Optimize Tissue Dissociation for Single-Cell Research

Single-cell secrets to research success Simplify your tissue dissociation process Improve cell viability in single-cell analysis cytiva.comContents 3. A note from Cytiva 4. Single-cell sequencing expands genomics research horizons 6. Webinar: The impact of tissue processing on single-cell sequencing 8. Comparison of VIA Extractor and gentleMACS in tissue dissociation for single-cell RNA sequencing analysis 11. Single-cell discussion guide 12. Gentle, efficient tissue disaggregation for optimized cell viability and yield 17. Expert Q&A 19. Master single-cell RNA sequencing with our free online course! 2 cytiva.comcytiva.com 3 Single cell magazine Forward A note from Cytiva Single-cell analysis offers researchers an unprecedented opportunity to understand individual cell populations and their behavior in health and disease. In contrast to traditional bulk analysis, which provides an average analysis of mixed-cell populations, single-cell studies allow for the examination of tens of thousands of individual cells from a single tissue sample. Consequently, single-cell analysis technologies have become a powerful tool for understanding cellular function, disease diagnosis, therapy response prediction and treatment selection. A crucial element of single-cell workflows is the dissociation of tissues into single cells. This process must be handled quickly and carefully to ensure high cell viability and accurate representation of the tissue’s cellular composition. Read our case study on page 7 to understand how tissue dissociation can affect the quality and reliability of downstream analyses. The VIA Extractor™ tissue disaggregator provides a standardized, semi-automated approach to tissue dissociation. Designed for high-throughput omics research, the VIA Extractor ensures high-quality single-cell suspensions with minimal cell damage. Read on to discover how your research could benefit from a gentler approach! We always appreciate your feedback, so please get in touch. We hope you enjoy this issue!4 cytiva.com Single cell magazine Article Single-cell sequencing expands genomics research horizons how genetic mutations, gene expression changes and protein dynamics contribute to disease progression and treatment responses. Multiomics allows for a detailed analysis of the tumor microenvironment, revealing how different cell types interact and influence each other. It can identify mechanisms of drug resistance, track cell differentiation pathways and map the complex network of molecular interactions within and across cell populations. Using big data and advanced analytical tools, multiomics enhances the precision and depth of single-cell research, accelerating discovery of new biomarkers, therapeutic targets and diagnostic tools. Traditional sequencing methods lack the sensitivity to analyze individual cell differences, resulting in a loss of cellular heterogeneity information. In contrast, single-cell sequencing characterizes individual cells, uncovering small sub-populations within larger groups. (1) Multiomics approach harnesses big data to accelerate discovery The power of single-cell sequencing is even greater when combined with multiomics. Integrating genomics, transcriptomics and proteomics provides a comprehensive view of cellular functions and interactions. Researchers can better understand the complexities of biological systems by uncoveringcytiva.com 5 Single cell magazine Forward Challenges and considerations for tissue disaggregation The disaggregation – or dissociation – of tissue samples into viable single cells is a critical step in single-cell sequencing. Manual tissue processing is time-consuming and highly variable, depending on an operator’s skills. Researchers typically prefer a mild disaggregation process that ensures high cell yield and integrity while maintaining the context of the parent sample. Key aspects of an optimal disaggregation process include: • Minimal time between tissue collection and disaggregation into single cells for downstream applications. • Protocol consistency to minimize variability throughout the entire workflow. • High yield and viability of the disaggregated single cells. • Representative and consistent cell yield and viability relative to the original tissue sample. Transitioning from a manual to an automated dissociation system greatly enhances consistency, improves reproducibility, and increases efficiency, making it a desirable advancement for researchers working with single cells. References: 1. Method of the Year 2013. Nat Methods. 2014;11(1):1-1. doi:10.1038/ nmeth.2801 Cytiva and the Drop logo are tradem The single-cell sequencing workflow Initially limited by high costs and technical challenges, single-cell sequencing is now robust and accessible throughout the research community. The most important factor is high-quality starting material, as it ensures accurate and reliable results. Preparing whole tissue samples for data analysis involves several steps, including disaggregating tissues into viable single cells, extracting nucleic acids, amplifying individual cells, and sequencing (Fig 1). However, tissue disaggregation remains a significant challenge. Fig 1. Overview of the single-cell sequencing workflow, including collection, isolation, amplification sequencing and data analysis.6 cytiva.com Single cell magazine Article Discover how solid tissue processing techniques impact cell viability and revolutionize singlecell sequencing applications in