The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Imaging-Based Efficacy Evaluation of Cancer Immunotherapy in Engineered Tumor Platforms and Tumor Organoids.

Cancer immunotherapy is used to treat tumors by modulating the immune system. Although the anticancer efficacy of cancer immunotherapy has been evaluated prior to clinical trials, conventional in vivo animal and endpoint models inadequately replicate the intricate process of tumor elimination and reflect human-specific immune systems. Therefore, more sophisticated models that mimic the complex tumor-immune microenvironment must be employed to assess the effectiveness of immunotherapy. Additionally, using real-time imaging technology, a step-by-step evaluation can be applied, allowing for a more precise assessment of treatment efficacy. Here, an overview of the various imaging-based evaluation platforms recently developed for cancer immunotherapeutic applications is presented. Specifically, a fundamental technique is discussed for stably observing immune cell-based tumor cell killing using direct imaging, a microwell that reproduces a confined space for spatial observation, a droplet assay that facilitates cell-cell interactions, and a 3D microphysiological system that reconstructs the vascular environment. Furthermore, it is suggested that future evaluation platforms pursue more human-like immune systems.

Understanding organotropism in cancer metastasis using microphysiological systems.

Cancer metastasis, the leading cause of cancer-related deaths, remains a complex challenge in medical science. Stephen Paget's "seed and soil theory" introduced the concept of organotropism, suggesting that metastatic success depends on specific organ microenvironments. Understanding organotropism not only offers potential for curbing metastasis but also novel treatment strategies. Microphysiological systems (MPS), especially organ-on-a-chip models, have emerged as transformative tools in this quest. These systems, blending microfluidics, biology, and engineering, grant precise control over cell interactions within organ-specific microenvironments. MPS enable real-time monitoring, morphological analysis, and protein quantification, enhancing our comprehension of cancer dynamics, including tumor migration, vascularization, and pre-metastatic niches. In this review, we explore innovative applications of MPS in investigating cancer metastasis, particularly focusing on organotropism. This interdisciplinary approach converges the field of science, engineering, and medicine, thereby illuminating a path toward groundbreaking discoveries in cancer research.

Patient-Derived Microphysiological Systems for Precision Medicine.

Patient-derived microphysiological systems (P-MPS) have emerged as powerful tools in precision medicine that provide valuable insight into individual patient characteristics. This review discusses the development of P-MPS as an integration of patient-derived samples, including patient-derived cells, organoids, and induced pluripotent stem cells, into well-defined MPSs. Emphasizing the necessity of P-MPS development, its significance as a nonclinical assessment approach that bridges the gap between traditional in vitro models and clinical outcomes is highlighted. Additionally, guidance is provided for engineering approaches to develop microfluidic devices and high-content analysis for P-MPSs, enabling high biological relevance and high-throughput experimentation. The practical implications of the P-MPS are further examined by exploring the clinically relevant outcomes obtained from various types of patient-derived samples. The construction and analysis of these diverse samples within the P-MPS have resulted in physiologically relevant data, paving the way for the development of personalized treatment strategies. This study describes the significance of the P-MPS in precision medicine, as well as its unique capacity to offer valuable insights into individual patient characteristics.

Atypical occurrence of anti-Ma2-associated encephalitis after breast cancer surgery and COVID-19.

In this report, we present a rare case of anti-Ma2-associated encephalitis concurrent with coronavirus disease 2019 (COVID-19) following breast cancer surgery. The patient exhibited minimal clinical symptoms of COVID-19 infection but developed seizures and altered mental status after surgery, leading to diagnosis of a classic paraneoplastic syndrome. This case highlights the possibility of paraneoplastic neurological syndrome even after cancer surgery and the need for careful consideration of post-acute infection syndromes when neurological symptoms occur following an infection.

Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

Integration of reconfigurable microchannels into aligned three-dimensional neural networks for spatially controllable neuromodulation.

