Image-based profiling and deep learning reveal morphological heterogeneity of colorectal cancer organoids.

Patient-derived organoids have proven to be a highly relevant model for evaluating of disease mechanisms and drug efficacies, as they closely recapitulate in vivo physiology. Colorectal cancer organoids, specifically, exhibit a diverse range of morphologies, which have been analyzed with image-based profiling. However, the relationship between morphological subtypes and functional parameters of the organoids remains underexplored. Here, we identified two distinct morphological subtypes ("cystic" and "solid") across 31360 bright field images using image-based profiling, which correlated differently with viability and apoptosis level of colorectal cancer organoids. Leveraging object detection neural networks, we were able to categorize single organoids achieving higher viability scores as "cystic" than "solid" subtype. Furthermore, a deep generative model was proposed to predict apoptosis intensity based on a apoptosis-featured dataset encompassing over 17000 bright field and matched fluorescent images. Notably, a significant correlation of 0.91 between the predicted value and ground truth was achived, underscoring the feasibility of this generative model as a potential means for assessing organoid functional parameters. The underlying cellular heterogeneity of the organoids, i.e., conserved colonic cell types and rare immune components, was also verified with scRNA sequencing, implying a compromised tumor microenvironment. Additionally, the "cystic" subtype was identified as a relapse phenotype featuring intestinal stem cell signatures, suggesting that this visually discernible relapse phenotype shows potential as a novel biomarker for colorectal cancer diagnosis and prognosis. In summary, our findings demonstrate that the morphological heterogeneity of colorectal cancer organoids explicitly recapitulate the association of phenotypic features and exogenous perturbations through the image-based profiling, providing new insights into disease mechanisms.

Vascularized organoid-on-a-chip: design, imaging, and analysis.

Vascularized organoid-on-a-chip (VOoC) models achieve substance exchange in deep layers of organoids and provide a more physiologically relevant system in vitro. Common designs for VOoC primarily involve two categories: self-assembly of endothelial cells (ECs) to form microvessels and pre-patterned vessel lumens, both of which include the hydrogel region for EC growth and allow for controlled fluid perfusion on the chip. Characterizing the vasculature of VOoC often relies on high-resolution microscopic imaging. However, the high scattering of turbid tissues can limit optical imaging depth. To overcome this limitation, tissue optical clearing (TOC) techniques have emerged, allowing for 3D visualization of VOoC in conjunction with optical imaging techniques. The acquisition of large-scale imaging data, coupled with high-resolution imaging in whole-mount preparations, necessitates the development of highly efficient analysis methods. In this review, we provide an overview of the chip designs and culturing strategies employed for VOoC, as well as the applicable optical imaging and TOC methods. Furthermore, we summarize the vascular analysis techniques employed in VOoC, including deep learning. Finally, we discuss the existing challenges in VOoC and vascular analysis methods and provide an outlook for future development.

Targeted Phosphoproteomics of Human Saliva Extracellular Vesicles via Multiple Reaction Monitoring Cubed (MRM3).

Oral squamous cell carcinoma (OSCC) has become a global health problem due to its increasing incidence and high mortality rate. Early intervention through monitoring of the diagnostic biomarker levels during OSCC treatment is critical. Extracellular vesicles (EVs) are emerging surrogates in intercellular communication through transporting biomolecule cargo and have recently been identified as a potential source of biomarkers such as phosphoproteins for many diseases. Here, we developed a multiple reaction monitoring cubed (MRM3) method coupled with a novel sample preparation strategy, extracellular vesicles to phosphoproteins (EVTOP), to quantify phosphoproteins using a minimal amount of saliva (50 µL) samples from OSCC patients with high specificity and sensitivity. Our results established differential patterns in the phosphopeptide content of healthy, presurgery, and postsurgery OSCC patient groups. Notably, we discovered significantly increased salivary phosphorylated alpha-amylase (AMY) in the postsurgery group compared to the presurgery group. We hereby present the first targeted MS method with extremely high sensitivity for measuring endogenous phosphoproteins in human saliva EVs.

Hierarchical Dendritic Photonic Crystal Beads for Efficient Isolation and Proteomic Analysis of Multiple Cell Types.

