Innovations in Cell Science: A New Era of Cellular Insight

SPONSORED BY INNOVATIONS IN CELL SCIENCE: A New Era of Cellular Insight The Future is Bright: Advances and Developments in Flow Cytometry Revealing How the Immune System Behaves in Real-Time With Immune Cell Imaging Growing Into Their Potential: The Innovations Driving Organoids Forward Credit: iStock/adventtr CONTENTS 5 Is Cryopreservation Frozen in Time? 8 Applications of Optical Tweezers in Modern Cell Biology 12 Quality Control in Cell Culture: Ensuring Safety, Reliability, and Reproducibility 16 Revealing How the Immune System Behaves in Real-Time With Immune Cell Imaging 21 Growing Into Their Potential: The Innovations Driving Organoids Forward 25 Single-Cell Technologies Are Redefining Our Understanding of Human Development 30 The Future Is Bright: Advances and Developments in Flow Cytometry 3 FOREWORD TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE Progress in cell science has always depended on how clearly researchers can see and understand individual cells. Today, that view is sharper than ever, as a wave of new technologies brings unprecedented insight into how cells function, interact, and change over time. This eBook explores the evolution of cell science. From single-cell and multiomics approaches that capture the unique molecular signatures of individual cells, to advanced imaging, flow cytometry, and precision manipulation techniques, the collection highlights tools that are redefining how biological systems are studied. It also considers the broader ecosystem that supports discovery, including robust cell culture practices and the emergence of more physiologically relevant models such as organoids and assembloids. As innovation continues to push the boundaries of what is possible, the work featured here underscores a simple but powerful idea: understanding cells more precisely is key to improving human health. The Technology Networks editorial team The FIRST manual pipette that repeat dispenses. Lowest plunger force. This familiar lab tool is anything but ordinary. With INTEGRA's SWITCH you choose; pipette manually or 'SWITCH' to repeat dispense mode and gain productivity while enjoying the lowest plunger force possible. SEE IT IN ACTION > 5 INNOVATIONS IN CELL SCIENCE Is Cryopreservation Frozen in Time? Alexandra Boussommier-Calleja Let’s break the ice on cryopreservation: much like many scientific breakthroughs, the field took off thanks to a mistake. In 1949, Christopher Polge returned from holiday to find unexpectedly good survival in his sperm-freezing experiments—only to realize he had accidentally frozen a sample containing glycerol, the compound he used to slow sperm during observation. That accident allowed the cells to survive freezing in liquid nitrogen. Seventy-six years later, although the field has made progress, cryopreservation remains a bottleneck in both research and clinical practice. Will the next breakthrough also happen by accident, or can we find ways to accelerate it? A crystal-clear problem Cryopreservation works by cooling biological material to slow or pause cellular metabolism for long-term preservation. With slow-freezing methods, ice crystals can form inside and outside of cells causing mechanical damage or osmotic stress. With vitrification, ice formation is avoided during cooling, but can occur during warming, leading to recrystallization. Historically, permeable cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO) have been used to reduce intracellular free water and limit ice formation. But this is a double-edged sword: DMSO, while effective, is cytotoxic. Credit: iStock/Panupat Ratanawechtrakul TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 6 Until now, researchers have learned to live with it, given that “the core technology for preservation has changed little since the 1970's,” as explained by Prof. Allison Hubel from the Department of Mechanical Engineering, University of Minnesota. “The entire field needs improvement and there is hardly an aspect of cryopreservation that would not benefit from improvement,” Hubel added. A bottleneck in and outside of research In addition to being potentially cytotoxic, cryopreservation is generally slow and labor-intensive. “When we work with our primary human cells, we often generate 100+ vials while freezing. This takes 2–3 people working together to get them ready in record time and ensure room temperature does not damage them,” said Dr. Cynthia Hajal, assistant professor in the Department of Mechanical and Industrial Engineering at Northeastern University. As Dr. Hajal explained, currently “The main issues are post-thaw viability and phenotypic changes that occur due to freezing: if the background and behavior of the cells is affected, we cannot replicate our experiments well nor get accurate results.” Post-thaw viability also matters beyond research, in cell therapy, which often consists of injecting cells back into patients after modifying and cryopreserving them. Here, DMSO cytotoxicity damages both the cells and, if not fully removed, can even cause adverse reactions in patients. If freezing cells reliably remains difficult, freezing tissues or organoids is even more challenging. Reliable preservation of patient- or animal-derived tissues is needed to enable asynchronous workflows between clinicians and researchers, so that tissues don’t always need to be shipped right away. Organ transplantation is also ripe for innovation: today, 25% to two-thirds of donated organs are discarded which could be largely prevented by increasing the time available to find suitable recipients through advancing preservation methods. Similarly, shipping biologics that must be delivered far from their source remains a problem as often the only solution to maximize their viability during transport is to cryopreserve them. Cool technologies to scale beyond research In research, these issues are often circumvented with protocols optimized from one lab to the other. These challenges, however, still hold back larger-scale industrial or clinical applications. In some fields, such as in vitro fertilization, moving from slow freezing to vitrification has already dramatically improved outcomes, with survival rates improving by up to 30 points in some studies. But for many other applications, problems persist. This has spurred innovation, including the use of anti-freeze proteins (AFPs), which are naturally found in organisms that survive extreme cold. These non-toxic, bio-inspired proteins have improved the post-cryopreservation viability of human embryonic kidney cells when added both intra- and extracellularly. A group has even shown that fish AFPs extended the preservation time of transplanted rat hearts. To enter clinical practice, engineering more diverse AFPs, including membranepermeating variants, will be essential. DMSO-free CPAs also generate great interest: glycerol or trehalose have been used, but no single molecule seems to be capable of replacing DMSO. Instead, osmolyte mixtures could be transformative, according "[Cryopreservation] is a platform technology for a variety of fields, not just cell and gene therapy." –Prof. Allison Hubel. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 7 to Prof. Hubel, whose lab has tested them on dozens of cell types, as they combine solutes that stabilize proteins, maintain membrane integrity and reduce osmotic shock with far lower toxicity. Another exciting direction is ice-free preservation through isochoric (rather than isobaric) conditions, where ~ 45% of the solution remains liquid. Biological samples can be stored in this liquid phase, protected from freezing damage while still benefiting from reduced metabolism at low temperature. Rat hearts were successfully cryopreserved this way for the first time in 2018. While energy-efficient, this method exposes tissues to higher pressures, which may introduce new risks. Hydrogels, long used to mimic the cell’s microenvironment, may also improve cryopreservation by providing a protective barrier, inhibiting ice crystal growth during freezing and warming, and buffering CPA diffusion to prevent apoptosis from transiently high CPA concentrations. Another line of innovation focuses on optimizing rewarming. Nanoparticles (NPs) can absorb energy from external physical fields and convert it into heat, enabling rapid and uniform heating of biological samples to inhibit crystal formation. However, NPs may be cytotoxic, and achieving uniform distribution remains challenging. Since no single solution is perfect, studies have begun combining approaches. For example, Tian et al. microencapsulated mouse preantral follicles (PAFs) in alginate using a microfluidic device before mixing them with NPs. Microencapsulation separated the PAFs from the nanoparticles, eliminating potential toxicity. Their study reported a birth rate following fertilization of the vitrified PAFs comparable to the control group. Clearly, innovation is happening—but as Hubel noted: “The biggest obstacle to translation into clinical or industrial applications is the lack of knowledge of or expertise in cryopreservation. When people do not understand the scientific basis for cryopreservation, they do not understand how to improve outcomes or evaluate new technologies.” In other words, the field may need to return to first principles before it can meaningfully scale. Not yet ready for prime time Improving cryopreservation might seem like a logistical and incremental problem today— but once it’s solved, it could unlock disproportionately exciting possibilities. As Hubel explained: “We need to continue to raise awareness of the importance of cryopreservation for a variety of fields. It is a platform technology for a variety of fields, not just cell and gene therapy.” Longevity and transhumanism are one such area: if we cannot yet extend lifespan dramatically, could we at least halt post-mortem aging through cryogenics, preserving ourselves for a future revival? This is the incredible bet that hundreds of people have made since 1967 as they lie cryopreserved until future medical breakthroughs enable the possibility of safe thawing. Once again, cell culture—the foundation of so much biomedical progress—stands to play a central role in shaping our medical future. MEET THE INTERVIEWEES: Prof. Allison Hubel is a professor in mechanical engineering at the University of Minnesota. She combines her interest in heat and mass transfer, material science, and biology to address issues related to preservation of cells and tissues for therapeutic applications. She uses imaging techniques to detect cell response to freezing, optimization algorithms to improve preservation outcome, and a variety of other tools to understand the behavior of water during freezing and molecular mechanisms of protection. Dr. Cynthia Hajal is an assistant professor at Northeastern University. Her research interests are at the intersection of engineering and cancer biology, developing tissue engineered models of tumors to study cancer progression and drug delivery. Her lab focuses on the design of engineered tumor models to investigate the role of the components and properties of the microenvironment in cancer progression and treatment outcomes. 