties – one could say that “the very act of observation alters the observation” - and we had to find a way around this problem.
You ended up choosing the MACSQuant® Tyto® Cell Sorter from Miltenyi Biotec: How did it overcome those challenges?
The Tyto is perfect: it’s accurate, gentle, and, unlike traditional droplet-based sorters, it doesn’t have the potential for damaging the cells we’re interested in. What’s unique about the Tyto is its closed-cartridge system: you load your sample into the sort chamber through an aseptically locked port, and then all sorting happens exclusively within the cartridge. The instrument itself uses low-pressure, filtered air to guide cells individually along a microfluidic channel, where it interrogates them via flow cytometry. A microchip-controlled magnetic gate within the cartridge then sorts them into target and non-target chambers, which are also part of the cartridge. It’s an incredibly gentle process that leaves our target cells unharmed not only for an accurate understanding of them, but for downstream functional applications too – which is especially important for us. Our previous experience is that monocytes that have been isolated from healthy volunteers by conventional cell sorters seem to be “activated” by the process of sorting and are “hyper-responsive” to practically any stimulus.
What other options did you consider before settling on the Tyto?
To be honest, there was never anything else in the frame. I’d seen various bits of information about the Tyto from 2019, so I was fully aware of its existence and capabilities. I always thought it looked like a “nice to have”, but since we already had access to some very high-end traditional cell sorters, it would be a very big funding ask to get a Tyto too. But when COVID-19 came into the frame, that triggered a big change in thinking: we had no idea in the beginning just how infective the virus would be, so it became imperative we had a way to isolate cells in a closed system. That needed a Tyto: no fluidics whatsoever within the instrument itself, so no risk of carryover contaminations but, more importantly, no aerosol formation, which made it completely safe – even in BSL 1 or 2 conditions.
Another thing that’s really comforting about the whole system is that, even if you make a critical mistake, you don’t ruin your sample: it goes into a chamber from where it can be rescued. Indeed, far from just rescuing you if you mess up, you can use that feature to chance to redo and optimize your approaches over and again.
Are there any plans for long-COVID research?
Absolutely. We’re currently embarking on a long-COVID project with several physicians in the private sector. They have referred a lot of patients who’ve had documented SARS-CoV-2 infection, but are plagued by persistent post-infective symptoms, and they have provided us with blood samples from these individuals. Their blood seems mostly normal, but they have subtle defects in effector T cell subsets, meaning T cells will be a key population to look into. The Tyto is proving a highly effective way of isolating T cells for further study.
And outside of COVID-19?
Originally, the Tyto was housed in our diagnostic facility because it was at first intended only for use with potentially highly infectious COVID-19 samples. We have subsequently moved it to the Cancer Institute, and right now we’re using it for a variety of applications: recently, colleagues who work on neurological cancers have been using it to isolate individual cancer cells from dissociated brain tissue – something that is almost impossible to do with conventional sorters as the cells are so delicate. In our own work, we have been using the Tyto to isolate HSCs for metabolic studies of mitochondrial function and glucose metabolism. Previously this work would have been done on HSCs isolated using conventional sorters, and an underlying concern of ours has always been whether the very act of cell-sorting affected HSC metabolism. Ultimately, we foresee that the Tyto will become general “go-to” device for several groups in the Cancer Institute – and every week more groups are cottoning on to it.
What have been your overall impressions of working with Miltenyi Biotec?
Where to start? Like most hematology departments, I have a longstanding relationship with Miltenyi Biotec because of CD34 isolation – I mean, you’re the gold standard: that alone buys you the time of day when you’ve got something new to talk about! Actually, I don’t know a stem cell research group anywhere that doesn’t use your products.
Finally, what gets you out of bed in the mornings in the face of the inevitable obstacles a day in research will throw up for you?
All scientists will tell you they want to get to the bottom of whatever problem they’re working on: We’re captivated by getting the right answers to the questions that we’re interested in by formulating and testing hypotheses. It’s the sheer joy of grappling with a problem that keeps us going!
Rajeev Gupta, PhD, FRCP, FRCPath is Associate Professor of Hematology at UCL Cancer Institute, Honorary Consultant Hematologist and Clinical Lead for Laboratory Hematology at University College London Hospitals, and Clinical Lead for Hematology at HSL Pathology Ltd.
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.
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.
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.
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.
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