Cell types with different morphology, and function collaborate to maintain organ function. As such, analyzing proteomic differences and connections between different types of cells forms the foundation for establishing functional connectomes and developing in vitro organoid simulation experiments. However, the efficiency of cell type isolation from organs is limited by time, equipment, and cost. Here, hierarchical dendritic photonic crystal beads (HDPCBs) featuring high-density functional groups via the self-assembly of dendritic mesoporous structure SiO2 nanoparticles (DM-SiO2 ) and grafting dendrimers onto the surface of dendritic mesoporous photonic crystal beads (DMPCBs) is developed. This platform integrates multitype cell separation with in situ protein cleavage processes. Efficient simultaneous isolation of Kupffer cells and Liver Sinusoidal Endothelial cells (LSECs) from liver, with high specificity and convenient operation in a short separation time are demonstrated. The results reveal 2832 and 3442 unique proteins identified in Kupffer cells and LSECs using only 50 HDPCBs, respectively. 764 and 629 over-expressed proteins associated with the function of Kupffer cells and LSECs are found, respectively. The work offers a new method for efficiently isolating multiple cell types from tissues and downstream proteomic analysis, ultimately facilitating the identification of primary cell compositions and functions.

Direct Laser Writing Photonic Crystal Hydrogels with a Supramolecular Sacrificial Scaffold.

Photonic crystal hydrogels (PCHs), with smart stimulus-responsive abilities, have been widely exploited as colorimetric sensors for years. However, the current fabrication technologies are mostly applicable to produce PCHs with simple geometries at the sub-millimeter scale, limiting the introduction of structural design into PCH sensors as well as the accompanied advanced applications. This paper reports the microfabrication of three-dimensional (3D) PCHs with the help of supramolecular agarose PCH as a sacrificial scaffold by two-photon lithography (TPL). The supramolecular PCHs, formulated with SiO2 colloidal nanoparticles and agarose aqueous solutions, show bright structural color and are degradable upon short-time dimethyl sulfoxide treatment. Leveraging the supramolecular PCH as a sacrificial scaffold, PCHs with precise 3D geometries can be fabricated in an economical and efficient way. This work demonstrates the application of such a strategy in the creation of structural-designed PCH mechanical microsensors that have not been explored before.

[Applications and challenges of wearable electroencephalogram signals in depression recognition and personalized music intervention].

Rapid and accurate identification and effective non-drug intervention are the worldwide challenges in the field of depression. Electroencephalogram (EEG) signals contain rich quantitative markers of depression, but whole-brain EEG signals acquisition process is too complicated to be applied on a large-scale population. Based on the wearable frontal lobe EEG monitoring device developed by the authors' laboratory, this study discussed the application of wearable EEG signal in depression recognition and intervention. The technical principle of wearable EEG signals monitoring device and the commonly used wearable EEG devices were introduced. Key technologies for wearable EEG signals-based depression recognition and the existing technical limitations were reviewed and discussed. Finally, a closed-loop brain-computer music interface system for personalized depression intervention was proposed, and the technical challenges were further discussed. This review paper may contribute to the transformation of relevant theories and technologies from basic research to application, and further advance the process of depression screening and personalized intervention.

Imaging cellular forces with photonic crystals.

Current techniques for visualizing and quantifying cellular forces have limitations in live cell imaging, throughput, and multi-scale analysis, which impede progress in cell force research and its practical applications. We developed a photonic crystal cellular force microscopy (PCCFM) to image vertical cell forces over a wide field of view (1.3 mm ⨯ 1.0 mm, a 10 ⨯ objective image) at high speed (about 20 frames per second) without references. The photonic crystal hydrogel substrate (PCS) converts micro-nano deformations into perceivable color changes, enabling in situ visualization and quantification of tiny vertical cell forces with high throughput. It enabled long-term, cross-scale monitoring from subcellular focal adhesions to tissue-level cell sheets and aggregates.

The mechano-chemical circuit drives skin organoid self-organization.

Stem cells in organoids self-organize into tissue patterns with unknown mechanisms. Here, we use skin organoids to analyze this process. Cell behavior videos show that the morphological transformation from multiple spheroidal units with morphogenesis competence (CMU) to planar skin is characterized by two abrupt cell motility-increasing events before calming down. The self-organizing processes are controlled by a morphogenetic module composed of molecular sensors, modulators, and executers. Increasing dermal stiffness provides the initial driving force (driver) which activates Yap1 (sensor) in epidermal cysts. Notch signaling (modulator 1) in epidermal cyst tunes the threshold of Yap1 activation. Activated Yap1 induces Wnts and MMPs (epidermal executers) in basal cells to facilitate cellular flows, allowing epidermal cells to protrude out from the CMU. Dermal cell-expressed Rock (dermal executer) generates a stiff force bridge between two CMU and accelerates tissue mixing via activating Laminin and ß1-integrin. Thus, this self-organizing coalescence process is controlled by a mechano-chemical circuit. Beyond skin, self-organization in organoids may use similar mechano-chemical circuit structures.