Anisotropically organized neural networks are indispensable routes for functional connectivity in the brain, which remains largely unknown. While prevailing animal models require additional preparation and stimulation-applying devices and have exhibited limited capabilities regarding localized stimulation, no in vitro platform exists that permits spatiotemporal control of chemo-stimulation in anisotropic three-dimensional (3D) neural networks. We present the integration of microchannels seamlessly into a fibril-aligned 3D scaffold by adapting a single fabrication principle. We investigated the underlying physics of elastic microchannels' ridges and interfacial sol-gel transition of collagen under compression to determine a critical window of geometry and strain. We demonstrated the spatiotemporally resolved neuromodulation in an aligned 3D neural network by local deliveries of KCl and Ca2+ signal inhibitors, such as tetrodotoxin, nifedipine, and mibefradil, and also visualized Ca2+ signal propagation with a speed of ~3.7 µm/s. We anticipate that our technology will pave the way to elucidate functional connectivity and neurological diseases associated with transsynaptic propagation.

Mechanobiological Adaptation to Hyperosmolarity Enhances Barrier Function in Human Vascular Microphysiological System.

In infectious disease such as sepsis and COVID-19, blood vessel leakage treatment is critical to prevent fatal progression into multi-organ failure and ultimately death, but the existing effective therapeutic modalities that improve vascular barrier function are limited. Here, this study reports that osmolarity modulation can significantly improve vascular barrier function, even in an inflammatory condition. 3D human vascular microphysiological systems and automated permeability quantification processes for high-throughput analysis of vascular barrier function are utilized. Vascular barrier function is enhanced by >7-folds with 24-48 h hyperosmotic exposure (time window of emergency care; >500 mOsm L-1 ) but is disrupted after hypo-osmotic exposure (<200 mOsm L-1 ). By integrating genetic and protein level analysis, it is shown that hyperosmolarity upregulates vascular endothelial-cadherin, cortical F-actin, and cell-cell junction tension, indicating that hyperosmotic adaptation mechanically stabilizes the vascular barrier. Importantly, improved vascular barrier function following hyperosmotic exposure is maintained even after chronic exposure to proinflammatory cytokines and iso-osmotic recovery via Yes-associated protein signaling pathways. This study suggests that osmolarity modulation may be a unique therapeutic strategy to proactively prevent infectious disease progression into severe stages via vascular barrier function protection.

Modulating and monitoring the functionality of corticostriatal circuits using an electrostimulable microfluidic device.

The central nervous system is organized into different neural circuits, each with particular functions and properties. Studying neural circuits is essential to understanding brain function and neuronal diseases. Microfluidic systems are widely used for reconstructing and studying neural circuits but still need improvement to allow modulation and monitoring of the physiological properties of circuits. In this study, we constructed an improved microfluidic device that supports the electrical modulation of neural circuits and proper reassembly. We demonstrated that our microfluidic device provides a platform for electrically modulating and monitoring the physiological function of neural circuits with genetic indicators for synaptic functionality in corticostriatal (CStr) circuits. In particular, our microfluidic device measures activity-driven Ca2+ dynamics using Ca2+ indicators (synaptophysin-GCaMP6f and Fluo5F-AM), as well as activity-driven synaptic transmission and retrieval using vGlut-pHluorin. Overall, our findings indicate that the improved microfluidic platform described here is an invaluable tool for studying the physiological properties of specific neural circuits.

3D engineered tissue models for studying human-specific infectious viral diseases.