8 INNOVATIONS IN CELL SCIENCE Applications of Optical Tweezers in Modern Cell Biology Héctor Zamora Carreras, PhD Since their introduction in the 1980s, optical tweezers have become valuable tools for studying biological systems at microscopic scales. Developed from Arthur Ashkin’s pioneering work on radiation pressure, they use highly focused laser beams to trap and manipulate microscopic particles while exerting forces in the piconewton range, making them ideally suited for probing cellular and molecular processes. Their noncontact nature enables the manipulation of cells, organelles, proteins, and nucleic acids under nearphysiological conditions. At the same time, advances in holography, fluorescence microscopy, and microfluidics have greatly expanded their capabilities. Today, optical tweezers play a central role in modern cell biology by enabling quantitative measurements of forces, biomolecular conformational changes, and cellular responses to mechanical stimuli. This listicle highlights four major applications of optical tweezers, illustrating how they are helping researchers investigate biological systems across multiple spatial scales, from individual molecules to complex tissues. Quantification of cellular forces and mechanotransduction One of the most important applications of optical tweezers in cell biology is the investigation of cellular Credit: iStock/d1sk TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 9 mechanics. Cells constantly experience mechanical forces from their surrounding environment and must dynamically respond to these stimuli to maintain proper function. Mechanotransduction, the process through which cells convert mechanical signals into biochemical responses, therefore plays a central role in processes such as migration, differentiation, tissue morphogenesis, and immune activation. Because many of these interactions occur within the piconewton force regime, optical tweezers are uniquely suited to probe them with high spatial and temporal precision. Optical tweezers are especially valuable because they can exert and measure highly controlled forces on cells and intracellular structures. By trapping dielectric microspheres attached to the plasma membrane or cytoskeletal components, researchers can quantify how cells mechanically respond to external perturbations. Bead displacement within the optical trap can be translated into quantitative force measurements through calibrated trap stiffness. Studies using these approaches have revealed how integrins, membrane receptors, and cytoskeletal networks transmit forces throughout the cell and regulate downstream signaling pathways. Beyond probing individual cells, optical tweezers have become powerful tools for quantifying cell–cell and cell–matrix adhesion forces. These measurements have provided insights into how immune cells establish stable contacts with their targets, how epithelial tissues maintain structural integrity, and how alterations in adhesion contribute to cancer invasion and metastasis. In immune mechanobiology, optical trapping experiments have further revealed that T-cell activation is influenced not only by biochemical recognition events but also by mechanical forces exerted at the immune synapse. Similar approaches have been used to investigate force transmission during tissue morphogenesis, revealing how local mechanical interactions contribute to large-scale tissue organization during embryonic development. These studies highlight the growing recognition that mechanics plays an active role in regulating developmental processes rather than simply being a consequence of them. Among the most established mechanobiological assays is membrane tether pulling, in which optically trapped beads are used to extract thin membrane tubes from living cells. These experiments provide direct measurements of membrane tension and bending rigidity, key parameters governing membrane viscoelasticity. Their application has expanded beyond membrane mechanics to include probing of intracellular mechanics. Optical tweezer–based microrheology enables direct investigation of the cytoplasm using internalized beads or endogenous organelles as probes. These studies have shown that the cytoplasm behaves not as a simple viscous fluid, but as a dynamic active matter system shaped by ATP-dependent processes, cytoskeletal remodeling, and intracellular transport. Beyond single-cell measurements, modern optical trapping platforms are extending these approaches to multicellular mechanics and tissue-scale interactions. By generating multiple independently controlled traps through holographic or time-shared multiplexing strategies, these systems can quantify intercellular adhesion forces and viscoelastic responses during cell binding and separation. Together, these developments are helping researchers better understand how mechanical forces regulate tissue organization, embryonic morphogenesis, wound healing, fibrosis, and cancer progression. Revealing the mechanics of single molecules Optical tweezers have revolutionized singlemolecule biophysics by enabling the manipulation of biomolecules such as DNA, RNA, and proteins. Traditional biochemical approaches generally measure averaged behaviors from large molecular populations, often masking transient conformations, stochastic fluctuations, and intermediate states. Optical tweezers overcome these limitations by individually studying TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 10 molecules and applying controlled mechanical forces while monitoring molecular responses in real time. One of the most established applications involves nucleic acids. By tethering DNA or RNA molecules between optically trapped beads through specific molecular linkages, researchers can stretch, twist, and unzip individual strands while measuring forceextension behavior with nanometer precision. These experiments have provided major insights into DNA elasticity, chromatin organization, transcriptional regulation, replication dynamics, and DNA repair mechanisms. Optical trapping has also enabled direct observation of nucleosome unwrapping and transcription under mechanical tension, revealing how force can influence genome accessibility. Another major application of optical tweezers is the study of molecular motors, including kinesin, dynein, myosin, helicases, and ATP-dependent proteases. Landmark experiments revealing the characteristic 8 nm stepping behavior of kinesin provided some of the clearest evidence of how molecular motors transport cargo within cells. Beyond nucleic acids and motor proteins, optical tweezers are frequently used to investigate protein folding and protein–protein interactions. Many proteins transition between multiple conformational states, and these structural rearrangements are often central to biological function. Optical trapping experiments can monitor unfolding and refolding trajectories in real time. Importantly, these measurements allow reconstruction of molecular energy landscapes, revealing how proteins transition between metastable states and how external forces reshape the energetic barriers that govern biological function. Manipulation of cells and their internal machinery Single-cell manipulation remains one of the most established applications of optical tweezers. Individual cells can be trapped, repositioned, rotated, stretched, or assembled into controlled configurations without physical contact, minimizing mechanical damage while preserving viability. This capability has proven valuable for studying cell–cell interactions, immune responses, circulation dynamics, and developmental processes. Intracellular manipulation represents another important application of optical trapping. Organelles such as mitochondria, vesicles, lipid droplets, and peroxisomes can be repositioned inside living cells to investigate intracellular transport pathways and cytoskeletal organization. Such experiments provide unique opportunities to study intracellular transport dynamics and the role of cytoskeletal networks in organizing the cellular interior. Adaptive optical trapping approaches have further improved intracellular manipulation by compensating for optical aberrations generated within living cells, enhancing trapping stability while revealing the heterogeneous physical properties of the cytoplasmic environment. The characterization of cellular deformability represents another important application of optical tweezers. Optical stretchers, which use counterpropagating laser beams to deform suspended cells under nearphysiological conditions, have emerged as powerful tools for studying cell viscoelasticity. Modern optical trapping systems have also evolved beyond conventional single-beam configurations. Many platforms can now generate multiple independently controlled traps through holographic beam shaping or time-shared multiplexing approaches, enabling the simultaneous manipulation of many particles or cells. These systems facilitate applications such as three-dimensional cellular assembly, selective cell sorting, and coordinated manipulation of multiple intracellular structures. Building on these capabilities, recent developments integrating optical tweezers with automation and microfluidics are expanding their use in cell sorting and rare-cell isolation, supporting emerging single-cell analysis workflows. Optical tweezers are frequently combined with advanced imaging modalities such as fluorescence microscopy, Raman spectroscopy, and microfluidic imaging platforms. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 11 These multimodal approaches allow researchers to correlate mechanical measurements with biochemical and structural information in real time. Recent studies have additionally explored in vivo optical trapping approaches for manipulating blood cells and probing vascular dynamics directly inside living organisms. Advancing disease research and biomedical engineering In disease research, optical tweezers also play a promising role in investigating pathogen–host interactions. Early experiments demonstrated the manipulation of bacteria and viruses, while more recent studies have focused on quantifying how pathogens attach to and mechanically interact with host cells. Direct measurements of host–pathogen adhesion forces have become another important application of optical trapping, complementing traditional biochemical approaches with quantitative mechanical measurements Cancer research represents another major area of application. Because cancer progression is frequently accompanied by changes in cell stiffness, adhesion, and force generation, optical tweezers have become valuable tools for cancer mechanobiology. Measurements of cell deformability, adhesion, and force generation have shown that metastatic cells often exhibit distinct mechanical signatures compared with non-invasive counterparts. These findings are helping establish biomechanical phenotyping as a complementary approach to traditional molecular biomarkers for cancer diagnosis and prognosis. Regenerative medicine, tissue engineering, and singlecell diagnostics represent another rapidly expanding area of application. Their ability to position cells with micrometric precision makes them attractive for constructing engineered tissues, studying cellular heterogeneity, and integrating with lab-on-a-chip technologies. This level of control allows the assembly of defined cellular architectures and the investigation of how cell positioning influences tissue development and function. The future for optical tweezers in cell biology From quantifying cellular forces to probing individual biomolecules and manipulating living cells, optical tweezers have become one of the most versatile tools in modern cell biology. Their unique ability to combine precise force measurements with non-contact manipulation has enabled researchers to investigate biological systems across multiple spatial scales, from single proteins to complex tissues. As advances in imaging, automation, and force spectroscopy continue to expand their capabilities, optical tweezers are poised to play a central role in addressing fundamental questions in cell biology and developing new approaches for biomedical research and diagnostics. 