Application of colloidal photonic crystals in study of organoids.

As alternative disease models, other than 2D cell lines and patient-derived xenografts, organoids have preferable in vivo physiological relevance. However, both endogenous and exogenous limitations impede the development and clinical translation of these organoids. Fortunately, colloidal photonic crystals (PCs), which benefit from favorable biocompatibility, brilliant optical manipulation, and facile chemical decoration, have been applied to the engineering of organoids and have achieved the desirable recapitulation of the ECM niche, well-defined geometrical onsets for initial culture, in situ multiphysiological parameter monitoring, single-cell biomechanical sensing, and high-throughput drug screening with versatile functional readouts. Herein, we review the latest progress in engineering organoids fabricated from colloidal PCs and provide inputs for future research.

A new approach of using organ-on-a-chip and fluid-structure interaction modeling to investigate biomechanical characteristics in tissue-engineered blood vessels.

The tissue-engineered blood vessel (TEBV) has been developed and used in cardiovascular disease modeling, preclinical drug screening, and for replacement of native diseased arteries. Increasing attention has been paid to biomechanical cues in TEBV and other tissue-engineered organs to better recapitulate the functional properties of the native organs. Currently, computational fluid dynamics models were employed to reveal the hydrodynamics in TEBV-on-a-chip. However, the biomechanical wall stress/strain conditions in the TEBV wall have never been investigated. In this paper, a straight cylindrical TEBV was placed into a polydimethylsiloxane-made microfluidic device to construct the TEBV-on-a-chip. The chip was then perfused with cell culture media flow driven by a peristaltic pump. A three-dimensional fluid-structure interaction (FSI) model was generated to simulate the biomechanical conditions in TEBV and mimic both the dynamic TEBV movement and pulsatile fluid flow. The material stiffness of the TEBV wall was determined by uniaxial tensile testing, while the viscosity of cell culture media was measured using a rheometer. Comparison analysis between the perfusion experiment and FSI model results showed that the average relative error in diameter expansion of TEBV from both approaches was 10.0% in one period. For fluid flow, the average flow velocity over a period was 2.52 cm/s from the FSI model, 10.5% higher than the average velocity of the observed cell clusters (2.28 mm/s) in the experiment. These results demonstrated the facility to apply the FSI modeling approach in TEBV to obtain more comprehensive biomechanical results for investigating mechanical mechanisms of cardiovascular disease development.

On-chip clearing for live imaging of 3D cell cultures.

Three-dimensional (3D) cell cultures provide an important model for various biological studies by bridging the gap between two-dimensional (2D) cell cultures and animal tissues. Microfluidics has recently provided controllable platforms for handling and analyzing 3D cell cultures. However, on-chip imaging of 3D cell cultures within microfluidic devices is hindered by the inherent high scattering of 3D tissues. Tissue optical clearing techniques have been used to address this concern but remain limited to fixed samples. As such, there is still a need for an on-chip clearing method for imaging live 3D cell cultures. Here, to achieve on-chip clearing for live imaging of 3D cell cultures, we conceived a simple microfluidic device by integrating a U-shaped concave for culture, parallel channels with micropillars, and differentiated surface treatment to enable on-chip 3D cell culture, clearing, and live imaging with minimal disturbance. The on-chip tissue clearing increased the imaging performance of live 3D spheroids with no influence on cell viability or spheroid proliferation and demonstrated robust compatibility with several commonly used cell probes. It allowed dynamic tracking of lysosomes in live tumor spheroids and enabled quantitative analysis of their motility in the deeper layer. Our proposed method of on-chip clearing for live imaging of 3D cell cultures provides an alternative for dynamic monitoring of deep tissue on a microfluidic device and has the potential to be used in 3D culture-based assays for high-throughput applications.

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation.