oncology. Join our webcast to gain insights into current and future trends in single-cell sequencing, tissue processing and cryopreservation techniques. Learn how these advancements affect cell viability and single-cell data quality. We will explore: • Current solid tissue processing approaches, considering different sample types, sizes and structures. • The role of new automated tissue processing devices in optimizing cell integrity and preserving context in high-throughput applications. • Recommended methods for tissue disaggregation and cryopreservation to enhance single-cell sequencing outcomes. Watch this video The impact of tissue processing on single-cell sequencing Videocytiva.com 7 Single cell magazine Forward Comparing VIA Extractor and gentleMACS for scRNA-seq Single-cell sequencing is a powerful tool for studying cellular heterogeneity. Advances in single-cell RNA sequencing (scRNA-seq) provide new insights into transcription profiles, allowing for identification of rare cell populations. High sensitivity in single-cell analyses requires careful handling and processing of tissue samples to minimize process-induced effects that could skew results. This study investigated the ability of the VIA Extractor™ disaggregator (Fig 2) to generate highquality single-cell suspensions from fresh tissue for scRNA-seq analysis, comparing its performance to the gentleMACS™ dissociator (Miltenyi Biotec). How do the protocols compare? Murine liver tissue was weighed, washed, and equally divided between the VIA Extractor cell processor and gentleMACS to ensure sample consistency. The tissue was then disaggregated using the gentleMACS or VIA Extractor protocol (Fig 3). Notably, the VIA Extractor cell processor completed the dissociation process in 10 minutes, while gentleMACS required 30 minutes. How do the results compare? The VIA Extractor disaggregator produced higher results of viable single cells compared to gentleMACS (Fig 4). Observations under a microscope, cell counting, and viability assays indicated superior performance for VIA Extractor disaggregator, with a higher percentage of viable cells (72% ± 7%) compared to gentleMACS (52% ± 3.6%). UMAP clustering showed more cells represented in the sequencing data from VIA Extractor8 cytiva.com Single cell magazine Article Fig 2. (A) The VIA Extractor tissue disaggregator provides fast, low-impact tissue dissociation into single cell suspensions. (B) The Omics pouch placed into the VIA Extractor disaggregator and held in place with the Omics clamp. (C) The VIA Extractor disaggregator placed into the top of the VIA Freeze Uno. Fig 4. Cell viability. Cell counts as determined after sequencing from Cell Ranger software were significantly higher when using the VIA Extractor disaggregator for tissue dissociation. Fig 3. The complete workflow for both the gentleMACS dissociation method and the VIA Extractor disaggregator dissociation method. REGATION FOR OPTIMIZED CELL VIABILITY AND YIELD ™tissue disaggregator Fig 1. (A) The VIA Extractor™ tissue disaggregator, (B) the Omics pouch placed into the VIA Extractor™ tissue disaggregator and held in place with the Omics clamp, and (C) the VIA Extractor™ tissue disaggregator placed into the top of the VIA Freeze™ Uno controlled-rate freezer. first-in-kind id tissue and tor™ tissue nsistently ty relative m provides omics giving reliable pplications. impact ngled n tle as controlledfreezer to s. the Omics ag. The ng the mics clamp further me solution g a syringe. the Extractor™ reeze™ Uno regation rate freezer erature ample ystem of o control the mechanical ension, The following data was generated to demonstrate the effectiveness of the VIA Extractor™ tissue disaggregator for disaggregation of biological tissues into a viable cell suspension that is suitable for use for a wide range of applications, including tumoroid growth, flow cytometry, and single cell sequencing and analysis. The VIA Extractor™ tissue disaggregator was used alongside the gentleMACSTM Dissociator (Miltenyi Biotec) and a popular manual method to disaggregate a selection of commonly studied tissues according to standard protocols to determine relative performance in generating viable cell suspensions. *For research use only (RUO). Not for diagnostic use. (A) (B) (C) 30 mins 10 minscytiva.com 9 Single cell magazine Forward disaggregator, with fewer cells displaying stressrelated transcription profiles compared to gentleMACS (Fig 5). The fraction of reads in cells was also higher for VIA Extractor disaggregator (59.7% ± 5.7%) compared to gentleMACS (49.8% ± 7.4%). Fig 5. UMAP clustering of cells and differential abundance of cells. (A) UMAP clustering of all cells by sample set (gentleMACS and VIA Extractor disaggregator). While the clustering is similar, there are significantly more cells in each cluster from tissue processed using the VIA Extractor disaggregator. (B) Proportional representation of cells from both sample sets in each of the clusters identified. This indicates that for samples processed using the VIA Extractor disaggregator there are significantly more cells present in most clusters except cluster 10. What can we learn from this? This study demonstrates that the VIA Extractor disaggregator provides a higher yield of viable cells in a shorter time, with fewer cells displaying stressrelated transcription profiles compared to gentleMACS. This highlights the importance of choosing low-impact technologies for generating single-cell suspensions in omics workflows.10 cytiva.com Single cell