12 INNOVATIONS IN CELL SCIENCE Quality Control in Cell Culture: Ensuring Safety, Reliability, and Reproducibility Debomita Chakraborty, PhD Quality control (QC) in cell culture or tissue culture refers to a set of quality control measures used to ensure that the in vitro cultures are authentic, healthy, uncontaminated, and grown under defined conditions. This is important for generating reproducible, consistent, and reliable data for improved research outcomes. Advanced translational research with potential therapeutic applications, including stem cell-derived products and CAR T cells, requires strict QC for regulatory approval to ensure safety and consistent efficacy before clinical use. This guide will provide you with important tips on establishing, maintaining, and monitoring standard cell culture process. Maintaining aseptic, hygienic conditions The age-old golden rule of cell culture is working under sterile conditions, which includes keeping the cell culture clean. This involves handling cells under laminar flow hoods and biosafety cabinets equipped with HEPA/ULPA filters, wearing proper lab coats and gloves, and following updated lab safety guidelines as outlined in ISO 14644-5:2025. Cleanrooms for cell culture typically meet ISO 5–7 (Class 100–10,000), which are separated from other lab areas to maintain ideal conditions, including temperature (20–24 °C), humidity (40–60%), and 20–30 air changes/hour, preferably with in-built Credit: iStock/Murat Photo TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 13 HVAC filters for reduced airborne microbial and particle contaminants. In most cases, cell cultures are maintained at a stable temperature (37±0.5 °C), CO₂ level (5±0.2%), and pH (7.2–7.4). Incubators maintaining proper cell culture conditions should be cleaned regularly using manual wiping, high-heat/auto-decontamination cycles, professional fumigation, hydrogen peroxide decontamination, or other manufacturerrecommended methods. Water pans in incubators and water baths for thawing media and cells should be added with approved disinfectants. Laminar flow hoods need to be wiped with disinfectants, most commonly 70% ethanol, before and after use, and they should be sterilized further using UV light at least once a day. Cell culture attire, such as lab coats, should be sterilized frequently and not worn elsewhere. Controlling cell culture conditions The physical and chemical parameters for culturing the cells should be regularly monitored and documented. The temperature, CO₂, pH, media composition, osmolarity, glucose, or metabolite levels, and any other relevant material/method should be regularly monitored and well documented. Extra care should be taken when optimizing and employing specialized complex media for advanced tissue cultures, stem cells, or organoids. The reagents and media used should be stored optimally following the manufacturer's instructions, and regular checks should be undertaken to ensure that the activity and function of the growth factors or other supplements is well preserved and that the media are not degraded during storage. Media supplements should be aliquoted, and repeated freeze-thawing should be avoided. For large-scale suspension cultures or bioreactors, additional parameters such as oxygen supply, agitation, hydrodynamic stress, and cell density should also be regularly tracked. Stringent safety protocols should be established in cell culture: always use clean gloves, use sterile pipettes and pipette tips, wear lab coats, and cover hair to protect both yourself and the cell or tissue systems from harmful contaminants. Monitoring critical quality attributes (CQAs) In regulatory terms, CQAs related to the biological characteristics of the cells or cell-derived products need to be within defined limits to ensure safety, consistency, and efficacy. For cell lines, CAR-T cells, stem cells, and stem cellderived products, the following CQAs need to be regularly documented: cell identity, morphology, sterility, viability, potency, genetic integrity, clonogenicity, and differentiation potential. Authentication, PCR-based tests, or sequencing should be performed every few months to confirm cell identity and genetic integrity of the cells or cell lines in use. Viability of cells can be measured using metabolic assays (such as MTT or resazurin), ATP-based luminescence assays, membrane integrity assays, and cell proliferation or clonogenic assays. Cell identity, clones, and differentiation can be tested with multiple cell markers using immunofluorescence/ immunohistochemistry staining or flow cytometry analysis. In vitro differentiation assays, cell killing assays for T cells, antigen binding or ELISpot assays, or other functional assays should be performed for validation of cell functions and potency. The regions of tissues or organs used for generating organoids or precision-cut slices should be imaged for immunohistochemical stainings and additional clinical markers for proper documentation. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 14 Keeping cell culture free of contaminants The most important quality attribute of cell culture is sterility; cell or tissue systems, cell-derived products, and cell culture reagents and media should be free of mycoplasma, bacteria, fungi or mold, and viruses, which affect cell behavior and experimental readouts. Poor aseptic conditions, fecal cross-contamination, or cultivation of clinical, surgical, or animal samples might lead to microbial contamination. In vitro cultures should be inspected closely every day for turbidity of the medium, pH changes, and signs of bacterial contamination. Bacteria and fungi The presence of bacteria, fungi, or molds can be observed regularly under a phase-contrast or brightfield microscope at 100–400× magnification; check for spores, round or elongated rod-like structures, elongated hyphae (thread-like filaments), budding yeast cells, or mycelial networks distinct from mammalian cells. Several detection methods, such as PCR kits, isothermal amplification, and staining for Grampositive and Gram-negative bacteria are available for confirmation of contamination. PCR is the most efficient way of detecting contamination even at low levels. Mycoplasma Mycoplasma are very small, wall-less bacteria that cause the most frequent contamination of biological samples. They are antibiotic-resistant and can barely be detected using normal observation or a brightfield microscope, making them persistent and challenging to eradicate. It is advised to carry out routine mycoplasma detection every 4–8 weeks in all cell culture samples. When data are generated, the biological material should have proof that no mycoplasma are present in the samples at several passages. Mycoplasma can be detected by DAPI or Hoechst staining to check for extranuclear DNA fluorescence, but PCR-based assays are the most reliable method. Viruses If handling patient samples known to contain a virus such as HPV, virus detection tests should be carried out frequently, and the positive samples should be identified and labeled for sample characteristics as well as for biosafety. Otherwise, PCR or titer-based kits are commercially available for the detection of common viral contaminants, which should be used at least once per batch of samples or every 3–4 months. Take preventative action Freshly isolated or received cells should first be tested for contaminants before freezing and storing. Commercially purchased biological material should be requested for data for the assurance of contamination-free samples and checked for contamination when received, as the contamination might occur during delivery. Sera used in cell culture should be filtered through 0.2 μm filters before use. Endotoxin levels should be maintained according to the strictly specified levels, which are generally below 0.1 EU/mg for all in vitro studies. It should be ensured by the providers and can also be checked by different techniques, including Limulus amebocyte lysate (LAL)-based or recombinant factor C assays, as well as newer electrochemical aptasensors, nanozyme‑, or fiber‑optic–based biosensors. Tested positive for a cell culture contaminant? It is recommended to discard the contaminated cultures and, if possible, completely and thoroughly disinfect and decontaminate the entire cell culture workspace and cleanroom to stop spreading contamination. Then, contamination-free vials should be thawed and checked again for contamination in early passages. For valuable research materials, antibiotics, antimycotics, or antiviral compounds may be added to the media, but the microbial titer should be measured continuously. Experiments may be performed once the TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 15 contamination levels sink to a minimum, and it should be considered when interpreting the outcomes. Viral contamination is generally difficult to remove, and such samples should be clearly labeled during the study for data integrity. Utilizing automation and artificial intelligence (AI) in cell culture QC The digital era makes life easier for researchers. Deep learning interfaces allow non-invasive, realtime monitoring of cell culture media, changes in pH, metabolite depletion, and cell densities. Small digital incubators that allow automated measurements of cell viability and density are commercially available. Automated imaging systems placed in incubators also facilitate monitoring changes in cell morphologies. Ongoing research focuses on combining machine/deep learning with sensor analytics and AI-driven image analyses to not only monitor CQAs but also integrate automated sampling for dynamic tracking of them. This has already been successful on a small scale for stem cell engineering by detecting and predicting early contamination risks, indicating morphology changes, and measuring physical parameters and media composition. Conclusion Cell culture builds the foundation of research and assays related to biological studies: a clean culture leads to credible results. When QC is weak, even beautiful data are just a well‑decorated artefact. Little everyday care should be taken to keep the cell culture sterile, prevent contamination, continuously monitor cell properties and identity, and—most importantly—to document and report every protocol carried out. Strong cell culture QC turns your incubator from a guessing game into a reliable factory for trustworthy science. 