Cancer organoids have improved our understanding of recapitulating the histology, genotypes, and drug response of patient tumors for personalized medicine. However, the existing cancer organoids are typically grown in animal-derived matrices (e.g., Matrigel), which suffer from poor reproducibility and low throughput due to uncontrollable origin of seed cells, undefined matrix, and manual manipulation. Here, we report a new strategy to massively generate uniform pancreatic cancer organoids (PCOs) in a droplet system from single cells. This system is composed of all-in-water fluids that allow to mildly encapsulate single tumor cell into isolated droplet, which subsequently proliferate and self-assemble into organoids, resembling the initial state of a tumor in the body. This high-throughput method can produce thousands of organoids in a single batch. The droplets can serve as templates for synthesizing defined microgels with proper stiffness similar to that of native tumors, facilitating functional expressions of PCOs. These organoids exhibit superior uniformity and controllability in terms of size and morphologies compared with organoids cultured in manually dropped Matrigel, due mainly to the controllable number of initiating cells and defined microgels. In addition, the established organoids maintain the key biomarkers of pancreatic tumor (e.g., KRT7, KRT19 and SOX9) and higher expression of genes associated with drug metabolism confirmed by RNA-seq and PCR analysis. Furthermore, they show distinguishing responses to four clinically used drugs in a reproducible manner in automatic pipetting workstation, indicating the feasibility of the proposed method in high-throughput drug testing. The established strategy has integrated the formation, 3D cultures, and analysis of PCOs derived from single cells in a whole system, which may provide a novel platform for advancing organoids research with standardized procedure in translational applications.

Brain organoids are an emerging in vitro 3D brain model that is integrated from pluripotent stem cells. This model mimics the human brain's developmental process and disease-related phenotypes to a certain extent while advancing the development of human brain-based biological intelligence. However, many limitations of brain organoid culture (e.g., lacking a functional vascular system, etc.) prevent in vitro-cultured organoids from truly replicating the human brain in terms of cell type and structure. To improve brain organoids' scalability, efficiency, and stability, this paper discusses important contributions of material biology and microprocessing technology in solving the related limitations of brain organoids and applying the latest imaging technology to make real-time imaging of brain organoids possible. In addition, the related applications of brain organoids, especially the development of organoid intelligence combined with artificial intelligence, are analyzed, which will help accelerate the rational design and guidance of brain organoids.

Organoids, 3D organ-like tissue cultures derived from stem cells, show promising potential for developmental biology, drug discovery, and regenerative medicine. However, the function and phenotype of current organoids, especially neural organoids, are still limited by insufficient diffusion of oxygen, nutrients, metabolites, signaling molecules, and drugs. Herein, we present vascular network-inspired diffusible (VID) scaffolds to mimic physiological diffusion physics for generating functional organoids and phenotyping their drug response. Specifically, the VID scaffolds, 3D-printed meshed tubular channel networks, successfully engineer human midbrain organoids almost without necrosis and hypoxia in commonly used well plates. Compared with conventional organoids, these engineered organoids develop more physiologically relevant features and functions, including midbrain-specific identity, oxygen metabolism, neuronal maturation, and network activity. Moreover, these engineered organoids also better recapitulate pharmacological responses, such as neural activity changes to fentanyl exposure, compared with conventional organoids with significant diffusion limits. This platform may provide insights for organoid development and therapeutic innovation.

Organoids are systems derived from pluripotent stem cells at the interface between traditional monolayer cultures and in vivo animal models. The structural and functional characteristics of organoids enable the modelling of early stages of brain development in a physiologically relevant 3D environment. Moreover, organoids constitute a tool with which to analyse how individual genetic variation contributes to the susceptibility and progression of neurodevelopmental disorders. This Roadmap article describes the features of brain organoids, focusing on the neocortex, and their advantages and limitations - in comparison with other model systems - for the study of brain development, evolution and disease. We highlight avenues for enhancing the physiological relevance of brain organoids by integrating bioengineering techniques and unbiased high-throughput analyses, and discuss future applications. As organoids advance in mimicking human brain functions, we address the ethical and societal implications of this technology.