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with TiIV ions and a membrane-penetrating peptide, octa-arginine R8 + , which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to TiIV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few µL of plasma and over 1200 phosphopeptides with 100 µL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.

Large-scale high-throughput 3D culture, imaging, and analysis of cell spheroids using microchip-enhanced light-sheet microscopy.

Light sheet microscopy combined with a microchip is an emerging tool in biomedical research that notably improves efficiency. However, microchip-enhanced light-sheet microscopy is limited by noticeable aberrations induced by the complex refractive indices in the chip. Herein, we report a droplet microchip that is specifically engineered to be capable of large-scale culture of 3D spheroids (over 600 samples per chip) and has a polymer index matched to water (difference <1%). When combined with a lab-built open-top light-sheet microscope, this microchip-enhanced microscopy technique allows 3D time-lapse imaging of the cultivated spheroids with ∼2.5-µm single-cell resolution and a high throughput of ∼120 spheroids per minute. This technique was validated by a comparative study on the proliferation and apoptosis rates of hundreds of spheroids with or without treatment with the apoptosis-inducing drug Staurosporine.

4D Printing of Butterfly Scale-Inspired Structures for Wide-Angle Directional Liquid Transport.

Unidirectional liquid transport has been extensively explored for water/fog harvesting, electrochemical sensing, and desalination. However, current research mainly focuses on linear liquid transport (transport angle α = 0°), which exhibits hindered lateral liquid spreading and low unidirectional transport efficiency. Inspired by the wide-angle (0° < α < 180°) liquid transport on butterfly wings, this work successfully achieves linear (α = 0°), wide-angle, and even ultra-wide-angle (α = 180°) liquid transport by four-dimensional (4D) printing of butterfly scale-inspired re-entrant structures. These asymmetric re-entrant structures can achieve unidirectional liquid transport, and their layout can control the Laplace pressure in the forward (structure-tilting) and lateral directions to adjust the transport angle. Specifically, high transport efficiency and programmable forward/lateral transport paths are simultaneously achieved by the ultra-wide-angle transport, where liquid fills the lateral path before being transported forward. Moreover, the ultra-wide-angle transport is also validated in 3D space, which provides an innovative platform for advanced biochemical microreaction, large-area evaporation, and self-propelled oil-water separation.

Epidermis-on-a-chip system to develop skin barrier and melanin mimicking model.

In vitro skin models are rapidly developing and have been widely used in various fields as an alternative to traditional animal experiments. However, most traditional static skin models are constructed on Transwell plates without a dynamic three-dimensional (3D) culture microenvironment. Compared with native human and animal skin, such in vitro skin models are not completely biomimetic, especially regarding their thickness and permeability. Therefore, there is an urgent need to develop an automated biomimetic human microphysiological system (MPS), which can be used to construct in vitro skin models and improve bionic performance. In this work, we describe the development of a triple-well microfluidic-based epidermis-on-a-chip (EoC) system, possessing epidermis barrier and melanin-mimicking functions, as well as being semi-solid specimen friendly. The special design of our EoC system allows pasty and semi-solid substances to be effectively utilized in testing, as well as allowing for long-term culturing and imaging. The epidermis in this EoC system is well-differentiated, including basal, spinous, granular, and cornified layers with appropriate epidermis marker (e.g. keratin-10, keratin-14, involucrin, loricrin, and filaggrin) expression levels in corresponding layers. We further demonstrate that this organotypic chip can prevent permeation of over 99.83% of cascade blue (a 607 Da fluorescent molecule), and prednisone acetate (PA) was applied to test percutaneous penetration in the EoC. Finally, we tested the whitening effect of a cosmetic on the proposed EoC, thus demonstrating its efficacy. In summary, we developed a biomimetic EoC system for epidermis recreation, which could potentially serve as a useful tool for skin irritation, permeability, cosmetic evaluation, and drug safety tests.

Emerging strategies of engineering retinal organoids and organoid-on-a-chip in modeling intraocular drug delivery: Current progress and future perspectives.