16 INNOVATIONS IN CELL SCIENCE Revealing How the Immune System Behaves in Real-Time With Immune Cell Imaging Cliff Dominy, PhD For decades, science has made progress in understanding the complexities of the immune system by inferring its activity from histological slides and biomarkers— snapshots frozen in time. Imagine trying to follow a story from a stack of photographs. You might guess the general plot, but exactly who went where, when, and with whom would remain a mystery. Today, immune cell imaging can deliver dynamic 3D movies of immune interactions in real-time. These advances have deepened our understanding of a patient's inflammatory profile, including their antigen and vaccine responses, as well as how their immune system either combats or succumbs to cancer. Immune cell imaging combines targeted probes, often antibody-bound, with genetically expressed fluorescent proteins, all visualized using several detection systems. These range from whole‑body positron emission tomography/computed tomography (PET/CT) to subcellular two‑photon intravital microscopy (2P‑IVM). These technologies enable researchers to track immune cell interactions over time and across space. These imaging modalities bring clear benefits to personalized medicine, potentially allowing clinicians to monitor individual patients and understand why the immune system can protect one patient, while failing in another. Credit: iStock/gorodenkoff TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 17 Seeing is believing: the immune system in action Understanding the mechanics of a “normal” immune response is fundamental to developing therapies for immune dysfunction. Two complementary “workhorse” techniques, immune PET/CT and 2P-IVM, can be used alongside several auxiliary techniques to study health issues such as autoimmunity and adverse therapeutic events. Dr. Ronald Germain, a National Institute of Health (NIH) distinguished investigator, was one of the early pioneers of 2P-IVM. The Germain group, and others, have used this imaging modality to characterize the details of neutrophil swarming, a key mechanism by which the immune system responds to cell damage or infection. By labeling neutrophils with fluorescent probes, Germain and colleagues observed that this mechanism was not “a random walk of cells that interacted, like balls in a pinball machine but rather a much more organized system that let us understand how rare cells find each other efficiently to make a good response.” 2P-IVM also revealed how populations of naïve T cells, dispersed throughout the body, can mount a coordinated attack at any given moment. Germain elaborated, “Our two-photon imaging showed that T cells move on the stromal network that exists in the lymph node, and the reason that's interesting is that the dendritic cells are presenting the antigen live on that same fiber network.” In other words, antigen presentation is not a chance meeting, but an inevitable encounter between key immune components travelling along the same cellular scaffold. Autoimmunity: beyond the biomarkers Once we understand the normal immune response, the next challenge is understanding what happens when things go wrong. Autoimmune conditions occur when the body recognizes its own components as foreign, triggering an immune response. The target tissue can vary, leading to presentations ranging from rheumatoid arthritis to inflammatory bowel disease. In clinical settings, immune PET imaging provides non-invasive visualization of these pathophysiological immune responses. Dr. Erik Aarntzen, a nuclear imaging specialist at the University Medical Center Groningen Netherlands, has emphasized PET’s key strength in viewing the immune system as an interconnected network rather than isolated tissue. As he explains, “It allows you to assess the immune system at the systems level—how different organs interact, how they’re interconnected—from local lymph nodes to the spleen and bone marrow. It really captures the ‘systemness’ of our immune system.” By adding CT to the analysis, Aarntzen and colleagues can get even more information. “It's mostly anatomical. It’s size and anatomical variation,” Aarntzen explains, “then you add on top of that the functional information [of PET imaging] that I think is very valuable to have.” No longer are physicians interpreting an abstract immune activation event, but now have a spatially resolved, quantifiable whole-body view of the affected sites that can guide diagnosis, improve patient risk‑stratification, and ultimately therapy selection. At the clinical level, PET/CT is highly sensitive. “In the case of patients admitted to hospital with symptoms “Light can provide exceptional temporal control over dynamic systems like biology, and that's why it's so powerful.” – Dr. John James. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 18 that indicate inflammation,” Aarntzen confirms,” I have hardly seen any scan that you do not see an abnormal signal.” Furthermore, advances in PET technology have facilitated faster acquisition times, reduced the need for lengthy scans, and have also enabled more granular tracking of the immune response over a given period. However, there are still key processes to consider. Aarntzen explained, “Time in the scanner is no longer limiting. What matters is matching tracer half-life to the biological process you want to study.” For instance, radio-labelled glucose-based tracers are useful for short-term snapshots of inflammation, while other, more specific, tracers are used to track immune cell populations over several days. Minimizing the risks and maximizing the benefits of immunotherapy Modern cancer treatment options range from monoclonal antibodies to chimeric antigen receptors (CAR), engineered from patient T cells to tackle several blood cancers. However, similarly to autoimmune disease, immune exuberance can occur, resulting in toxicity and adverse events. All told, 5–10% of patients will die from treatment complications. One approach to rapidly identify inf lammatory responses to immunotherapy is immune PET/CT imaging. In a clinical setting, this guides dosage adjustments, determines therapeutic response, and facilitates earlier recognition of adverse events, potentially mitigating the risk of unfavorable outcomes. With regard to CAR T specifically, optogenetic control may offer a strategy to minimize adverse events. Optogenetics uses a naturally occurring light switch from a family of proteins found in plants and some bacteria. The proteins are dormant in the dark but, when exposed to specific wavelengths of light, undergo a conformational change to become active. Researchers have fused this technology into the CAR T gene cassette. They hypothesize patients could be infused with an inactive therapy that is selectively activated at the target site by light exposure. Dr. John James, a biochemist at the University of Warwick, is investigating CAR T optogenetic control systems. “Light can provide exceptional temporal control over dynamic systems like biology, and that's why it's so powerful.” If successful, a switchable CAR T delivery system could allow clinicians to control drug doses with light, keeping adverse events in check while preserving therapeutic efficacy. However, depending on the wavelength used, visible light only penetrates approximately 1–3 mm into tissue. Therefore, optogenetic systems are only applicable to surface or near-surface conditions, such as melanoma and arthritis. Although if successful, clinicians could consider using optical windows or laparoscopic surgery to access deeper tumors. Revealing more in every frame with multiplexing Experts agree that limited multiplexing capabilities are a significant barrier to a greater understanding of immune interactions. Multiplexing enables the real-time tracking of multiple signals from distinct immune components. The more spotlights you have on the ice rink, the more skaters you will see. Similarly with imaging, increasing the number of detectors to highlight all the participants in an immune response would be like placing floodlights on the arena. However, nuclear imaging groups have faced challenges in simultaneous signal identification because all positrons emit at the same energy level, making this impossible. Research is underway on TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 19 two-isotope hybrid PET studies, which have reported success in simultaneous imaging of radiotracers. On the other hand, optical techniques benefit from a broader range of visible light, and 2P-IVM can distinguish several fluorescent probes within images. Though there are still limitations on the number of signals that can be identified compared to the number present. Germain’s group has sidestepped this multiplexing constraint by using a single fluorophore during in vivo imaging. Using a two-stage in vivo/in vitro approach called correlative microscopy, his group tracks immune system interactions in vivo before preserving the tissue for staining. “What you can do is watch the cells move, stop the system, and then ask in the preserved sample what proteins they expressed and what their transcriptome looked like in that exact spatial context.” The preserved section can be re-probed with different reagents to detect distinct target molecules, and signal positions can be mapped onto the 3D tissue structure. This approach has improved the multiplexing capabilities to track up to eight components, with further improvements in development. A completely in vivo approach is the goal of high-plex 3D imaging, though it may take some time to come to fruition. Germain confirms that “Real‑time transcriptomics in a living tissue is not yet here.” Spatial biology is the future Aarntzen looks forward to the integration of PET imaging with genomic and proteomic data, but says he “would not see it happening in the near future.” Prior to this integration, several hurdles must be addressed, including heterogeneity amongst human immune responses and the scarcity of complete datasets for various clinical conditions. Furthermore, reconciling genomic and proteomic profiles with downstream immune phenotypes will be challenging, as well as defining a “healthy” response. Nevertheless, it is a worthwhile goal. “True progress can only come from a better understanding of health and disease,” Aarntzen said. On the optical side, the spotlight is on the fusion of dynamic immune cell imaging with high‑plex spatial transcriptomics. Germain anticipates a future in which imaging modalities, assisted by high‑plex 3D technology, are merged with spatial transcriptomic data to train AI models on immune response prediction. He says that this would provide “a truly multiomic, spatiotemporal map of immune tissue”. “Multiomics”, he concluded, “is where this is all going”. When asked if computer bandwidth might be a problem, he shares a photograph of the expansive computing facility at NIH “You don't do this on a desktop anymore,” he laughed. MEET THE INTERVIEWEES: Dr. Ronald N. Germain received his MD and PhD from Harvard University in 1976. Since then he has investigated basic immunobiology, first on the faculty at Harvard, then at NIAID, NIH. He has contributed to understanding MHC class II molecules, antigen processing, and T cell recognition, more recently pioneering analysis of the immune system using dynamic and static in situ microscopy. Dr. Erik Aarntzen is a nuclear imaging specialist at UMC Groningen with a research interest in onco-immunology. His research uses multiple tracers targeting the immune system, like nanoparticles, antibodies/minibodies and small peptides, to better understand immune responses. His work with PET scanning technology, in conjunction with the development of new tracers, is focused on translating laboratory research into meaningful improvements in the treatment of cancer and immune mediated