The viscoelastic properties of tissues influence their morphology and cellular behavior, yet little is known about changes in these properties during brain malformations. Lissencephaly, a severe cortical malformation caused by LIS1 mutations, results in a smooth cortex. Here, we show that human-derived brain organoids with LIS1 mutation exhibit increased stiffness compared to controls at multiple developmental stages. This stiffening correlates with abnormal extracellular matrix (ECM) expression and organization, as well as elevated water content, measured by diffusion-weighted MRI. Short-term MMP9 treatment reduces both stiffness and water diffusion levels to control values. Additionally, a computational microstructure mechanical model predicts mechanical changes based on ECM organization. These findings suggest that LIS1 plays a critical role in ECM regulation during brain development and that its mutation leads to significant viscoelastic alterations.

Neocortical folding (i.e., gyrification) is a key evolutionary and developmental feature of the brain, facilitating cortical surface expansion and enhanced cognitive function. However, the precise strategies and mechanisms underlying cortical folding remain incompletely understood. In this study, we systematically investigated the dynamic formation of neocortical folding in the ferret. Our findings reveal significant differences in neurogenesis and neuronal migration between the developing lateral gyrus (LG) and adjacent lateral sulcus (LS) of the ferret neocortex. Specifically, progenitors in the LG exhibited higher mitosis activity and a shorter S-phase duration compared to those in the LS. Additionally, immature neurons in the LG followed a fan-like migration pattern, whereas those in the LS exhibited a flower bud-like pattern. Organotypic slice cultures and time-lapse imaging further demonstrated that the migration trajectory of immature neurons to the neocortex is more straightforward in the LG than in the LS. Together, these results highlight distinct cellular behaviors between the developing gyrus and sulcus, providing novel insights into cellular mechanisms underlying cortex folding.

The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform-the organoids-on-a-chip technology-shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.

Organoids are stem-cell derived tissue structures mimicking specific structural and functional characteristics of human organs. Despite significant advancements in the field over the last decade, challenges like limited long-term functional culture and lack of maturation are hampering the implementation of organoids in biomedical research. Culture of organoids in microfluidic chips is being used to tackle these challenges through dynamic and precise control over the organoid microenvironment. This review highlights the significant breakthroughs that have been made in the innovative field of "organoids-on-chip," demonstrating how these have contributed to advancing organoid models. We focus on the incorporation of organoids representative for various tissues into chips and discuss the latest findings in multi-organoids-on-chip approaches. Additionally, we examine current limitations and challenges of the field towards the development of reproducible organoids-on-chip systems. Finally, we discuss the potential of organoids-on-chip technology for both in vitro and in vivo applications.

Organ morphogenesis is multifaceted, multiscale, and fundamentally a robust process. Despite the complex and dynamic nature of embryonic development, organs are built with reproducible size, shape, and function, allowing them to support organismal growth and life. This striking reproducibility of tissue form exists because morphogenesis is not entirely hardwired. Instead, it is an emergent product of mechanochemical information flow, operating across spatial and temporal scales-from local cellular deformations to organ-scale form and function, and back. In this review, we address the mechanical basis of organ morphogenesis, as understood by observations and experiments in living embryos. To this end, we discuss how mechanical information controls the emergence of a highly conserved set of structural motifs that shape organ architectures across the animal kingdom: folds and loops, tubes and lumens, buds, branches, and networks. Moving forward, we advocate for a holistic conceptual framework for the study of organ morphogenesis, which rests on an interdisciplinary toolkit and brings the embryo center stage.