magazine Article Do you work in single-cell research? What steps make up your current workflow? What instruments and brands do you use for each step? Examples of labs performing single-cell research: (genomics, proteomics, metabolomics) and single-cell sequencing • Undertaking high throughput (20–40 samples per week) and medium throughput (5–20 samples per week) tissue disaggregation • Using Miltenyi GentleMAC tissue disaggregator • Using 10X Genomics platform (BGI) • Using illumina® platforms • Using Flow Cytometry platforms VIA Extractor tissue disaggregator • High-throughout • Gentle processing • Semi-automated • Reduces sample to sample variation • Maximize sample viability • Preserves cell content • Wide range of effective temperatures for maximum variability Example challenges: • Cell viability • Cell stress • Cell aggregation or clumping • Reproducible results • Personnel training • Disaggregation at higher temperatures damaging cells • Cost • Time Example applications: • Genomics analyses • Primary cell culture • Cell imaging • Proteomics • scRNA sequencing (single-cell) • Organoids/Tumoroids • Patient-derived xenografts (PDX) Can you describe your downstream applications? What is your current tissue disaggregation approach? Can you describe some challenges you encounter with your current method? Are you impacted by low sample availability? Manual disaggregation Automated disaggregation Does sample-to-sample and technician-to-technician variability impact your work? How many samples do you process per week? Do you often have sample backlog? Would a higher throughput be advantageous? Yes No Single-cell discussion guideSingle cell magazine Forward cytiva.com 11 Looking to improve cell viability in singlecell analysis Introducing the VIA Extractor tissue disaggregator* • Mild processing optimizes yield, cell viability, and preserves content relative to the parent sample. • Standardized, automated process reduces sample-tosample variation. • Disaggregates solid tissue to single cell suspension in as little as ten minutes. • Process up to three samples in parallel in single-use pouches, minimizing the risk of contamination. *For research use only (RUO). Not for diagnostic use. cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corp. or an affiliate doing business as Cytiva. VIA Extractor is a trademark of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. *For Research Use Only (RUO). Not for diagnostic use. ©2022 Cytiva For local office contact information, visit cytiva.com/contact CY32942-03Oct22-DA Looking to improve cell viability in single-cell analysis? Introducing the VIA Extractor™ tissue disaggregator* • Mild processing optimizes cell viability and yield and preserves content relative to the parent sample. • Standardized, automated process reduces sample-to-sample variation. • Disaggregates solid tissue to single cell suspension in as little as ten minutes. • Process up to three samples in parallel in single-use pouches, minimizing the risk of contamination. *For research use only (RUO). Not for diagnostic use. CY32942-03Nov23- cytiva.com Request a demo12 cytiva.com Single cell magazine Article The VIA Extractor tissue disaggregator is a novel device for the disaggregation of human and animal solid tissue and tumor samples into viable single cells. The VIA Extractor disaggregator uses a mild processing approach for consistently high cell viability, yield, and preservation of cell integrity relative to the parent sample. The standardized, closed system provides a semi-automated process for use in high-throughput omics research (genomics, proteomics, metabolomics, etc.) giving reliable results in single-cell sequencing and flow cytometry applications. Key features: • Gentle: Optimizes cell viability and yield with low-impact disaggregation. • Standardized: Consistent process reduces sample-to-sample variation. • Semi-automated: Simplifies tissue dissociation with fewer steps. • Fast: Processes tissue to single cell suspension in as little as 10 minutes. Gentle, efficient tissue disaggregation for optimized cell viability and yieldcytiva.com 13 Single cell magazine Forward Comparative performance of tissue dissociation methods To compare the efficacy of the VIA Extractor disaggregator with gentleMACS and manual disaggregation methods, we conducted experiments on mouse liver, lung, kidney, and brain tissues. Across all tissue types, the VIA Extractor disaggregator demonstrated higher yields and viability with fewer undissociated clumps compared to gentleMACS and manual methods. 13 cytiva.com VIA Extractor disaggregator protocol: • Weigh and wash tissue. • Place tissue in an Omics pouch with enzyme cocktail (Fig 6). • Process in VIA Extractor disaggregator for 10 minutes at 37°C. • Centrifuge, remove red blood cells, and dilute cells to 800 cells/µL for analysis. gentleMACS protocol: • Weigh and wash tissue with pre-warmed DMEM. • Add tissue to a C tube with enzyme cocktail. • Process using gentleMACS for 30 minutes at 37°C, followed by additional dissociation. • Centrifuge, remove red blood cells, and dilute cells to 800 cells/µL for analysis. Manual disaggregation: • Weigh and wash tissue. • Mince tissue using scalpel blades and scissors. • Filter cell suspension through 70 μm cell strainers. • Centrifuge, remove red blood cells, and dilute cells to 800 cells/µL for analysis. Fig 6. Omics pouch placed into the VIA Extractor and held in place with the Omics clamp.Single cell magazine Article 14 cytiva.com Table 1. Comparative performance