inflammatory diseases in patients. Dr. John James is an associate professor at the University of Warwick. James’ research is focused on how T cells, an essential component of our immune system, interpret and respond to signals from potentially infected cells within the body. His research priorities include uncovering how the dynamic signaling network inside T cells is capable of computing the most appropriate response. To achieve this, the James lab makes use of chemical and optogenetic (light) control techniques to probe this intracellular network in a quantitative manner without disrupting it. A longer-term goal of this research is to develop therapeutic control strategies to fine-tune T-cell responses in patients. Single-cell analysis techniques Single-cell analysis encompasses a suite of powerful techniques used by researchers to examine multiple aspects of individual cells with unprecedented resolution. Single-cell genomics Uses single-cell whole genome sequencing to identify genetic differences and heterogeneity across single cells.4 Single-cell transcriptomics Measures gene expression by analyzing RNA in individual cells, typically using single-cell RNA sequencing (scRNA-seq).5 Single-cell proteomics Analyzes cellular proteins through techniques like mass spectrometry or single-cell antibody sequencing (scAb-seq), which profiles proteins at single-cell resolution, to understand cell function.5 Single-cell epigenomics Uses a single-cell chromatin accessibility assay (scATAC-seq) to measure open chromatin regions and DNA methylation (scDNA-Metseq) to detect methylated DNA sites, allowing analysis of epigenetic DNA modifications in individual cells that regulate gene expression.5 Single-cell metabolomics Measures metabolites in individual cells to assess their metabolic state and function using mass spectrometry.5 Single-cell multiomics Combines multiple omics layers (genomics, transcriptomics, proteomics) from the same cells for comprehensive analysis.6 Single-cell spatial biology Maps molecular features (RNA, proteins, or metabolites) in intact tissues to preserve spatial context and cell–cell interactions.7 Single-cell imaging Uses microscopy-based techniques, including mass spectrometry imaging (MSI), to visualize cells, structures, and molecular markers at high resolution, linking cellular phenotype with molecular information.7 Click here to view the full infographic 21 INNOVATIONS IN CELL SCIENCE Growing Into Their Potential: The Innovations Driving Organoids Forward Laura Elizabeth Lansdowne In 2009, Dutch molecular geneticist Hans Clevers achieved something extraordinary. By isolating a single stem cell from a mouse intestine and embedding it in a nutrient-rich bio-gel, he, along with his postdoc Toshiro Sato, coaxed the cell into organizing itself into a mini organ. Within days, the Petri dish contained a tiny three-dimensional structure—an intestinal organoid— that survived for several months. Researchers could now grow small, lab-made structures from stem cells that mimicked the architecture and function of real organs. Since then, organoids have transformed biomedical research, helping to study disease, test drugs, and explore human development. But as these in vitro models have become more sophisticated and more widely used, their limitations have become increasingly evident. Now efforts are shifting toward overcoming these challenges. In this article, we explore some key innovations that are helping these mini organ models achieve enormous potential. Credit: iStock/Meletios Verras TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 22 What is an organoid? An organoid is a small, three-dimensional tissue model grown in the laboratory from stem cells. These cells self-organize into structures that resemble features of real organs, allowing organoids to replicate aspects of organ structure and function. Breaking organoid size barriers with vasculature Without a vascular network to deliver oxygen and nutrients, organoids often struggle to grow beyond small clusters, preventing them from fully reproducing the complexity of living organs. In the human body, a cell is typically no more than 50–100 μm away from a capillary; but in the case of organoids, those cells located at the very center can suffocate. “Traditional organoids are fundamentally limited by their lack of vasculature,” explained Dr. Joseph C. Wu, director of the Stanford Cardiovascular Institute. “Without blood vessels, oxygen and nutrients cannot penetrate the core, leading to necrosis and restricting organoid size and fidelity.” Dr. Wu’s lab has developed a method to overcome this by using spatially micropatterned human pluripotent stem cells (hPSCs). These cells self-organize into structures that mimic the earliest stages of human heart development. Their approach relied on them being able to identify a specific “cocktail” of growth factors and small molecules that could support the co-differentiation of various cardiac and neuronal cells alongside a branched, lumenized vascular network from a single starting population of hPSCs. Using four fluorescent reporter lines, Wu’s team could monitor cell fate decisions in real time as the organoid developed. Single-cell transcriptomics and high-resolution 3D imaging revealed that the cellular composition of the vascularized organoids closely mirrored a human heart at about 6.5 weeks post-conception. “Incorporating vasculature dramatically improves physiological relevance,” noted Wu. “It enables the nutrient and signaling gradients that shape organ patterning, and allows us to study how the vasculature communicates with and instructs neighboring organspecific cells.” What’s exciting is that this approach is not organspecific. The researchers discovered that it worked for liver progenitors just as effectively as for the heart. “This suggests a conserved developmental program underlies vascularization across organ systems,” Wu said, “an insight that would have been almost impossible to obtain from animal models or early human embryos.” Steps towards scaling the use of vascularized organoids Wu is confident that vascularized organoids could transform preclinical testing, but he notes that scaling them remains challenging. Wu elaborates: “The micropatterning approach we use is powerful but requires careful optimization of geometry, cell density, and timing. Scaling this to a high-throughput format, such as 96-well or 384-well plates, while maintaining consistency in vascular network formation is an active engineering challenge.” Wu adds that the vessels are not yet perfused with flow—an important factor for vessel maturation: “its absence means the vessels lack some features of mature vasculature... To bring vascularized organoids to scale, we must integrate fluid flow, supporting cells, and robust quality standards—only then can these systems truly mimic real human tissue and accelerate drug discovery." Looking beyond a single organ: Enter the assembloid As researchers strive to capture greater complexity, a new term has emerged—the “assembloid”. While an organoid is a 3D structure that recapitulates TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 23 a single organ, an assembloid is a more complex system that combines multiple organoids, spheroids, or cultured cells, allowing researchers to model more complex interactions. The assembloid’s origin The term “assembloid” was coined in 2017 by Stanford’s Sergiu Pasca, who had recognized that so many biological processes rely on integrated circuits of communication and interaction—the brain is a great example—and that a single spheroid or organoid could not capture this adequately. At the time, his lab was interested in understanding how connections between cells go awry in the brains of people with psychiatric conditions. Pasca’s team began “fusing” discrete units together—such as a cortical organoid (the brain’s “thinking” center) and a subcortical organoid (the “relay” station). This allowed them to study conditions like autism or schizophrenia, known as “circuitopathies”. In these disorders, individual parts of the brain may seem to be structurally sound, but the signaling between them has suffered a systemic breakdown. Beyond the brain: Realizing the broader potential of assembloids “Traditional organoids model a compartment. Assembloids model relationships,” stated Associate Prof. Shafagh Waters, a Scientia Associate Professor at the University of New South Wales and co-lead of the Non-Animal Technologies Network. Waters highlights how assembloids can model the boundaries between tissues—the “zones” where different cell types meet and interact. “By integrating epithelial, mesenchymal, and sometimes vascular or neural elements, we begin to recreate tissue interfaces—the epithelial–stromal boundary, immune– epithelial contact zones, or vascular–parenchymal exchange surfaces,” she noted. This approach is particularly useful for studying complex chronic diseases, where interactions between cells drive pathology. For example, in airway disease, epithelial dysfunction is shaped by its crosstalk with immune cells, fibroblasts, and the tissue environment. While many assembloid systems remain experimental, Waters’ own work focuses on patient-derived airway models that can help guide treatment decisions. She develops patient-derived airway models to test treatments and develop personalized therapies for cystic fibrosis and other airway conditions. In a study published in Thorax, her team grew “mini-me” organoids from children’s airway cells to predict how each patient would respond to drugs targeting the faulty protein (CFTR) that causes cystic fibrosis. Wired for growth: Engineering cyborg organoids One of the biggest barriers facing organoid research is maturation. As Wu flags, “most organoids today resemble fetal tissue rather than fully developed adult organs, limiting their usefulness for studying conditions such as heart failure, cirrhosis, or neurodegenerative disease.” A new approach aims to tackle this challenge by integrating soft electronics directly into developing tissues. The method involves embedding flexible mesh nanoelectronics during the formation of the organoid so that the device becomes part of the tissue as it grows and differentiates—essentially creating a “cyborg” model. “Organoids have become increasingly sophisticated models for human disease modeling and drug discovery, yet our ability to measure and control their real-time functional dynamics during development and maturation has lagged. As a result, a major limitation for the current organoids is that- they lack functional maturation compared to their in vivo counterparts,” said Dr. Jia Liu, assistant professor in the School of Engineering and Applied Sciences at Harvard University. