Advancing in vitro models of immune tissues is a major focus toward answering lingering questions about immunity and developing physiologically relevant drug testing platforms. The composition and structure of the extracellular matrix is an essential consideration in these models as these physical cues can modulate immune cell behavior and ability to mount an immune response. In immune tissues, stromal cells deposit and maintain the extracellular matrix, yet they are often overlooked in existing engineered models. Within, three major strategies for tuning the extracellular matrix in vitro for immune tissue engineering are explored: tunable hydrogel platforms, decellularized tissues, and in-situ deposition of matrix by stromal cells. Overall, the challenge of recapitulating in vivo microenvironments is vast, but the inclusion of stromal cells in the study of extracellular matrix-immune cell dynamics is a vital step forward.
Fluid flow is ubiquitous throughout the immune system. Immune cells exhibit sensitivity to fluid shear stress, altering their activation and behavior in response to the presence, magnitude, and timing of fluid flow. Therefore, understanding how flow regulates the immune response is crucial for understanding the immune system's contribution to homeostasis, disease progression, and therapeutic interventions. Recent developments have indicated Piezo1, a mechanosensitive ion channel protein, is a crucial mechanism for cell sensing and response to shear, but our understanding of how shear impacts immune function is far from complete. Open questions including: how cells respond to acute vs chronic shear stress exposure, how shear influences cell-cell communication, and direct molecular mechanisms require further investigation. In vitro modeling provides one promising approach to fill this gap, and many systems have been recently developed that incorporate flow to investigate the immune response in model organs and disease states. This review discusses recent advances in our understanding of fluid shear stress's impact on immune cell behavior, and we provide a landscape of in vitro models integrating flow and immune cells. To guide future development, we evaluate five critical design considerations: flow directionality, channel cross-section, culture substrate, recirculation strategies, and TEER compatibility.
The translational gap in oncology, particularly in immunotherapy, is largely driven by the inability of traditional preclinical models to accurately represent the dynamic physical and biological aspects of human tumor microenvironment (TME). This review presents the evolution of microphysiological systems, so-called organ-on-a-chip systems from 2021 to 2025 as predictive and analytical tools for immunotherapy efficacy in TME. We highlight advancements in engineered vasculature models that move beyond angiogenesis assays to screen vascular normalization strategies, such as anti-vascular endothelial growth factor and tyrosine kinase inhibitors (TKIs) and dissect immune trafficking mechanisms. Furthermore, we discuss the integration of patient-derived organoids (PDOs) into TME-on-a-chip platform, creating autologous immune organoids that retain donor heterogeneity to predict clinical responses to checkpoint inhibitors and combination therapies. Additional emphasis is placed on the critical shift from static end-point analysis to real-time, noninvasive readouts via integrated biosensors and artificial intelligence-driven high-content analysis, which enable the longitudinal noninvasive tracking of metabolic and physical TME dynamics. Finally, we address the regulatory impact of the 2025 Food and Drug Administration guidance supporting the adoption of non-animal alternatives. In conclusion, we observed how, by recapitulating critical TME factors such as interstitial fluid pressure, hypoxia, and stromal stiffness, these platforms are transitioning from platforms capable of optimizing therapeutic regimens before clinical application.
Biomechanics play a critical role in modeling tissues to untherapy. This includes inherent properties, such as stiffness, as well as physiological capabilities, such as ventilation. In addition, biomechanics is a key contributor in the guidance of interventional procedures for cancer treatment, holding considerable importance in treatment planning and optimization and image registration. Here, we describe recent biomechanical innovations to characterize solid malignancies, such as elastography obtained via magnetic resonance or ultrasound, and evaluate normal tissues including physiologic function and structural components to both optimize and understand the impact of therapy. In addition, the role of biomechanics to optimize cancer therapy through applications such as ventilation mapping, guiding surgical interventions via finite element modeling, and enhancing image registration accuracy in multimodal and longitudinal imaging by incorporating biomechanics into deformable image registration, is explored. We also evaluate how emerging and future applications can have a significant impact on the detection, diagnosis, treatment, and response assessment of cancer malignancies.