Reproducible pattern and form generation during embryogenesis is poorly understood. Intestinal organoid morphogenesis involves a number of mechanochemical regulators such as cell-type-specific cytoskeletal forces and osmotically driven lumen volume changes. It is unclear how these forces are coordinated in time and space to ensure robust morphogenesis. Here we show how mechanosensitive feedback on cytoskeletal tension gives rise to morphological bistability in a minimal model of organoid morphogenesis. In the model, lumen volume changes can impact the epithelial shape via both direct mechanical and indirect mechanosensitive mechanisms. We find that both bulged and budded crypt states are possible and dependent on the history of volume changes. We test key modelling assumptions via biophysical and pharmacological experiments to demonstrate how bistability can explain experimental observations, such as the importance of the timing of lumen shrinkage and robustness of the final morphogenetic state to mechanical perturbations. This suggests that bistability arising from feedback between cellular tensions and fluid pressure could be a general mechanism that coordinates multicellular shape changes in developing systems.

Brain organoids are important 3D models for studying human brain development, disease, and evolution. To overcome some of the existing limitations that affect organoid quality, reproducibility, characteristics, and in vivo resemblance, current efforts are directed to improve their physiological relevance by exploring different, yet interconnected, routes. In this review, these approaches and their latest developments are discussed, including stem cell optimization, refining morphogen administration strategies, altering the extracellular matrix (ECM) niche, and manipulating tissue architecture to mimic in vivo brain morphogenesis. Additionally, strategies to increase cell diversity and enhance organoid maturation, such as establishing co-cultures, assembloids, and organoid in vivo xenotransplantation, are reviewed. We explore how these various factors can be tuned and intermingled and speculate on future avenues towards even more physiologically-advanced brain organoids.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSCs) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives an unprecedented opportunity to explore human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e. Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy syndrome, tuberous sclerosis syndrome) and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems in the realm of personalized medicine.

Organoids, as 3D in vitro models derived from stem cells, have unparalleled advantages over traditional cell and animal models for studying organogenesis, disease mechanisms, drug screening, and personalized diagnosis and treatment. Despite the tremendous progress made in organoid technology, the translational application of organoids still presents enormous challenges due to the complex structure and function of human organs. In this review, the limitations of the translational application of traditional organoid technologies are first described. Next, we explore ways to address many of the limitations of traditional organoid cultures by engineering various dimensions of organoid systems. Finally, we discuss future directions in the field, including potential roles in drug screening, simulated microphysiology system and personalized diagnosis and treatment. We hope that this review inspires future research into organoids and microphysiology system.

Culturing living cells in three-dimensional environments increases the biological relevance of laboratory experiments, but requires solutes to overcome a diffusion barrier to reach the centre of cellular constructs. We present a theoretical and numerical investigation that brings a mechanistic understanding of how microfluidic culture conditions, including chamber size, inlet fluid velocity and spatial confinement, affect solute distribution within three-dimensional cellular constructs. Contact with the chamber substrate reduces the maximally achievable construct radius by 15%. In practice, finite diffusion and convection kinetics in the microfluidic chamber further lower that limit. The benefits of external convection are greater if transport rates across diffusion-dominated areas are high. Those are omnipresent and include the diffusive boundary layer growing from the fluid-construct interface and regions near corners where fluid is recirculating. Such regions multiply the required convection to achieve a given solute penetration by up to 100, so chip designs ought to minimize them. Our results define conditions where complete solute transport into an avascular three-dimensional cell construct is achievable and applies to real chambers without needing to simulate their exact geometries.

Organoids-on-chips is an emerging innovative integration of stem cell-derived organoids with advanced organ-on-chip technology, providing a novel platform for the in vitro construction of biomimetic micro-physiological systems. The synergistic merger transcends the limitations of traditional drug screening and safety assessment methodologies, such as 2D cell cultures and animal models. In this review, we examine the prevailing challenges and prerequisites of preclinical models utilized for drug screening and safety evaluations. We highlighted the salient features and merits of organoids-on-chip, elucidating their capability to authentically replicate human physiology, thereby addressing contemporary impediments. We comprehensively overviewed the recent endeavors where organoids-on-chips have been harnessed for drug screening and safety assessment and delved into potential opportunities and challenges for evolving sophisticated, near-physiological organoids-on-chips. Based on current achievements, we further discuss how to enhance the practicality of organoids-on-chips and accelerate the translation from preclinical to clinical stages in healthcare and industry by utilizing multidisciplinary convergent innovation.