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 24 Liu’s approach turns traditional organoids from largely descriptive models into continuously trackable and controllable biological systems, allowing researchers to quantify and guide their functional maturation. In pancreatic islet organoids, this approach enables researchers to track electrical activity from individual alpha and beta cells and observe how glucose responsiveness changes over time. Electrical stimulation can also be used to tune cellular activity, giving researchers a way to guide functional development in real time. Liu’s team is already applying this approach to other organ systems. “We have developed cyborg cardiac organoids and cyborg brain organoids to track electrical activity chronologically, during in vitro maturation. “We believe cyborg organoids represent a broadly applicable platform for advancing organoid research and regenerative medicine,” Liu explained. Looking ahead, Liu thinks that cyborg organoid technology could expand beyond electrical sensing to incorporate a broader suite of bioelectronic sensors, capable of tracking metabolites, oxygen levels, and other physiological signals. This would allow researchers to build a more comprehensive functional profile of stem cell–derived organoids and even primary human tissues. In addition, by implementing AI-enabled closed-loop recording and control, the system is expected to provide a precise modulation of the functional development and maturation of the organoids. Beyond the lab By incorporating vasculature, fusing multiple organoids, and embedding bioelectronic sensors, these miniature systems (Figure 1) are evolving from static models into dynamic, trackable, and clinically relevant platforms. Alongside these advances, regulatory momentum is creating new opportunities: the US Food and Drug Administration Modernization Act 2.0/3.0 and similar initiatives worldwide now support the use of non-animal models for preclinical testing, provided platforms are predictive and reproducible. “Trust is built through evidence and transparency. Regulatory approval will follow when organoid platforms are demonstrated to be reliable, reproducible, and predictive across independent centers... with the right investment, for many applications, human-relevant models can become not just alternatives—but the preferred standard,” concluded Waters. MEET THE INTERVIEWEES: Associate Professor Shafagh Waters is a translational stem cell biologist and leader in organoid-based precision medicine at UNSW Sydney. She co-leads the NSW Non-Animal Technologies Network, a cross-sector initiative coordinating Australia’s transition toward validated human-relevant research platforms. Her research integrates patient-derived airway models, functional electrophysiology, and clinical translation to advance personalized therapies for cystic fibrosis and develop regenerative and geneedited airway strategies for sinus disease. She has authored more than 60 publications and leads national collaborations across academia, clinical centers, industry, and government. Dr. Joseph Wu is director of the Stanford Cardiovascular Institute and the Simon H. Stertzer, MD, Professor of Medicine and Radiology at Stanford University. He earned his MD from Yale University and his PhD in molecular and medical pharmacology from the University of California, Los Angeles. He currently serves as president of the Association of University Cardiologists (2026). Wu’s research integrates genomics, stem cells and organoids, artificial intelligence, and drug discovery to understand disease mechanisms, advance precision medicine, and accelerate therapeutic development through new alternative methodologies and the “clinical trial in a dish” concept. Dr. Jia Liu received his PhD in Chemistry from Harvard University in 2014 and completed postdoctoral training at Stanford University in 2018. He joined the Harvard School of Engineering and Applied Sciences as an assistant professor in 2019. His laboratory develops tissue-integrated bioelectronic systems that combine flexible, soft electronics with living tissues, alongside multimodal in situ characterization and AI-driven analysis to decode and control biological processes. His research spans tissue-like bioelectronics, cyborg organoids, and AI-enabled multimodal approaches to study neural dynamics, organ function, and disease mechanisms. 25 INNOVATIONS IN CELL SCIENCE Single-Cell Technologies Are Redefining Our Understanding of Human Development Kaja Ritzau-Reid, PhD Early pregnancy has long been described as a “black box” in human development. Key biological events that happen at this time determine whether a pregnancy will be successful or not. Our understanding of this extraordinary developmental journey has remained limited due to the difficulty of accessing the developing embryo (both practically and ethically) and the limited tools available for studying this process. As a result, common pregnancy complications like recurrent miscarriages and pre-eclampsia are still poorly understood, with limited treatment options. A new revolution in single-cell technologies is changing this. Up until recently, researchers have characterized cells by their structure, function and location, but have been kept in the dark about their individual molecular profiles. This means that essential details, like the intricate set of instructions sent between cells, are missed. Single-cell technologies now allow researchers to isolate and profile each cell individually, capturing its unique molecular signature. This has opened up a whole new toolkit for understanding complex biological systems – such as piecing together the biological events of early pregnancy. Dr. Roser Vento-Tormo, a group leader at the Sanger Wellcome Institute, heads a research team investigating women’s reproductive health and the developmental trajectory of early pregnancy using single-cell technologies. “The maternal–fetal interface embodies a delicate equilibrium between growth, invasion and immune tolerance – processes that are Credit: iStock/Tempura TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 26 fascinating at a fundamental biological level but also central to many pregnancy complications,” explained Vento-Tormo. ‘‘The biology itself adds complexity and drawing robust conclusions requires larger sample sizes than in more static tissues, as well as experimental and computational technologies that enable us to disentangle this complexity.” The single-cell analysis toolkit The fundamental shift in sequencing technology was kick-started in 2009, when a pioneering study performed the first single-cell genome-wide mRNA sequencing on a mouse blastomere. This was largely enabled by the emergence of next-generation sequencing. The field has rapidly developed ever since, and today, single-cell RNA sequencing (scRNAseq) is a go-to tool for many researchers. In a landmark study published in Nature in 2018, Vento- Tormo and her team used scRNAseq to investigate the early stage of pregnancy, where placental cells detach and migrate into the maternal uterus, forming branch-like structures that attach to the uterus. “These placental cells remodel the maternal arteries to ensure adequate nutrient delivery to the embryo,” explained Vento-Tormo. “For decades, we lacked a human-specific, cellular-resolution view of the maternal uterine–fetal placental interface to understand what ‘normal’ implantation and placentation looks like. Our 2018 single-cell atlas of the decidua and placenta provided that baseline and a window into how maternal and fetal cells interact.” These high-resolution maps of gene expression are now becoming a blueprint for the multiomics era. Where scRNAseq allowed researchers to define a cells identity and state, essentially creating a “parts-list” of a tissue, multiomics opens the possibility to ask the question Figure 1: Single-cell sequencing can be used to measure the genome (scDNA-seq), the DNA-methylome or the transcriptome (scRNA-seq) of each cell from a population of cells. Credit: Technology Networks TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 27 “why is the cell in that particular state?” and “how is the cell being regulated?”. By simultaneously profiling the genome, epigenome or proteome alongside the transcriptome, researchers can examine different cell states in parallel, capturing a much more dynamic and comprehensive picture of cell behavior (Figure 1). For example, examining epigenetic regulation of cells alongside gene expression can help explain why certain genes are turned on or off by examining epigenetic factors like chromatin and DNA methylation. Spatial transcriptomics is also changing the landscape of single-cell technologies. Single-cell methods require cells to be dissociated first, losing information about where they are in relation to other cells. Huge advances in this field mean that it is now possible to keep this positional information by attaching unique spatial barcodes to the RNA, allowing researchers to map where different genes are being expressed within the tissue after sequencing. In a recent paper published in Nature, Vento-Tormo and her team built on their previous research and used multiomics to define the full developmental trajectory of the placental cells migrating to and interacting with the uterus. They also used spatial transcriptomics to provide spatial context, mapping gene expression to its specific location in the tissue. “To understand what goes wrong in disease, we first need a clear reference for what ‘healthy’ looks like,” explained Vento-Tormo. “In our 2023 study, we were able to describe the microenvironments affecting cellular behavior across the full maternal-fetal interface, including the layer of smooth muscle under the endometrium, and how the immune cells have a key role in modulating placental invasion.” Creating a human cell atlas Vento-Tormo is also a key contributor to the Human Cell Atlas (HCA) – a global consortium of scientists with a mission to map every cell type in the human body. The rapid rise of single-cell and multiomics technologies has paved the way for this ambitious project. The HCA was co-founded in 2016 by Sarah Teichmann, professor of stem cell medicine at the University of Cambridge and former head of cellular genetics at the Sanger Institute. “This ‘reference map’ allows us to better understand how healthy cells function and communicate, and how they work synergistically to form tissues and organs,” explained Teichmann. “As we add disease datasets into the atlas, we begin to better understand pathology and support development of new diagnostics and treatments.” Formed of over 3,600 scientists globally, the HCA community has already made significant and impactful discoveries. Teichman’s research team recently published a paper in Nature, in collaboration with others from the HCA Heart BioNetwork, that sought to map the cells of the cardiac conduction system. The team “Working at the crossroads of genomics, computation and human biology allows us to explore these processes in unprecedented detail, and to translate those insights into meaningful improvements in maternal and reproductive health.” – Dr. Roser Vento-Tormo. TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 28 made the unexpected finding that human pacemaker cells (which set the pace of our heartbeats) express a particular receptor that is the target of a gut hormone, and its synthetic analogues, which are found in diabetes drugs. As Teichmann explained, this supports a direct mechanism for increased heart rate when taking these medications, which was observed clinically but not explained at a molecular mechanistic level before. This work shows how the HCA has already been instrumental in describing how different organs talk to each other, and how it can help to interpret the effects of drugs. The HCA is currently integrating this vast collection of data towards a first draft atlas of cells in the human body. Big data As the cell atlas moves into three dimensions by incorporating multiple layers of data from the different omics modalities, the