Retinal diseases are a rising concern as major causes of blindness in an aging society; therapeutic options are limited, and the precise pathogenesis of these diseases remains largely unknown. Intraocular drug delivery and nanomedicines offering targeted, sustained, and controllable delivery are the most challenging and popular topics in ocular drug development and toxicological evaluation. Retinal organoids (ROs) and organoid-on-a-chip (ROoC) are both emerging as promising in-vitro models to faithfully recapitulate human eyes for retinal research in the replacement of experimental animals and primary cells. In this study, we review the generation and application of ROs resembling the human retina in cell subtypes and laminated structures and introduce the emerging engineered ROoC as a technological opportunity to address critical issues. On-chip vascularization, perfusion, and close inter-tissue interactions recreate physiological environments in vitro, whilst integrating with biosensors facilitates real-time analysis and monitoring during organogenesis of the retina representing engineering efforts in ROoC models. We also emphasize that ROs and ROoCs hold the potential for applications in modeling intraocular drug delivery in vitro and developing next-generation retinal drug delivery strategies.

Advances in In Vitro Models of Neuromuscular Junction: Focusing on Organ-on-a-Chip, Organoids, and Biohybrid Robotics.

The neuromuscular junction (NMJ) is a peripheral synaptic connection between presynaptic motor neurons and postsynaptic skeletal muscle fibers that enables muscle contraction and voluntary motor movement. Many traumatic, neurodegenerative, and neuroimmunological diseases are classically believed to mainly affect either the neuronal or the muscle side of the NMJ, and treatment options are lacking. Recent advances in novel techniques have helped develop in vitro physiological and pathophysiological models of the NMJ as well as enable precise control and evaluation of its functions. This paper reviews the recent developments in in vitro NMJ models with 2D or 3D cultures, from organ-on-a-chip and organoids to biohybrid robotics. Related derivative techniques are introduced for functional analysis of the NMJ, such as the patch-clamp technique, microelectrode arrays, calcium imaging, and stimulus methods, particularly optogenetic-mediated light stimulation, microelectrode-mediated electrical stimulation, and biochemical stimulation. Finally, the applications of the in vitro NMJ models as disease models or for drug screening related to suitable neuromuscular diseases are summarized and their future development trends and challenges are discussed.

OOCDB: A Comprehensive, Systematic, and Real-time Organs-on-a-chip Database.

Organs-on-a-chip is a microfluidic microphysiological system that uses microfluidic technology to analyze the structure and function of living human cells at the tissue and organ levels in vitro. Organs-on-a-chip technology, as opposed to traditional two-dimensional cell culture and animal models, can more closely simulate pathologic and toxicologic interactions between different organs or tissues and reflect the collaborative response of multiple organs to drugs. Despite the fact that many organs-on-a-chip-related data have been published, none of the current databases have all of the following functions: searching, downloading, as well as analyzing data and results from the literature on organs-on-a-chip. Therefore, we created an organs-on-a-chip database (OOCDB) as a platform to integrate information about organs-on-a-chip from various sources, including literature, patents, raw data from microarray and transcriptome sequencing, several open-access datasets of organs-on-a-chip and organoids, and data generated in our laboratory. OOCDB contains dozens of sub-databases and analysis tools, and each sub-database contains various data associated with organs-on-a-chip, with the goal of providing researchers with a comprehensive, systematic, and convenient search engine. Furthermore, it offers a variety of other functions, such as mathematical modeling, three-dimensional modeling, and citation mapping, to meet the needs of researchers and promote the development of organs-on-a-chip. The OOCDB is available at http://www.organchip.cn.

Organoids revealed: morphological analysis of the profound next generation in-vitro model with artificial intelligence.

In modern terminology, "organoids" refer to cells that grow in a specific three-dimensional (3D) environment in vitro, sharing similar structures with their source organs or tissues. Observing the morphology or growth characteristics of organoids through a microscope is a commonly used method of organoid analysis. However, it is difficult, time-consuming, and inaccurate to screen and analyze organoids only manually, a problem which cannot be easily solved with traditional technology. Artificial intelligence (AI) technology has proven to be effective in many biological and medical research fields, especially in the analysis of single-cell or hematoxylin/eosin stained tissue slices. When used to analyze organoids, AI should also provide more efficient, quantitative, accurate, and fast solutions. In this review, we will first briefly outline the application areas of organoids and then discuss the shortcomings of traditional organoid measurement and analysis methods. Secondly, we will summarize the development from machine learning to deep learning and the advantages of the latter, and then describe how to utilize a convolutional neural network to solve the challenges in organoid observation and analysis. Finally, we will discuss the limitations of current AI used in organoid research, as well as opportunities and future research directions.

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes.

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.