Symmetry breaking leading to axis formation and spatial patterning is crucial for achieving more accurate recapitulation of human development in organoids. While these processes can occur spontaneously by self-organizing capabilities of pluripotent stem cells, they can often result in variation in structure and composition of cell types within organoids. To address this limitation, bioengineering techniques that utilize geometric, topological and stiffness factors are increasingly employed to enhance control and consistency. Here, we review how spontaneous manners and engineering tools such as micropattern, microfluidics, biomaterials, etc. can facilitate the process of symmetry breaking leading to germ layer patterning and the formation of anteroposterior and dorsoventral axes in blastoids, gastruloids, neuruloids and neural organoids. Furthermore, brain assembloids, which are composed of multiple brain regions through fusion processes are discussed. The overview of organoid polarization in terms of patterning tools can offer valuable insights for enhancing the physiological relevance of organoid system.

Advanced brain organoids provide promising platforms for deciphering the cellular and molecular processes of human neural development and diseases. Although various studies and reviews have described developments and advancements in brain organoids, few studies have comprehensively summarized and analyzed the global trends in this area of neuroscience. To identify and further facilitate the development of cerebral organoids, we utilized bibliometrics and visualization methods to analyze the global trends and evolution of brain organoids in the last 10 years. First, annual publications, countries/regions, organizations, journals, authors, co-citations, and keywords relating to brain organoids were identified. The hotspots in this field were also systematically identified. Subsequently, current applications for brain organoids in neuroscience, including human neural development, neural disorders, infectious diseases, regenerative medicine, drug discovery, and toxicity assessment studies, are comprehensively discussed. Towards that end, several considerations regarding the current challenges in brain organoid research and future strategies to advance neuroscience will be presented to further promote their application in neurological research.

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Perfluorooctane sulfonate (PFOS), a class of synthetic chemicals detected in various environmental compartments, has been associated with dysfunctions of the human central nervous system (CNS). However, the underlying neurotoxicology of PFOS exposure is largely understudied due to the lack of relevant human models. Here, we report bioengineered human midbrain organoid microphysiological systems (hMO-MPSs) to recapitulate the response of a fetal human brain to multiple concurrent PFOS exposure conditions. Each hMO-MPS consists of an hMO on a fully 3D printed holder device with a perfusable organoid adhesion layer for enhancing air-liquid interface culturing. Leveraging the unique, simply-fabricated holder devices, hMO-MPSs are scalable, easy to use, and compatible with conventional well-plates, and allow easy transfer onto a multiple-electrode array (MEA) system for plug-and-play measurement of neural activity. Interestingly, the neural activity of hMO-MPSs initially increased and subsequently decreased by exposure to a concentration range of 0, 30, 100, to 300 μM of PFOS. Furthermore, PFOS exposure impaired neural development and promoted neuroinflammation in the engineered hMO-MPSs. Along with PFOS, our platform is broadly applicable for studies toxicology of various other environmental pollutants.

Organoids, three-dimensional in vitro organ-like tissue cultures derived from stem cells, show promising potential for developmental biology, drug discovery, and regenerative medicine. However, the function and phenotype of current organoids, especially neural organoids, are still limited by insufficient diffusion of oxygen, nutrients, metabolites, signaling molecules, and drugs. Herein, we present Vascular network-Inspired Diffusible (VID) scaffolds to fully recapture the benefits of physiological diffusion physics for generating functional organoids and phenotyping their drug response. In a proof-of-concept application, the VID scaffolds, 3D-printed meshed tubular channel networks, support the successful generation of engineered human midbrain organoids almost without necrosis and hypoxia in commonly used well-plates. Compared to conventional organoids, these engineered organoids develop with more physiologically relevant features and functions including midbrain-specific identity, oxygen metabolism, neuronal maturation, and network activity. Moreover, these engineered organoids also better recapitulate pharmacological responses, such as neural activity changes to fentanyl exposure, compared to conventional organoids with significant diffusion limits. Combining these unique scaffolds and engineered organoids may provide insights for organoid development and therapeutic innovation.