role of computational tools can’t be underestimated. The shift from single-cell to multiomics technologies has created a data deluge—vast datasets that span a huge number of cells, with billions of potential data points. Advanced computational tools that can extract biologically meaningful data from this are essential. Machine learning (ML) tools can integrate high-dimensional data, learning the complex relationships between the different modalities. ML is also the primary tool for pattern recognition, like identifying cellular “neighborhoods” or microenvironments within a tissue or detecting anomalies in the data. The role of computational biologists and bioinformaticians in the biology lab has never been more critical. To delve deeper into the developmental journey of placental cells, Vento-Tormo and her team developed a computational method to reconstruct the differentiation of these cells by combining the cells’ gene expression similarity and proximity in space. Marie Moullet is a PhD student working in Vento-Tormo’s group, and focuses on developing deep learning models for single-cell omics data. “This method allowed us to investigate branches and decision points during the differentiation process of the placenta cells,” Moullet said. “In general, machine learning helps us to learn structure from high-dimensional data and to standardize analyses across cohort, platform and laboratories.” “Computation is at the heart of everything we do, from preprocessing and integration of multi-modal data to inference of cell-cell communication and spatial patterning,” explained Vento-Tormo. Machine learning tools are also being increasingly developed for predictive analysis and are already used to help reconstruct dynamic processes, such as developmental pathways, or predict cell-cell interactions. As the field continues to develop, ML tools will shift towards a more predictive and simulative role, with deep learning models predicting clinical outcomes, such as drug efficacy and optimal dosage. This could have a significant clinical impact, shifting from reactive treatments (based on symptoms having already appeared) to a more predictive and preventive healthcare model, as ML models utilize cell data to forecast the risk of disease. “We are in an exciting time in this domain due to the opportunities from deep learning, generative AI and AI agents,” said Teichmann. Looking ahead to the future of single-cell technologies Looking towards the next decade, both Teichmann and Vento-Tormo predict that technologies will become cheaper, higher-throughput and more readily available. “I expect multiomics at cellular resolution on clinically sized cohorts will become routine,” said Vento-Tormo. Teichmann adds that access to large clinical sample sets will further accelerate understanding of disease pathways and treatment responses. Advances in computational approaches will also increasingly be used to optimize experimental designs, potentially refining in vitro model systems and making them more physiologically accurate. As more computational tools and artificial intelligence/ ML models become increasingly available, one of the challenges researchers will face is navigating this new, TECHNOLOGYNETWORKS.COM CANCER RESEARCH 29 vast landscape of advanced technology. The challenge of choosing the “right” tool to analyze the data will shift the bottleneck of discovery from data generation to data interpretation. Some groups are already performing large-scale benchmarking studies to help create more standardization in the field. Clinically, the application of single-cell multiomics is still in its early stages, but discoveries from the HCA community have already provided new insights into cancer, COVID-19, lung diseases such as cystic fibrosis, bowel disease and heart disease, among others. As cellular maps move into three dimensions, scientists can start addressing questions that have so far remained a mystery—like unravelling the biological mysteries of early pregnancy to shed light on common and devastating complications like pre-eclampsia and miscarriage. “Deeper understanding of complex disease biology can translate into real improvements for patients” said Vento-Tormo. “Working at the crossroads of genomics, computation and human biology allows us to explore these processes in unprecedented detail, and to translate those insights into meaningful improvements in maternal and reproductive health.” Undoubtedly, single-cell technologies are not just a new technique, but an entire shift in how we can ask and answer questions about human biology. MEET THE INTERVIEWEES: Dr. Roser Vento-Tormo is a group leader at the Sanger Wellcome Institute. Her research investigates the female reproductive system and women’s health, with particular focus on the maternal-fetal interface during pregnancy. Vento-Tormo has a background in immunology and completed her postdoctoral work with Professor Sarah Teichmann. Marie Moullet is completing her PhD in Dr Roser Vento-Tormo’s group, and her research looks at machine learning methods to characterize in vivo and in vitro cell states in women’s health and disease. Dr. Sarah Teichmann, is a professor of stem cell medicine at the University of Cambridge, the co-founder of the Human Cell Atlas and the former head of cellular genetics at the Sanger Institute. Her research utilizes genomics and artificial intelligence methods to investigate gene regulation and protein interactions in the immune system. 30 INNOVATIONS IN CELL SCIENCE The Future Is Bright: Advances and Developments in Flow Cytometry Kate Harrison, PhD For almost six decades, flow cytometry (FC) has been a cornerstone of clinical and laboratory research, with applications in a wide range of fields, including immunephenotyping, diagnostics, cell counting, and cell cycle analysis. FC is a powerful tool for multi-parametric cell characterization, even in complex, mixed samples, due to its ability to assess cell properties by light scatter and fluorescence. Cells suspended in a saline solution flow past laser light sources in a single-file manner, known as hydrodynamic focusing. When the laser hits the cells, any visible light scattered forwards (forward scatter—FSC) gives information about the cell diameter, while light scattered sideways (side scatter—SSC) provides information about the granularity of the cell. Further phenotypical characterization of the cell can be achieved with fluorescent dyes or fluorophoreconjugated antibodies. Flow cytometers can also be used for cell sorting, i.e., the physical separation of cells into distinct populations, known as fluorescenceactivated cell sorting (FACS). For many years, FC advances were defined by increasing the number of parameters that could be simultaneously measured, though the fundamental processes of fluorescence-based, polychromatic FC remained largely unchanged. While early flow cytometers could only analyze one or two parameters, modern fluorescence-based instruments can simultaneously measure up to 30 parameters. However, this meant that FC reached a technological ceiling—since each color channel requires its own detector, the number of detectors in a single machine Credit: iStock/anamejia18 TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 31 became the limiting factor. Other challenges in traditional FC techniques include relatively high limits of detection, subjective interpretation of results, unstable fluorophores, and the need for large numbers of controls. A range of recent developments are tackling these challenges and helping to advance the field of FC significantly, including spectral FC, nanoparticle cytometry, and droplet cytometry. These advances are improving the accuracy and efficiency of FC and opening up new research and clinical applications. Looking at the full spectral picture: Spectral flow cytometry In traditional, polychromatic FC, fluorescent light signals are produced when fluorophore-labelled probes are excited by lasers of a corresponding wavelength range. The peak emission spectrum from each fluorophore is isolated by a series of dichroic mirrors and bandpass filters and captured by a single detector. As a consequence, the number of detectors available becomes the limiting factor. In addition, as the number of parameters increases, the more individual fluorophores must be used and the closer their emission peaks become, making experiments extremely complex. Spectral cytometry is a rapidly growing technology that captures the full emission spectra of each fluorophore using an array of detectors, overcoming these limitations. “It became apparent that there was a physical limitation of about 28–30 colors for polychromatic FC,” says J. Paul Robinson, distinguished professor of cytometry at Purdue University. “However, this is not the case with spectral cytometry. We don’t talk about instruments being restricted by colors, because we don’t yet know the upper limits of how many different colors a spectral cytometer can collect.” Robinson was involved in the development of spectral cytometry technology in the early 2000s, and his lab filed a patent on it in 2004. This patent was then the basis of the first commercially available spectral flow cytometer. Spectral cytometry captures the emission spectra for each fluorophore across multiple detectors, allowing it to differentiate between fluorochromes with overlapping emission peaks. In order to quantify the signal of each fluorophore, the spectra must be separated, using a spectral unmixing algorithm that fits and identifies individual fluorophores by comparison with single-stained controls. One of the benefits of spectral cytometry is that it is less reliant on subjective, manual compensation. “Compensation is a very personal thing, dependent on experience. The math of spectral unmixing is essentially the same as the math of compensation,” explains Robinson. “It’s just performed in an automated manner, which gives it much better consistency and greater accuracy.” The applications of spectral cytometry have already shown enormous potential. Spectral FC is able to calculate cellular autofluorescence and treat it as another channel. Thus, noise in complex samples is reduced, and identification of rare cell subsets in highly autofluorescent samples such as blood is improved. Its role in advancing cancer research has also become apparent, having been used to study subtle changes in immune signatures and characterize complex tumor infiltrates in response to novel treatments. Robinson believes that spectral cytometry has even more to offer. “Spectral FC has already achieved around 50 colors in one experiment, and I suspect it will achieve 100 eventually”, he says. “As the numbers of potential parameters increases, we could be able to look at enough biomarkers to predict when a patient’s physiological environment has changed, even before symptoms appear.” Life in nanoscale: Nanoparticle flow cytometry FC is instrumental in characterizing cells and continues to enable significant advances across clinical and life sciences. However, the sensitivity limitations of most conventional flow cytometers and assays mean that observations are limited to biomarkers present either intracellularly, or on the surface of cells. Secreted, extracellular biomarkers such as proteins, metabolites, and molecules contained in extracellular vesicles (EVs) TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 32 generally fall below the