of tissue dissociation methods. Tissue type Viability (%) Yield (cells/mg) Liver Results Both instruments produced similarly high viability percentages. Both instruments produced similarly high yield per mg of tissue. Lung Results The viability of lung tissue was variable using the gentleMACS, but consistently above 80% using the VIA Extractor disaggregator. The VIA Extractor disaggregator produced a higher yield of viable cells compared to gentleMACS.cytiva.com 15 Single cell magazine Forward Tissue type Viability (%) Yield (cells/mg) Kidney Results Both instruments produced equally high cell viability. The VIA Extractor produced higher viable cell counts compared to gentleMACS for both small and large tissue sizes. Lung Results The cell viability is significantly better using the VIA Extractor disaggregator compared to manual techniques. The cell yield is significantly better using the VIA Extractor disaggregator compared to manual techniques.Single cell magazine Article 16 cytiva.com Conclusion The data described shows that the VIA Extractor™ tissue disaggregator: • Uses a simple and efficient technique to generate viable cells in suspension for downstream applications • Produces higher single-cell yield than a manual method in 5–10 minutes and minimizes any stress-induced effects that can interfere with downstream analysis • Is gentle and semi-automated for standardized and consistent dissociation which results in single viable cells and reduction in sample-tosample variation. For more information, read our case study Discover the future of oncology with single-cell sequencing Our comprehensive white paper reveals how singlecell sequencing is revolutionizing oncology by providing unprecedented insights into tumor biology and cellular function. Download your copy todaycytiva.com 17 Single cell magazine Forward for high-throughput research like genomics, proteomics, and metabolomics. Q: What kinds of tissue samples can the system handle? A: The system is designed to break down mammalian tissues, tumors, and small organs. It’s been tested on various mouse organs like the kidney, liver, muscle, lungs, brain, and spleen, as well as on human renal cell carcinoma. Q: What settings can be adjusted on the device to control tissue breakdown? A: You can tweak several settings depending on the tissue type: • Time: Decide how long you want the digestion process to run. • Speed: You can set the paddle rotation speed anywhere between 50 to 240 rpm. • Mode: Pick between continuous paddle beating or an on-and-off rotation (one minute on, one minute off). • Temperature: You can set the temperature for digestion from 0°C to 37°C, depending on what you need. Angeliki Achimastou is the product manager for single cell within the Genomics and Diagnostic solutions business at Cytiva. Angeliki has been working in the genomics sector for over a decade in academic, research, and commercial settings, in varying roles including Field Application Specialist, Modality Specialist and Life Science Specialist. She completed her BSc at King’s College London and her MSc at Imperial College in London. Angeliki went on to gain her PhD and post-doctorate in Molecular Neurobiology at the National Institute for Medical Research in the UK. Q: What is the VIA Extractor tissue disaggregator? A: The VIA Extractor is a device that breaks down human and animal tissues, including tumors, into a uniform mix of live single cells. It’s a closed system that’s semi-automated, making it great Q&A The VIA Extractor tissue disaggregator Angeliki Achimastou, PhD, Product Manager, Single Cell18 cytiva.com Single cell magazine Article Q: What’s the minimum and maximum amount of tissue the system can handle? A: The system can handle tissue samples as small as 30 mg and as large as 1.2 g, depending on the tissue type. Q: Besides cells, can we also isolate nuclei? A: That hasn’t been tested yet. Q: How does the system optimize cell viability? A: The VIA Extractor uses a gentle, semi-automated process that helps keep cells intact and preserves their content, similar to the original tissue. Q: Can the cells be preserved for use at a later stage? A: Yes, you can preserve the cells using the VIA Freeze™ Uno controlled-rate freezer, which maintains cell viability for future applications. Q: Lastly, if I go ahead and get the VIA Extractor and VIA freezer for my lab, what else will I need to buy? A: Here’s a list of everything you’ll need: • VIA Extractor tissue disaggregator • VIA Freeze Uno controlled-rate freezer • Omics clamp • Omics pouch • Omics applicator • Heat sealer (to prevent sample contamination) • Forceps or tweezers • Luer-lock 5 mL syringe (helps with getting the sample in and protects both the sample and the bag) If you want to keep it simple, there’s also the Omics bundle, which comes with the VIA Extractor, VIA Freeze Uno, and the Omics clamp all together. Enhance your research Master single-cell RNA sequencing with our free online course! Enroll in our 30-minute course to gain comprehensive insights into single-cell analysis, focusing on the single-cell RNA sequencing (scRNA-seq) workflow. Start now Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. VIA Extractor and VIA Freeze are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. gentleMACs is a trademark of Miltenyi Biotec GmbH. illumina is a trademark of illumina Inc. Any other third-party trademarks are the property of their respective owners. © 2024 Cytiva For local office contact information, visit cytiva.com/contact cytiva.com