Neurodegenerative diseases (NDDs) affect more than 50 million people worldwide, posing a significant global health challenge as well as a high socioeconomic burden. With aging constituting one of the main risk factors for some NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD), this societal toll is expected to rise considering the predicted increase in the aging population as well as the limited progress in the development of effective therapeutics. To address the high failure rates in clinical trials, legislative changes permitting the use of alternatives to traditional pre-clinical in vivo models are implemented. In this regard, microphysiological systems (MPS) such as organ-on-a-chip (OoC) platforms constitute a promising tool, due to their ability to mimic complex and human-specific tissue niches in vitro. This review summarizes the current progress in modeling NDDs using OoC technology and discusses five critical aspects still insufficiently addressed in OoC models to date. Taking these aspects into consideration in the future MPS will advance the modeling of NDDs in vitro and increase their translational value in the clinical setting.

Organoids have emerged as major technological breakthroughs and novel organ models that have revolutionized biomedical research by recapitulating the key structural and functional complexities of their in vivo counterparts. The combination of organoid systems and microfluidic technologies has opened new frontiers in organoid engineering and offers great opportunities to address the current challenges of existing organoid systems and broaden their biomedical applications. In this review, the key features of the existing organoids, including their origins, development, design principles, and limitations, are described. Then the recent progress in integrating organoids into microfluidic systems is highlighted, involving microarrays for high-throughput organoid manipulation, microreactors for organoid hydrogel scaffold fabrication, and microfluidic chips for functional organoid culture. The opportunities in the nascent combination of organoids and microfluidics that lie ahead to accelerate research in organ development, disease studies, drug screening, and regenerative medicine are also discussed. Finally, the challenges and future perspectives in the development of advanced microfluidic platforms and modified technologies for building organoids with higher fidelity and standardization are envisioned.

Brain organoids are three-dimensional aggregates of self-organized differentiated stem cells that mimic the structure and function of human brain regions. Organoids bridge the gaps between conventional drug screening models such as planar mammalian cell culture, animal studies, and clinical trials. They can revolutionize the fields of developmental biology, neuroscience, toxicology, and computer engineering. Conventional microinstrumentation for conventional cellular engineering, such as planar microfluidic chips; microelectrode arrays (MEAs); and optical, magnetic, and acoustic techniques, has limitations when applied to three-dimensional (3D) organoids, primarily due to their limits with inherently two-dimensional geometry and interfacing. Hence, there is an urgent need to develop new instrumentation compatible with live cell culture techniques and with scalable 3D formats relevant to organoids. This review discusses conventional planar approaches and emerging 3D microinstrumentation necessary for advanced organoid-machine interfaces. Specifically, this article surveys recently developed microinstrumentation, including 3D printed and curved microfluidics, 3D and fast-scan optical techniques, buckling and self-folding MEAs, 3D interfaces for electrochemical measurements, and 3D spatially controllable magnetic and acoustic technologies relevant to two-way information transfer with brain organoids. This article highlights key challenges that must be addressed for robust organoid culture and reliable 3D spatiotemporal information transfer.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Unveiling the intricate interplay of cells in their native environment lies at the heart of understanding fundamental biological processes and unraveling disease mechanisms, particularly in complex diseases like cancer. Spatial transcriptomics (ST) offers a revolutionary lens into the spatial organization of gene expression within tissues, empowering researchers to study both cell heterogeneity and microenvironments in health and disease. However, current ST technologies often face limitations in either resolution or the number of genes profiled simultaneously. Integrating ST data with complementary sources, such as single-cell transcriptomics and detailed tissue staining images, presents a powerful solution to overcome these limitations. This review delves into the computational approaches driving the integration of spatial transcriptomics with other data types. By illuminating the key challenges and outlining the current algorithmic solutions, we aim to highlight the immense potential of these methods to revolutionize our understanding of cancer biology.