lower limits of light scatter detection of conventional flow cytometers, with far dimmer signals. EVs are nano-sized, cargo-carrying vesicles secreted from cells into the extracellular environment. They are becoming increasingly recognized as both important biomarkers in a range of physiological functions and processes such as aging and the immune response, and for their potential clinical applications. Traditional methods of EV analysis such as polymerase chain reaction (PCR) or mass spectrometry can measure the total amount of EV components in a sample, but cannot give a comprehensive picture of the distribution of those components. “These methods are limited in their ability to identify informative biomarkers or bioactive cargo, two key translational goals of EV research,” explains Prof. John Nolan, of the Scintillon Research Institute. Nolan’s lab develops new tools for analysis of EVs, their formation and their interactions with cells—including custom-built nanoparticle flow cytometers along with optimized and validated assays. “A new generation of more sensitive [FC] instruments and optimized assays with improved specificity and reproducibility make it possible to measure even the smallest EVs and their cargo, down to < 40 nm and 10 or fewer molecules,” he says. However, there are still challenges; even for more sensitive machines, detection efficiencies can vary and create inconsistencies in reporting. To combat this, Nolan is part of a working group developing a framework for standardizing EV FC experiment reporting, known as MIFlowCyt-EV. “By designing and optimizing assays that include the proper positive and negative controls, calibrating instruments and reporting results in absolute units of number and brightness, we can address these challenges, enabling results to be compared across instruments and between labs,” explains Nolan. “Standardization is essential for the effective translation of findings from basic research labs into new clinical biomarkers and therapeutics.” Teaching an old cytometer new tricks: Double droplet cytometry Although advances in FC technology continue to expand the field, the importance of standard flow cytometers and FACS machines for research advances shouldn’t be overlooked. Droplet microfluidics allows the highthroughput analysis of millions of individual cells or proteins, in volumes as small as 10-15 liters. While these droplets and their cargo can be phenotypically sorted, traditional single-layer water-in-oil droplets require custom, technically demanding microfluidics devices, which tend to be slower than standard FACS machines, with limited color channels. Dr. Polly Fordyce, associate professor of bioengineering and genetics at Stanford University, has been developing double emulsion droplet technology. The contents of double emulsion droplets can be phenotyped and sorted using a standard flow cytometer, allowing far higher throughput and multiparametric analysis. “A double emulsion droplet is an aqueous droplet, inside an oil droplet that is surrounded again by an aqueous buffer, similar to a cell membrane,” explains Fordyce. “To a FACS machine, these droplets just look like large cells, so they can be analyzed using a normal flow cytometer.” However, there are still challenges. “We’ve spent a lot of time figuring out how to stabilize the oil– water interfaces, how to keep the droplets in suspension and how to calibrate the FACS machine,” says Fordyce. The Fordyce lab has been using machine learning to characterize the effect of different variables on droplets, to create a model that can predict the droplets produced by given flow rates and surfactants, and then design devices to create droplets with specific parameters. “These are all the small things that will push the science forward and make the technology more accessible,” she says. The double emulsion droplets provide a level of protection from the shear forces of the FC fluidics, allowing examination of more temporal or spatial phenotypes. “The droplet creates a shell around a cell, which enables you to examine secreted proteins that would usually diffuse away. You can encapsulate cell pairs to examine cell–cell interactions or encapsulate TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE 33 enzymes with their substrates to examine enzymatic activity,” explains Fordyce. For Fordyce’s lab, the aim is to use double droplet technology and FC to understand how the sequence of a protein encodes its functions. “Our main goal,” she says, “is pioneering new ways to make thousands to millions of protein variants, and then quantify what they do. For example, understanding how the sequences of enzymes encode the catalytic efficiency, their specificity and the different parts of their enzymatic cycle.” By encapsulating these protein variants in double emulsion droplets and using fluorescent tags, FC and FACS can then be used as a high-throughput methods to sort and identify the active, functional variants. Is the future fluorescent? Even considering all the recent developments in the field, the advancement of FC shows no signs of slowing down. Robinson believes the next steps for FC may be quantum. “Technology advances to a true generation of quantum cytometers is likely to emerge within 2–3 years”. Quantum measurement could allow the detection of single fluorophores, in turn enabling the identification of extremely rare biomarkers. This, combined with single photon detectors, could see the transition of FC from a somewhat subjective, qualitative technology to a fully quantitative technology. Nolan, too, believes the measurement of single molecules to be a powerful tool for the future of research. “New needs and applications tend to drive the development of new techniques, rather than the other way around.” He says “In EV research, the capabilities of FC will help researchers test hypotheses about how these systems work, and help us to understand, predict, and control cells to improve health.” However, as rapidly as FC advances, we cannot forget about the basic principles that brought us to this point. Novel technologies cannot flourish without labs and institutions investing in the foundations of high-quality FC, and the highly-skilled operators that perform it. “Shared, core facilities with trained operators have been key in turning FC into the powerful tool that it is today,” says Fordyce, “and I hope to see more institutions understanding the value of these central facilities in the future.” MEET THE INTERVIEWEES: Dr. Polly Fordyce is an Associate Professor of Bioengineering and Genetics and Institute Scholar of ChEM-H at Stanford, where her lab develops and applies new microfluidic platforms for quantitative and high-throughput biophysics, biochemistry and single-cell biology. She graduated from the University of Colorado at Boulder with undergraduate degrees in physics and biology before moving to Stanford University, where she earned a PhD in physics for work with Professor Steve Block developing instrumentation and assays for single-molecule studies of kinesin motor proteins. Prof. John Nolan received BS degrees in biology and chemistry from the University of Illinois and a PhD in biochemistry from Penn State University. He did post-doctoral research at Los Alamos National Laboratory and was the director of the National Flow Cytometry Resource there. He is an elected Fellow of the American Institute for Medical and Biological Engineering, past-president of the International Society for Advancement of Cytometry and founder of Cellarcus Biosciences, which provides products and services for extracellular vesicle research. Dr. J. Paul Robinson received his early education at the University of NSW, Sydney, Australia where he received BSc (Hons), MSc and PhD degrees. He was a postdoctoral fellow in the University of Michigan Medical School. Dr. Robinson is currently the Distinguished Professor of Cytometry and professor of biomedical engineering in the Weldon School of Biomedical Engineering. 34 TECHNOLOGYNETWORKS.COM INNOVATIONS IN CELL SCIENCE CONTRIBUTORS Alexandra Boussommier- Calleja, PhD Alexandra Boussommier-Calleja is an entrepreneur trained in biomedical engineering with extensive experience in biomechanical research and skilled in 2D and 3D cell culture, microfluidic models, in vitro and in vivo assay development. After spending years learning to manipulate tissues and cells to mimic life in the lab, she became fascinated with the ability to manipulate cells to make life happen directly in the lab with in vitro fertilization (IVF). Cliff Dominy, PhD Cliff Dominy is a freelance science writer and medical journalist based in rural Ontario, Canada. He earned his PhD in Molecular and Cell Biology and completed postdoctoral fellowships at the University of Guelph and McMaster University in Canada. His writing covers all of the biomedical sciences, with a particular interest in how digital technologies are enhancing our understanding of chronic disease. Debomita Chakraborty, PhD Dr. Debomita Chakraborty works as researcher in the field of translational medicine and novel therapies in immuno-oncology, inflammatory, and fibrotic diseases in different University hospitals in Germany. Her work focuses on identifying aberrant regulatory mechanisms, cell‑signalling pathways, and molecular targets in disease, enabling her to develop and use patient‑derived samples, animal models, and advanced culture systems such as tumoroids and organoids. Héctor Zamora Carreras, PhD Héctor earned undergraduate degrees in biochemistry and chemistry before completing a PhD in biochemistry at the Blas Cabrera Institute for Physical Chemistry (IQF, Madrid). After research appointments at the Karlsruhe Institute of Technology, the Complutense University of Madrid, and the Center for Biological Research (CIB, Madrid), where he worked on optical tweezers, cell mechanics, and nanoelectromechanical devices for intracellular measurements, he is currently a lecturer at the Valencia International University (VIU). Kaja Ritzau-Reid, PhD Kaja attained a BSc in genetics (2014), MRes in neurotechnology (2017) and a PhD in bioengineering (2022) at Imperial College London. During her PhD she worked on bioengineering organoids and spent part of her time working in an organoid group in Vienna, Austria. Here, she developed a microfibrous scaffold platform to guide brain organoid growth, which was patented for commercialization. She is currently working as a freelance science writer. Kate Harrison, PhD Kate has a BSc in Microbiology from the University of Manchester and a PhD in virology from the University of Edinburgh. Following this, she completed a post-doc at the Jenner Institute, University of Oxford, developing vaccines for neglected tropical diseases including dengue and zika viruses. Realising that her passion lay in science communication and public engagement rather than in the lab, Kate made the leap into science writing in 2022. Laura Elizabeth Lansdowne As managing editor, Laura works with the editorial director to create, inform and maintain the overall editorial strategy for Technology Networks. After obtaining a first-class honors degree in biology, Laura worked as a quality assurance technologist before joining the Wellcome Sanger Institute and undertaking an additional qualification in clinical chemistry. In 2015, she left to pursue pharmaceutical and editorial-based roles, before joining Technology Networks in 2017.