Viral infections cause damage to various organ systems by inducing organ-specific symptoms or systemic multi-organ damage. Depending on the infection route and virus type, infectious diseases are classified as respiratory, nervous, immune, digestive, or skin infections. Since these infectious diseases can widely spread in the community and their catastrophic effects are severe, identification of their causative agent and mechanisms underlying their pathogenesis is an urgent necessity. Although infection-associated mechanisms have been studied in two-dimensional (2D) cell culture models and animal models, they have shown limitations in organ-specific or human-associated pathogenesis, and the development of a human-organ-mimetic system is required. Recently, three-dimensional (3D) engineered tissue models, which can present human organ-like physiology in terms of the 3D structure, utilization of human-originated cells, recapitulation of physiological stimuli, and tight cell-cell interactions, were developed. Furthermore, recent studies have shown that these models can recapitulate infection-associated pathologies. In this review, we summarized the recent advances in 3D engineered tissue models that mimic organ-specific viral infections. First, we briefly described the limitations of the current 2D and animal models in recapitulating human-specific viral infection pathology. Next, we provided an overview of recently reported viral infection models, focusing particularly on organ-specific infection pathologies. Finally, a future perspective that must be pursued to reconstitute more human-specific infectious diseases is presented.

Brain organoid-on-a-chip: A next-generation human brain avatar for recapitulating human brain physiology and pathology.

Neurodegenerative diseases and neurodevelopmental disorders have become increasingly prevalent; however, the development of new pharmaceuticals to treat these diseases has lagged. Animal models have been extensively utilized to identify underlying mechanisms and to validate drug efficacies, but they possess inherent limitations including genetic heterogeneity with humans. To overcome these limitations, human cell-based in vitro brain models including brain-on-a-chip and brain organoids have been developed. Each technique has distinct advantages and disadvantages in terms of the mimicry of structure and microenvironment, but each technique could not fully mimic the structure and functional aspects of the brain tissue. Recently, a brain organoid-on-a-chip (BOoC) platform has emerged, which merges brain-on-a-chip and brain organoids. BOoC can potentially reflect the detailed structure of the brain tissue, vascular structure, and circulation of fluid. Hence, we summarize recent advances in BOoC as a human brain avatar and discuss future perspectives. BOoC platform can pave the way for mechanistic studies and the development of pharmaceuticals to treat brain diseases in future.

Hyperglycemic Neurovasculature-On-A-Chip to Study the Effect of SIRT1-Targeted Therapy for the Type 3 Diabetes "Alzheimer's Disease".

Diabetes mellitus (DM) is closely related to Alzheimer's disease (AD), but individual cellular changes and the possibilities of recovery through molecular level regulation have not been investigated. Here, a neurovasculature-on-a-chip (NV chip) model is presented in which the perfusable brain microvasculature is surrounded by the neurons. Under hyperglycemic conditions, the brain microvasculature shows disruption of barrier function and reduced expression of junctional markers. The neurons show Tau pathology and amyloid-beta (Aß) accumulation. Endothelial cells and neurons in the NV chip show sirtuin 1 (SIRT1) downregulation under hyperglycemic conditions, suggesting SIRT1 as a key regulator of hyperglycemia-induced AD. The recovery of glucose levels rescue SIRT1 expression, suggesting that this type of intervention may rescue the progression of hyperglycemia-mediated AD. Furthermore, the short hairpin RNA (shRNA)-, clustered regularly interspaced short palindromic repeats (CRISPR)-, and pharmaceutics-mediated regulation of SIRT1 regulate the pathophysiology of the brain endothelium and neurons at the functional and molecular levels.

Cleavage-Responsive Biofactory T Cells Suppress Infectious Diseases-Associated Hypercytokinemia.

Severe infectious diseases, such as coronavirus disease 2019 (COVID-19), can induce hypercytokinemia and multiple organ failure. In spite of the growing demand for peptide therapeutics against infectious diseases, current small molecule-based strategies still require frequent administration due to limited half-life and enzymatic digestion in blood. To overcome this challenge, a strategy to continuously express multi-level therapeutic peptide drugs on the surface of immune cells, is established. Here, chimeric T cells stably expressing therapeutic peptides are presented for treatment of severe infectious diseases. Using lentiviral system, T cells are engineered to express multi-level therapeutic peptides with matrix metallopeptidases- (MMP-) and tumor necrosis factor alpha converting enzyme- (TACE-) responsive cleavage sites on the surface. The enzymatic cleavage releases γ-carboxyglutamic acid of protein C (PC-Gla) domain and thrombin receptor agonist peptide (TRAP), which activate endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), respectively. These chimeric T cells prevent vascular damage in tissue-engineered blood vessel and suppress hypercytokinemia and lung tissue damages in vivo, demonstrating promise for use of engineered T cells against sepsis and other infectious-related diseases.