Brain organoids hold great potential for modeling human brain development and pathogenesis. They recapitulate certain aspects of the transcriptional trajectory, cellular diversity, tissue architecture and functions of the developing brain. In this review, we explore the engineering strategies to control the molecular-, cellular- and tissue-level inputs to achieve high-fidelity brain organoids. We review the application of brain organoids in neural disorder modeling and emerging bioengineering methods to improve data collection and feature extraction at multiscale. The integration of multiscale engineering strategies and analytical methods has significant potential to advance insight into neurological disorders and accelerate drug development.

Brain-on-Chip devices, which facilitate on-chip cultures of neurons to simulate brain functions, are receiving tremendous attention from both fundamental and clinical research. Consequently, microsensors are being developed to accomplish real-time monitoring of neurotransmitters, which are the benchmarks for neuron network operation. Among these, electrochemical sensors have emerged as promising candidates for detecting a critical neurotransmitter, dopamine. However, current state-of-the-art electrochemical dopamine sensors are suffering from issues like limited sensitivity and cumbersome fabrication. Here, a novel route in monolithically microfabricating vertically aligned carbon nanofiber electrochemical dopamine microsensors is reported with an anti-blistering slow cooling process. Thanks to the microfabrication process, microsensors is created with complete insulation and large surface areas. The champion device shows extremely high sensitivity of 4.52× 104 µAµM-1·cm-2, which is two-orders-of-magnitude higher than current devices, and a highly competitive limit of detection of 0.243 nM. These remarkable figures-of-merit will open new windows for applications such as electrochemical recording from a single neuron.

Local cells can actively create reverse bending (evagination) in invaginated epithelia, which plays a crucial role in the formation of elaborate organisms. However, the precise physical mechanism driving the evagination remains elusive. Here, we present a three-dimensional vertex model, incorporating the intrinsic cell polarity, to explore the complex morphogenesis induced by local mechanical modulations. We find that invaginated tissues can spontaneously generate local reverse bending due to the shift of the apicobasal polarity. Their exact shapes can be analytically determined by the local apicobasal differential tension and the internal stress. Our continuum theory exhibits three regions in a phase diagram controlled by these two parameters, showing curvature transitions from ordered to disordered states. Additionally, we delve into epithelial curvature transition induced by the nucleus repositioning, revealing its active contribution to the apicobasal force generation. The uncovered mechanical principles could potentially guide more studies on epithelial folding in diverse systems.

Neural organoids consist of three-dimensional tissue derived from pluripotent stem cells that could recapitulate key features of the human brain. During the past decade, organoid technology has evolved in the field of human brain science by increasing the quality and applicability of its products. Among them, a novel approach involving the design of neural organoids engineered by mechanical forces has emerged. This review describes previous approaches for the generation of neural organoids, the engineering of neural organoids by mechanical forces, and future challenges for the application of mechanical forces in the design of neural organoids.

Organoid cultures offer a valuable platform for studying organ-level biology, allowing for a closer mimicry of human physiology compared to traditional two-dimensional cell culture systems or non-primate animal models. While many organoid cultures use cell aggregates or decellularized extracellular matrices as scaffolds, they often lack precise biochemical and biophysical microenvironments. In contrast, three-dimensional (3D) bioprinting allows precise placement of organoids or spheroids, providing enhanced spatial control and facilitating the direct fusion for the formation of large-scale functional tissues in vitro. In addition, 3D bioprinting enables fine tuning of biochemical and biophysical cues to support organoid development and maturation. With advances in the organoid technology and its potential applications across diverse research fields such as cell biology, developmental biology, disease pathology, precision medicine, drug toxicology, and tissue engineering, organoid imaging has become a crucial aspect of physiological and pathological studies. This review highlights the recent advancements in imaging technologies that have significantly contributed to organoid research. Additionally, we discuss various bioprinting techniques, emphasizing their applications in organoid bioprinting. Integrating 3D imaging tools into a bioprinting platform allows real-time visualization while facilitating quality control, optimization, and comprehensive bioprinting assessment. Similarly, combining imaging technologies with organoid bioprinting can provide valuable insights into tissue formation, maturation, functions, and therapeutic responses. This approach not only improves the reproducibility of physiologically relevant tissues but also enhances understanding of complex biological processes. Thus, careful selection of bioprinting modalities, coupled with appropriate imaging techniques, holds the potential to create a versatile platform capable of addressing existing challenges and harnessing opportunities in these rapidly evolving fields.