The lymphatic system is central to immune surveillance, coordinating antigen transport, and immune cell trafficking. While in vivo studies have revealed key aspects of lymphatic and lymph node biology, their complexity has motivated the development of in vitro platforms that enable controlled interrogation of specific immune processes. This review presents a tiered framework for organizing engineered lymphatic and lymph node models based on increasing architectural and functional complexity. Tier 1 models employ two-dimensional lymphatic endothelial monolayers to investigate immune cell docking, chemokine presentation, and junctional regulation. Tier 2 systems incorporate three-dimensional matrices and stromal organization to recreate lymphoid microenvironments, including lymph node scaffolds and immune organoids that support lymphocyte positioning and antigen handling under static conditions. Tier 3 platforms integrate microfluidic perfusion and compartmentalization to model lymphatic transport, flow-dependent endothelial behavior, and immune trafficking in dynamic lymphatic systems. Collectively, these models define the experimental landscape of in vitro lymphatic immunity.
Chromatin-modifying enzymes (CMEs) have traditionally been studied in their nuclear context for regulating gene expression. However, recent evidence points to the significant non-canonical functions that they perform in the cytoplasm, mitochondria, and plasma membrane, which can contribute to disease progression and alter cell phenotypes. This review surveys emerging engineering approaches to control protein localization, which could be applied to CMEs, particularly histone-modifying enzymes. Natural regulatory mechanisms include nuclear import/export signals and mechanical force-mediated translocation. Engineering strategies encompass diverse approaches: synthetic localization signals for directional transport, RNA editing systems like SNAP-ADAR, and small molecule platforms including bifunctional compounds, self-localizing ligands, and nanobody-mediated translocation. Optogenetic tools provide spatiotemporal control through light-inducible trapping, while inducible condensates enable reversible protein sequestration. Additional tools provide extra control via protease-based cleavage mechanisms and endogenous secondary messenger coupling. Despite significant advances in protein relocalization technologies, their application to CMEs remains largely unexplored, which would allow us to decode mechanisms of disease and develop targeted therapeutic interventions for those diseases. Future applications of these tools to CMEs will elucidate our understanding of epigenetic regulation and expand how we conceptualize CMEs.
CRISPR/Cas-based epigenome editing technologies hold great promise for identifying novel therapeutic targets, improving gene and cell therapies, and directly addressing the underlying issues in many diseases, all while minimizing risks of genotoxicity often associated with conventional genome editing. Exciting recent advances in CRISPR/Cas-based epigenome editing technologies have drastically enhanced the ability to precisely control the timing, levels, and durations of endogenous gene expression and reprogram epigenetic states in human cells. As a result, epigenome editing is now poised to unlock new biomedical discoveries and treatments for diseases driven by transcriptional and epigenetic dysregulation as well as those stemming from aberrantly repetitive genomic regions or complex genomic arrangements that are difficult to target using conventional genome editing. Additionally, the power of epigenome editors is generating new strategies to control cell fate and function, which has direct and important implications for cell therapies and regenerative medicines. Here, as the first wave of CRISPR/Cas-based epigenome editors move into clinical trials, we cover recent advances as the field looks to address pressing hurdles facing widespread clinical deployment of epigenome editing technologies including delivery, performance, and safety. For instance, the discovery of compact Cas chassis, engineering efforts to reduce effector sizes for efficient delivery, and campaigns to tailor the targeting discrimination of epigenome editors are rapidly progressing, as is research into the development of new effector domains with high specificity, robust performance, and a lack of immunogenicity and cytotoxicity. This exciting progress is quickly moving the community closer to fulfilling the promise of CRISPR/Cas-based epigenome editing as a powerful class of platform technologies for biological discoveries, biotechnological innovations, and medicines.
