Real-time imaging technology is reshaping the future of health by providing researchers with deeper insights into cell interactions, spatial context of cells, and morphological features.
by Keefe Chee
Real-time imaging spectral flow cytometry (RTI-SFC), a technological advancement in cell sorting and analysis, is facilitating highparameter experiments by empowering scientists to identify previously unclassifiable cells in real time, bridging a long-standing gap in biomedical research and cell-based therapeutic development.
A Vital Tool for Immunologists
The cell sorter is now ubiquitous in immunology research and clinical laboratories around the world. It has assisted with advancing research and clinical care, including the treatment of AIDS, and several infectious diseases. For example, the vital role of immunologists was recently highlighted during the COVID-19 pandemic, as scientists around the world raced to develop vaccines. Advanced vaccination development work conducted by immunologists has saved more lives globally than any other medical intervention as a defense against emerging pathogens.
Immunological research has also been crucial to understanding how to treat significant health issues essential for clinical and commercial application. The breakthrough discovery of monoclonal antibodies was derived from this research, providing scientists and researchers with vitally important research techniques and tools, for example, antibody technology and flow cytometry.
The Fluorescence Activated Cell Sorter (FACS), which was first introduced commercially in the early 1970s, is a technology that has been constantly evolving. Exciting new developments in RTI-SFC are enabling researchers to see and sort cells at speeds never before possible, including the innovation of the BD FACSDiscover™ S8, the world’s first real-time imaging, spectral flow cytometer that provides customers experimental power and additional insights through the ability to sort on image and spectral flow cytometry, eliminating the need for scientists to continue performing these processes separately.
Cell Isolation and Characterisation
When implementing orthogonal frequency domain multiplexing, BD CellView™ Image Technology performs camera-free imaging to picture cells with the electronic and optical components used in flow cytometers. This unique technology enables real-time imaging for analysis and sorting, and all image files can be analysed using the CellView™ Lens plugin on the peer-leading FlowJo Software.
Sorting by image features enables new-to-world cell sorting capabilities that add spatial, and morphological insights to make sort decisions and facilitate accurate gating strategies in flow cytometry experiments. For example, gating with both scatter parameters and BD CellView™ image features can improve the identification and screening of doublets and unwanted events.
Additionally, the ability to inspect images of multiple events at once without separate microscopy workflow, makes quality checks easier and more efficient.
Deeper phenotypic and functional insights can also be obtained during sorting and can be combined with downstream assays (such as next-generation sequencing) for more comprehensive cell profiling. The advanced features of BD CellView™ Image Technology enable these completely new capabilities and open new possibilities that make existing post-analysis applications accessible and easier to develop.
Enhancing Flow Cytometry Experimental Insights
By identifying cell-to-cell interaction and revealing the spatial context of cells using image feature analysis, researchers can identify combinations of engaged cells. This means they can distinguish between two cells that are coincident and true doublets.
These morphological insights allow scientists to interrogate and sort cell types that were previously unidentifiable and unable to be isolated. This can be achieved through a flexible sorting process using BD’s innovation.
Full spectrum cytometry maximises the palate of colours for reagent flexibility and simplifies the panel design process. The BD SpectralFX™ comprises a 78-detector full spectrum optimised hardware, coupled with next-generation QC of in-built LEDs and QC bead. These features measure noise, perform gain calibration, and provide real-time signal/noise hardware information.
When supported by the proprietary system-aware algorithm, the unmixing process adapts to the system noise, sample quality and detector gain to manage spillover spreading. These work in unison to provide excellent levels of quality control and unrivalled resolution power of biological samples.
Empowering Discovery Through Advanced Spectral Resolution and Image-Based Sorting
Guided BD FACSChorus™ Software, automated stream, and sort setup on the BD FACSDiscover™ S8, allows users to learn quickly and use best practices in high-parameter experimental setup.
The software is also compatible with FlowJo™ Software import and export using HyperFinder to generate de novo gates for populations or optimise an existing gating strategy using the algorithm.
BD FACSDiscover™ S8 Cell Sorter with BD CellView™ Image Technology and SpectralFX™ Technology is an innovative high-parameter spectral sorter that offers more ways to classify cells. It has six imaging detectors with seven image feature analyses, including fluorescence location and label-free discrimination on top of the 78 fluorescent detectors across five lasers.
The BD FACSDiscover™ S8 allows the correlation of cell images, traditional flow data and downstream analysis (index sorting), which provides a comprehensive profile at the single-cell level that has the potential to enable insights into highly complex diseases and systems.
Unlocking Biomedical Innovation
Advanced spectral cell technology provides the opportunity to make a positive impact on a wide range of research, here we consider three key areas:
Cancer immunotherapy research has had unprecedented bench-to-bedside clinical success and continues to grow. This area of immunological research has revolutionised oncology, extending the life of patients with fatal cancers. The use of immune-based cancer treatments is accelerating, and it is set to expand rapidly.