Spinal cord organoids are three-dimensional tissues derived from stem cells that recapitulate the primary morphological and functional characteristics of the spinal cord in vivo. As emerging bioengineering methods have led to the optimization of cell culture protocols, spinal cord organoids technology has made remarkable advancements in the past decade. Our literature search found that current spinal cord organoids do not only dynamically simulate neural tube formation but also exhibit diverse cytoarchitecture along the dorsal-ventral and rostral-caudal axes. Moreover, fused organoids that integrate motor neurons and other regionally specific organoids exhibit intricate neural circuits that allows for functional assessment. These qualities make spinal cord organoids valuable tools for disease modeling, drug screening, and tissue regeneration. By utilizing this emergent technology, researchers have made significant progress in investigating the pathogenesis and potential therapeutic targets of spinal cord diseases. However, at present, spinal cord organoid technology remains in its infancy and has not been widely applied in translational medicine. Establishment of the next generation of spinal cord organoids will depend on good manufacturing practice standards and needs to focus on diverse cell phenotypes and electrophysiological functionality evaluation.

Two-dimensional (2D) cultures do not fully reflect the human organs' physiology and the real effectiveness of the used therapy. Therefore, three-dimensional (3D) models are increasingly used in bioanalytical science. Organ-on-a-chip systems are used to obtain cellular in vitro models, better reflecting the human body's in vivo characteristics and allowing us to obtain more reliable results than standard preclinical models. Such 3D models can be used to understand the behavior of tissues/organs in response to selected biophysical and biochemical factors, pathological conditions (the mechanisms of their formation), drug screening, or inter-organ interactions. This review characterizes 3D models obtained in microfluidic systems. These include spheroids/aggregates, hydrogel cultures, multilayers, organoids, or cultures on biomaterials. Next, the methods of formation of different 3D cultures in Organ-on-a-chip systems are presented, and examples of such Organ-on-a-chip systems are discussed. Finally, current applications of 3D cell-on-a-chip systems and future perspectives are covered.

Synthetic organ models such as organoids and organ-on-a-chip have been receiving recognition from administrative agencies. Despite the proven success of organoids in predicting drug efficacy on laboratory scales, their translational advances have not fully satisfied the expectations for both clinical implementation and commercial applications. The transition from laboratory settings to clinical applications continues to encounter challenges. Employing engineering methodologies to facilitate the bridging of this gap for organoids represents one of the key directions for future advancement. The main measures to bridge the gap include environmental and phenotypic recapitulation, 3D patterning, matrix engineering, and multi-modality information acquisition and processing. Pilot whole-process clinical/pharmaceutical applications with fast and standardized organoid models will continuously offer convincing frontline optimization clues and driving forces to the organoid community, which is a promising path to translational organoid technologies.

The symbiotic interplay of organoid technology and advanced imaging strategies yields innovative breakthroughs in research and clinical applications. Organoids, intricate three-dimensional cell cultures derived from pluripotent or adult stem/progenitor cells, have emerged as potent tools for in vitro modeling, reflecting in vivo organs and advancing our grasp of tissue physiology and disease. Concurrently, advanced imaging technologies such as confocal, light-sheet, and two-photon microscopy ignite fresh explorations, uncovering rich organoid information. Combined with advanced imaging technologies and the power of artificial intelligence, organoids provide new insights that bridge experimental models and real-world clinical scenarios. This review explores exemplary research that embodies this technological synergy and how organoids reshape personalized medicine and therapeutics.