Chromatin and transcriptional regulators are critical in neuronal development, function, and plasticity by shaping how, when and, where genes are expressed. Recent advances in DNA-targeting technologies have enabled precise manipulation of chromatin states through direct modification of DNA and histone marks by chromatin editors, and of transcriptional regulation using engineered activators or repressors. While both approaches have the potential to modulate gene expression, they operate via distinct mechanisms with potentially different implications for therapeutic durability and reversibility. To complement these advances, it is important to also implement these technologies within cellular models that capture the complexity of heritable epigenetic processes and human-specific information for modeling and therapeutic development. Here, we highlight recent work that leveraged chromatin editing and transcriptional regulatory tools to model and potentially treat neurological disorders. We also discuss work in developing advanced cellular models of human neurological diseases and the challenges and limitations of such systems.
Transcriptional heterogeneity reflects the inherent variability in gene expression among genetically identical cells. This variability stems from subtle molecular fluctuations including those affecting preinitiation complex assembly and RNA polymerase progression rates. As a result, differences in transcription timing can emerge, driving phenotypic diversity at the population-level. Such heterogeneity can play a critical role in key biological processes including differentiation, selection, adaptation, and disease progression, most notably in cancer. At the core of this variability lies epigenetic regulation, a major determinant of cell-type-specific gene expression patterns. Multiple interconnected mechanisms contribute to epigenetic gene regulation, including DNA methylation, nucleosomal positioning, histone modifications, chromatin compaction, and nuclear organization. Together, these mechanisms establish and maintain cell-type-specific transcriptional programs while preserving a remarkable degree of plasticity. Importantly, epigenetic regulation is highly dynamic, with continuous remodeling and fine-tuning transcriptional output in response to internal and external cues. In this review, we focus on the dynamics in distinct layers of epigenetic regulation and how these regulatory layers interact. We discuss how their interactions impact transcriptional heterogeneity and we highlight recent technological advances that have enabled deeper insights into these complex regulatory processes.
Nature uses epigenetics to establish gene expression patterns in cells that, while reversible, are stable over the lifetime of an individual. If we could appropriately harness the rules of epigenetics, we should be able to use short term interventions to create lifelong therapeutic effects on gene expression. Such epigenetic edits would be comparatively safer (compared to creating double-strand breaks), reversible, and potentially tunable. However, key challenges have impeded the realization of these ambitions in vivo, especially in the brain. Efforts to overcome these challenges have established the basic paradigms driving research and innovations over the past several years. This chapter will describe these basic paradigms and how they are being applied to achieve long-term epigenetic editing in the brains of animal models in vivo.
In the last 25 years, epigenetics has become a mature independent discipline, rooted in fundamental research in developmental biology and advancing toward clinical tools such as Despite Food and Drug Administration (FDA)-approval, such ing, rewriting chromatin biochemical marks in a genetargeted fashion, has received academic and commercial interest. Indeed, quickly upon the discovery of CRISPR-Cas9 as a flexible gene editing tool, rapid developments in genetargeting technologies, including base editing and prime editing, have expanded the genome engineering toolbox. Importantly, this platform has also been repurposed for nuclease activity-free editing of gene states, enabling precise reprogramming of gene function rather than changing DNA sequences. Within the following decade, the gene-targeting field has achieved significant progress, with the first FDAapproved CRISPR-Cas9-based therapeutic and promising technologies going beyond wildtype Cas9. In this review, we discuss the state-of-the-art in epigenetic tools, including their therapeutic potential translating to clinical trials and current challenges.