Nanodiamond as a Cytokine Sponge in Infectious Diseases.

Cytokine release syndrome (CRS) is a systemic inflammatory response resulting in overexpression of cytokines in serum and tissues, which leads to multiple-organ failure. Due to rapid aggravation of symptoms, timely intervention is paramount; however, current therapies are limited in their capacity to address CRS. Here, we find that the intravenous injection of highly purified detonation-synthesized nanodiamonds (DND) can act as a therapeutic agent for treating CRS by adsorbing inflammatory cytokines. Highly purified DNDs successfully inactivated various key cytokines in plasma from CRS patients with pneumonia, septic shock, and coronavirus disease 2019 pandemic (COVID-19). The intravenous injection of the DND samples in a mouse sepsis model by cecal ligation and puncture significantly improved survival rates and prevented tissue damage by reducing the circulating inflammatory cytokines. The results of this study suggest that the clinical application of highly purified DND can provide survival benefits for CRS patients by adsorbing inflammatory cytokines.

Vascularized Lung Cancer Model for Evaluating the Promoted Transport of Anticancer Drugs and Immune Cells in an Engineered Tumor Microenvironment.

The tumor microenvironment (TME) is the environment around the tumor, including blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM). Owing to its component interactions, the TME influences tumor growth and drug delivery in a highly complex manner. Although several vascularized cancer models are developed to mimic the TME in vitro, these models cannot comprehensively reflect blood vessel-tumor spheroid interactions. Here, a method for inducing controlled tumor angiogenesis by engineering the microenvironment is presented. The interstitial flow direction regulates the direction of capillary sprouting, showing that angiogenesis occurs in the opposite direction of flow, while the existence of lung fibroblasts affects the continuity and lumen formation of sprouted capillaries. The vascularized tumor model shows enhanced delivery of anticancer drugs and immune cells to the tumor spheroids because of the perfusable vascular networks. The possibility of capillary embolism using anticancer drug-conjugated liquid metal nanoparticles is investigated using the vascularized tumor model. This vascularized tumor platform can aid in the development of effective anticancer drugs and cancer immunotherapy.

Brain physiome: A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain.

In the last few decades, adverse reactions to pharmaceuticals have been evaluated using 2D in vitro models and animal models. However, with increasing computational power, and as the key drivers of cellular behavior have been identified, in silico models have emerged. These models are time-efficient and cost-effective, but the prediction of adverse reactions to unknown drugs using these models requires relevant experimental input. Accordingly, the physiome concept has emerged to bridge experimental datasets with in silico models. The brain physiome describes the systemic interactions of its components, which are organized into a multilevel hierarchy. Because of the limitations in obtaining experimental data corresponding to each physiome component from 2D in vitro models and animal models, 3D in vitro brain models, including brain organoids and brain-on-a-chip, have been developed. In this review, we present the concept of the brain physiome and its hierarchical organization, including cell- and tissue-level organizations. We also summarize recently developed 3D in vitro brain models and link them with the elements of the brain physiome as a guideline for dataset collection. The connection between in vitro 3D brain models and in silico modeling will lead to the establishment of cost-effective and time-efficient in silico models for the prediction of the safety of unknown drugs.

Three-Dimensional Axotomy and Regeneration on Open-Access Microfluidic Platform.