Developing diagnostics and treatments for neurodegenerative diseases (NDs) is challenging due to multifactorial pathogenesis that progresses gradually. Advanced in vitro systems that recapitulate patient-like pathophysiology are emerging as alternatives to conventional animal-based models. In this review, we explore the interconnected pathogenic features of different types of ND, discuss the general strategy to modelling NDs using a microfluidic chip, and introduce the organoid-on-a-chip as the next advanced relevant model. Lastly, we overview how these models are being applied in academic and industrial drug development. The integration of microfluidic chips, stem cells, and biotechnological devices promises to provide valuable insights for biomedical research and developing diagnostic and therapeutic solutions for NDs.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

Understanding the signals from the physical microenvironment is critical for deciphering the processes of neurogenesis and neurodevelopment. The discovery of how surrounding physical signals shape human developing neurons is hindered by the bottleneck of conventional cell culture and animal models. Notwithstanding neural organoids provide a promising platform for recapitulating human neurogenesis and neurodevelopment, building neuronal physical microenvironment that accurately mimics the native neurophysical features is largely ignored in current organoid technologies. Here, it is discussed how the physical microenvironment modulates critical events during the periods of neurogenesis and neurodevelopment, such as neural stem cell fates, neural tube closure, neuronal migration, axonal guidance, optic cup formation, and cortical folding. Although animal models are widely used to investigate the impacts of physical factors on neurodevelopment and neuropathy, the important roles of human stem cell-derived neural organoids in this field are particularly highlighted. Considering the great promise of human organoids, building neural organoid microenvironments with mechanical forces, electrophysiological microsystems, and light manipulation will help to fully understand the physical cues in neurodevelopmental processes. Neural organoids combined with cutting-edge techniques, such as advanced atomic force microscopes, microrobots, and structural color biomaterials might promote the development of neural organoid-based research and neuroscience.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

The complex structure and function of the human central nervous system that develops from the neural tube made in vitro modeling quite challenging until the discovery of brain organoids. Human-induced pluripotent stem cells-derived brain organoids offer recapitulation of the features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation into mature neurons and micro-macroglial cells, as well as the complex interactions among these diverse cell types of the developing brain. Recent advancements in brain organoids, microfluidic systems, real-time sensing technologies, and their cutting-edge integrated use provide excellent models and tools for emulation of fundamental neurodevelopmental processes, the pathology of neurological disorders, personalized transplantation therapy, and high-throughput neurotoxicity testing by bridging the gap between two-dimensional models and the complex three-dimensional environment in vivo. In this review, we summarize how bioengineering approaches are applied to mitigate the limitations of brain organoids for biomedical and clinical research. We further provide an extensive overview and future perspectives of the humanized brain organoids-on-chip platforms with integrated sensors toward brain organoid intelligence and biocomputing studies. Such approaches might pave the way for increasing approvable clinical applications by solving their current limitations.

The creation of a suitable scaffold is a crucial step in the process of bone tissue engineering (BTE). The scaffold, acting as an artificial extracellular matrix, plays a significant role in determining the fate of cells by affecting their proliferation and differentiation in BTE. Therefore, careful consideration should be given to the fabrication approach and materials used for scaffold preparation. Natural polypeptides such as gelatin and collagen have been widely used for this purpose. The unique properties of nanoparticles, which vary depending on their size, charge, and physicochemical properties, have demonstrated potential in solving various challenges encountered in BTE. Therefore, nanocomposite biomaterials consisting of polymers and nanoparticles have been extensively used for BTE. Gelatin has also been utilized in combination with other nanomaterials to apply for this purpose. Composites of gelatin with various types of nanoparticles are particularly promising for creating scaffolds with superior biological and physicochemical properties. This review explores the use of nanocomposite biomaterials based on gelatin and various types of nanoparticles together for applications in bone tissue engineering.

Brain development in humans is achieved through precise spatiotemporal genetic control, the mechanisms of which remain largely elusive. Recently, integration of technological advances in human stem cell-based modelling with genome editing has emerged as a powerful platform to establish causative links between genotypes and phenotypes directly in the human system. Here, we review our current knowledge of complex genetic regulation of each key step of human brain development through the lens of evolutionary specialization and neurodevelopmental disorders and highlight the use of human stem cell-derived 2D cultures and 3D brain organoids to investigate human-enriched features and disease mechanisms. We also discuss opportunities and challenges of integrating new technologies to reveal the genetic architecture of human brain development and disorders.