In difficult-to-treat tumours, the effect sizes observed in clinical trials of checkpoint blockade agents, ATC transfer therapies and cancer vaccines, have been far higher than the most effective chemotherapeutic agents. Despite some immune-related adverse effects, immune-targeting therapies are better tolerated than traditional chemotherapeutic agents.1
We know cancer biomarkers enable better-targeted therapies, and the importance of being able to identify and isolate tumour-associated antigens (TAA) is crucial in developing new cancer therapies.2
Advances in flow cytometry with fluorescence imaging and image-based decisioning, empower researchers to isolate and view cells with specific, observable traits of interest at speeds not previously achieved.
A major advance in cancer immunology has been the development of Chimeric antigen receptor-engineered T cells (CAR -T). High-speed image-based decisioning has the potential to rapidly accelerate the discovery and manufacture of new and improved cancer-specific biomarkers, heralding advances in targeted cancer therapies.
Recent advances in technology such as CRISPR/Cas9 and spectral cell sorter with high-speed imaging are set to catapult gene therapy to the forefront of both medical research and therapeutics.
Gene therapy can involve the insertion of a copy of a new gene, modifying or inactivating a gene, or correcting a gene mutation. This is done with the help of a vector derived from a genetically modified virus. Several different viral vectors are now used for this purpose.3
Although known since the 1960s as a treatment, gene therapy was first licenced in 2003 in China. It was almost another decade before a European licence was granted. Used successfully in the treatment of HIV-1, inherited eye disease, ADA-SCID, haemophilia B and leukaemia. In the past five years development has rapidly increased and in August 2022, the FDA approved gene therapy for the blood disorder beta-thalassemia. Gene therapy has the potential to treat many types of cancer and recently demonstrated some success in treating spinal muscular atrophy.4
The latest gene editing technique is CRISPR/Cas9. It is more precise, with a high level of flexibility when cutting and pasting DNA, which reduces cost and increases efficiency. It allows the introduction or removal of more than one gene at a time, making it possible to manipulate many different genes in a cell line, slashing the development time in this area from years to weeks.
Flow cytometry is a vital tool in the development of cell and gene therapies and widely used to manufacture Advanced Therapy Medicinal Products (ATMPs) as well as tissue engineered products to measure cell product characteristics.
Traditional manual data analysis can lead to wide variation that can reduce the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis.5 It is a cornerstone tool for cellular therapy. As noted, software for the latest RTI-SFC simplifies set-up and automates analysis. This software development, along with vastly improved speed and accuracy, will increase workflow, vital in such a fast-moving field of medicine.
Typical ATMP drug product critical quality attributes (CQAs) evaluated by flow cytometry include identity, purity, potency, quantity, and viability. Since flow cytometry plays a critical role in ATMP manufacturing, the need for continual development of best practices, along with standardisation within the field is well recognised.6
Stem Cell Research
The potential for stem cell research is considerable; from testing new treatments for efficacy and understanding the causes of disease to regenerative medicine – growing new tissue, bones, and even organs. This could offer hope to millions of people with conditions as diverse as heart disease, cancer, diabetes, burns, Alzheimer’s, and even spinal cord injuries.
Stem cells, unspecialised cells able to change into any cell of an organism, have the ability to self-renew.7 There are two main types of human and animal stem cells, embryonic stem cells and somatic (adult) stem cells. Embryonic cells are of particular interest with their potential to repair damaged tissue as they can develop into specific cells such as blood cells, brain, or bone cells. It was thought that adult cells could not change but that is now being questioned.
Induced Pluripotent Stem Cells (iPSCs) are genetically modified from adult stem cells. These reprogrammed cells are used in the development of cellular drugs. FACS sorting is used in all areas of stem cell research because it ensures cells are kept viable, pure, and functional throughout the process.
Cell-based medicines are fragile, so cell line identity and characterisation and cell culture and differentiation process protocols are vital parts of good manufacturing practice, ensuring efficacy and safety are maintained.8 The latest combination of advanced spectral flow cytometry and novel image technology will enable detailed microscopic image analysis of stem cells at high sort speed.
There is a high possibility for new subsets and expanded research boundaries in the future. With the constant search for breakthrough discoveries, technology that facilitates the performance of complex cellular analysis and improves functional and disease mechanism research will enable such discoveries in less time and with greater confidence. This could in turn provide the opportunity to uncover new applications that can help shape the future of health. [APBN]
This article was first published in the September & December 2023 print version of Asia-Pacific Biotech News.
About the Author
Keefe Chee, Segment Marketing Lead, BD Life Sciences, Singapore
With a deep expertise encompassing flow cytometry, single cell multiomics, molecular diagnostics, and microbiology, Keefe plays a pivotal role in driving the business forward. Keefe’s adept fusion of tech expertise, strategic planning, and technical consultation has been pivotal in shaping regional single-cell research.