An increasing number of patients are suffering from central nervous system (CNS) injury, including spinal cord injury. However, no suitable treatment is available for such patients as yet. Various platforms have been utilized to recapitulate CNS injuries. However, animal models and in vitro two-dimensional (2D)-based cell culture platforms have limitations, such as genetic heterogeneity and loss of the neural-circuit ultrastructure. To overcome these limitations, we developed a method for performing axotomy on an open-access three-dimensional (3D) neuron-culture platform. In this platform, the 3D alignment of axons in the brain tissue was recapitulated. For direct access to the cultured axons, the bottom of the 3D neuron-culture device was disassembled, enabling exposure of the neuron-laden Matrigel to the outside. The mechanical damage to the axons was recapitulated by puncturing the neuron-laden Matrigel using a pin. Thus, precise axotomy of three-dimensionally aligned axons could be performed. Furthermore, it was possible to fill the punctuated area by re-injecting Matrigel. Consequently, neurites regenerated into re-injected Matrigel. Moreover, it was confirmed that astrocytes can be co-cultured on this open-access platform without interfering with the axon alignment. The proposed open-access platform is expected to be useful for developing treatment techniques for CNS injuries.

Effect of carbon nanomaterial dimension on the functional activity and degeneration of neurons.

Despite growing concerns regarding the threat of airborne nanoparticle-mediated brain degeneration, the underlying pathological mechanisms remain unclear. Carbon nanomaterials, the main components of airborne nanoparticles, have multi-dimensional structures. Therefore, the dimensional effect of carbon-based nanomaterials on the regulation of neural function in brain disorders requires additional clarification. Herein, we report the interaction between zero-to three-dimensional carbon nanostructures and the amyloid-beta protein, which can either activate or interrupt neuronal functions, depending on the dimension of the carbon nanostructures. The carbon nanomaterials induced significant cellular activation by short-term exposure, while prolonged exposure eventually caused neuronal cell death. Such dimension-dependent activation or degeneration was more evident in the higher-dimension carbon nanomaterials, as confirmed by the increases in neurotransmitter secretion and synapse-related protein levels to more than five times at 72 h of monitoring and calcium signaling in the neurons. The inclusion of amyloid-beta proteins ameliorated the cytotoxic effects of carbon nanomaterials in higher-dimensional carbon nanomaterials by regulating 333 genes. We found that the ɑ-synuclein gene is the key factor in carbon-induced abnormal neuronal function. Therefore, through biological analyses and in vitro feasibility studies, this new insight may contribute toward understanding the pathological mechanism and finding a new target for therapy in human brain pathologies.

Engineered neural circuits for modeling brain physiology and neuropathology.

The neural circuits of the central nervous system are the regulatory pathways for feeling, motion control, learning, and memory, and their dysfunction is closely related to various neurodegenerative diseases. Despite the growing demand for the unraveling of the physiology and functional connectivity of the neural circuits, their fundamental investigation is hampered because of the inability to access the components of neural circuits and the complex microenvironment. As an alternative approach, in vitro human neural circuits show principles of in vivo human neuronal circuit function. They allow access to the cellular compartment and permit real-time monitoring of neural circuits. In this review, we summarize recent advances in reconstituted in vitro neural circuits using engineering techniques. To this end, we provide an overview of the fabrication techniques and methods for stimulation and measurement of in vitro neural circuits. Subsequently, representative examples of in vitro neural circuits are reviewed with a particular focus on the recapitulation of structures and functions observed in vivo, and we summarize their application in the study of various brain diseases. We believe that the in vitro neural circuits can help neuroscience and the neuropharmacology. STATEMENT OF SIGNIFICANCE: Despite the growing demand to unravel the physiology and functional connectivity of the neural circuits, the studies on the in vivo neural circuits are frequently limited due to the poor accessibility. Furthermore, single neuron-based analysis has an inherent limitation in that it does not reflect the full spectrum of the neural circuit physiology. As an alternative approach, in vitro engineered neural circuit models have arisen because they can recapitulate the structural and functional characteristics of in vivo neural circuits. These in vitro neural circuits allow the mimicking of dysregulation of the neural circuits, including neurodegenerative diseases and traumatic brain injury. Emerging in vitro engineered neural circuits will provide a better understanding of the (patho-)physiology of neural circuits.