Epigenome editing, the site-specific rewriting of chromatin modifications, provides a powerful approach to modulate gene regulation in basic research, biotechnology and pre-clinical settings. Synthetic epigenome editors (EpiEditors) contain a programmable DNA-binding module (CRISPR/dCas systems, zinc finger, and transcription activator-like effector proteins), combined with effector modules derived from chromatin-modifying enzymes (such as DNA methyltransferases or Ten-eleven translocation methylcytosine dioxygenases), or recruitment domains (such as Kr & uuml;ppelassociated box proteins). Substantial progress has been made in recent years in improving the specificity, stability, and functional robustness of epigenome editing technologies. Advances include optimized effector domains with reduced off-target activity, toxicity, and size, expanded CRISPR/Cas toolkits, combinatorial and modular editor designs, and increasingly efficient delivery strategies based on viral vectors, lipid nanoparticles, virus-like particles, and engineered exosomes. Epigenome editing allows the dissection of causal relationships between chromatin states and gene regulation, revealing context-dependent and combinatorial effects of epigenome modifications. Beyond basic research, a growing number of pre-clinical applications demonstrate durable repression or activation of disease-relevant genes in models of neurodegenerative disorders, imprinting diseases, cancer, and metabolic disorders such as hypercholesterolemia. These studies highlight both the therapeutic potential of epigenome editing and the importance of understanding chromatin context, downstream signaling, and cell type-dependencies. They also demonstrated the potential reversibility of the epigenome editing interrogation. Despite significant progress, key challenges remain, including the reliable prediction of editing outcomes, understanding the mechanistic basis of long-term stability, and the development of safe, efficient, and powerful delivery systems. Continued methodological development and systematic comparative studies are expected to further advance epigenome editing toward precision medicine applications.
The interplay between diverse cell types and their extracellular matrix (ECM) is fundamental for multicellular life. The ECM is a complex meshwork of fibrillar proteins and soluble factors. Cells and their surrounding ECM interact bidirectionally, whereby cells deposit their tissue-specific ECM and remodel it enzymatically and by exerting contractile forces. The ECM in turn modulates cellular functions like gene expression, proliferation, and motility. A careful balance of this interaction is key for homeostasis, and is lost during cancer progression. Different cell types constituting a tumor including cancer and stromal cells, contribute to an imbalanced cell-ECM crosstalk within the tumor. Cumulatively, this leads to a tumor ECM characterized by particular features like increased stiffness and viscoelasticity, altered alignment, bundled fibers, etc. In this review, we discuss the advances in our understanding of the tumor ECM architecture and the multicellular interactions that help achieve it, with a special focus on increasing granularity in disentangling the contributions of individual tumor ECM features in disease progression.
In recent years, there have been remarkable advancements in artificial intelligence (AI) techniques, particularly in their application to biomedical imaging. This integration has opened up new possibilities for early and improved diagnosis, automation, and interoperability across various medical applications. This review explores the key developments in AI-driven biomedical imaging, examining the techniques and applications that have evolved. We highlight recent enhancements in various areas, such as early-stage diagnostics and explainability. Additionally, we address the challenges and limitations while shedding light on potential research directions to further integrate AI into clinical imaging, thereby enhancing patient-centered care. By synthesizing these key advancements and ongoing challenges, we aim to underscore AI's potential to transform biomedical imaging practices.
DNA methylation and hydroxymethylation are two key epigenetic modifications increasingly regarded as viable targets for gene therapy. Dysregulation of these marks in specific regions of the genome, such as regulatory regions, alters the expression of genes in various human disease settings, including cancer, obesity, infectious diseases, and other biological processes such as stem cell reprogramming and aging. Here, we summarize recent approaches in editing DNA methylation and discuss the state-of-the-art epigenome engineering strategies aimed at reprogramming DNA methylation in specific genomic sequences for therapeutic purposes. In addition, we outline major challenges for the preclinical and clinical implementation of epigenome editors - particularly CRISPR-based epigenetic modifiers - including the optimization of epigenetic editors, co-effector requirements, the risk of off-target activity, and the obstacles associated with achieving efficient in vivo tissue-specific delivery.
The eukaryotic epigenome plays a central role in regulating gene expression, cellular identity, and development through dynamic, multilayered biochemical modifications to DNA, histones, and chromatin architecture. Disruption of these regulatory mechanisms contributes to a wide range of human diseases, including cancer, neurodegenerative disorders, and immunological conditions. Targeted epigenome editing offers promising discovery and therapeutic strategies by enabling the correction of aberrant epigenetic states without the need for permanent changes to the DNA sequence. The catalytically inactive CRISPR-Cas (dCas) molecule fused to epigenetic effector domains has emerged as a versatile platform for programmable, locus-specific modulation of chromatin states. These CRISPR-based epigenetic editors can deposit or remove desired epigenetic marks and alter three-dimensional genome organization to fine-tune gene expression with high specificity. Recent developments have expanded the CRISPR epigenome editing toolbox by introducing new effector domains, improving multiplexing capabilities, and enabling large-scale genetic screening, leading to novel insights into the functional genomics across various cellular contexts. However, clinical translation remains challenged by inefficient delivery and suboptimal editing efficacy in vivo. This review highlights recent advances in CRISPR-based epigenetic editing, with a focus on applications in primary cells, new tool development, and the translational potential of epigenome modulation for safe, durable, and precise therapies.
Poorly designed medical devices and digital health tools can disproportionately affect their accessibility, adoption, and effectiveness for marginalized populations, such as older adults and Black and Indigenous people of colour. Inequalities in adoption and adherence to these medical technologies can be influenced by their usability; therefore, usability testing throughout all stages of development is significant to ensure that these solutions are safe, efficient, and easy to use for diverse user populations. However, traditional usability testing methods are often resource-intensive and inconsistent, raising concerns about their effectiveness, especially for addressing the needs of vulnerable groups. With the increasing integration of artificial intelligence (AI) in usability testing, questions remain about its ability to represent the perspectives of marginalized populations. This narrative review of 35 articles examined the current state of AI integration into usability testing processes, its usefulness and ease of use for evaluating the usability of medical devices and digital health tools, and its potential impact on including marginalized community perspectives. 20 articles provided AI-informed usability testing tools exclusively for digital products, such as mobile applications. These tools primarily supported usability analysts by automating specific usability testing tasks, such as identifying usability issues, performing sentiment analysis, and scoring interfaces. Three studies demonstrated AI's use in simulating human participants or replacing evaluators under specific conditions. However, equity considerations were limited: 16 studies did not address how their tools would impact equitable usability testing practices, and 12 provided limited acknowledgment in their discussion sections. Only three sources in the literature explicitly explored AI-supported usability tools with marginalized communities. While AI-informed usability tools show promise for formative evaluations of digital health tools, their application in diverse contexts remains limited. Future priorities include validating on-the-market and literature AI-supported usability tools with various healthcare solutions and user groups, aligning AI-informed usability practice development with usability analyst workflows, and integrating equity considerations into usability testing frameworks. Developing guidelines for both traditional and AI-informed usability methods through collaboration with experts from AI, human factors, medical, and ethics fields is critical to ensuring equitable outcomes in medical device and digital health tool evaluation.
Technologies for editing epigenetic modifications and controlling transcription in mammalian cells have revolutionized targeted gene perturbation, functional genomics, and basic research. By avoiding the generation of DNA breaks, epigenome editing serves as a safe and precise approach for altering gene expression and has emerged as a promising platform for therapeutic applications. The advent of CRISPR has contributed significantly to the expansion of the existing toolkit for programmable modulation of epigenetic and transcriptional states. This review highlights recent discoveries in engineering novel tools for epigenome editing and transcriptional modulation through rational design, high throughput screening methods, and mutational scans, which leverage the endogenous reservoir of chromatin and transcriptional effectors for targeted gene repression and activation. We also discuss the therapeutic potential of epigenome modulators and highlight the key challenges that need to be addressed to improve their safety and efficacy. Advancing our understanding of the complex mechanisms driving gene expression and overcoming current limitations will pave the way for the development of novel technologies that advance fundamental research and translational applications.
