Photoresist Development for 3D Printing of Conductive Microstructures via Two-Photon Polymerization.

The advancement of electronic devices necessitates the development of three-dimensional (3D) high-precision conductive microstructures, which have extensive applications in bio-electronic interfaces, soft robots, and electronic skins. Two-photon polymerization (TPP) based 3D printing is a critical technique that offers unparalleled fabrication resolution in 3D space for intricate conductive structures. While substantial progress has been made in this field, this review summarizes recent advances in the 3D printing of conductive microstructures via TPP, mainly focusing on the essential criteria of photoresist resins suitable for TPP. Further preparation strategies of these photoresists and methods for constructing 3D conductive microstructures via TPP are discussed. The application prospects of 3D conductive microstructures in various fields are discussed, highlighting the imperative to advance their additive manufacturing technology. Finally, strategic recommendations are offered to enhance the construction of 3D conductive microstructures using TPP, addressing prevailing challenges and fostering significant advancements in manufacturing technology.

Artificial Meshed Vessel-Induced Dimensional Breaking Growth of Human Brain Organoids and Multiregional Assembloids.

Brain organoids are widely used to model brain development and diseases. However, a major challenge in their application is the insufficient supply of oxygen and nutrients to the core region, restricting the size and maturation of the organoids. In order to vascularize brain organoids and enhance the nutritional supply to their core areas, two-photon polymerization (TPP) 3D printing is employed to fabricate high-resolution meshed vessels in this study. These vessels made of photoresist with densely distributed micropores with a diameter of 20 µm on the sidewall, are cocultured with brain organoids to facilitate the diffusion of culture medium into the organoids. The vascularized organoids exhibit dimensional breaking growth and enhanced proliferation, reduced hypoxia and apoptosis, suggesting that the 3D-printed meshed vessels partially mimic vascular function to promote the culture of organoids. Furthermore, cortical, striatal and medial ganglionic eminence (MGE) organoids are respectively differentiated to generate Cortico-Striatal-MGE assembloids by 3D-printed vessels. The enhanced migration, projection and excitatory signaling transduction are observed between different brain regional organoids in the assembloids. This study presents an approach using TPP 3D printing to construct vascularized brain organoids and assembloids for enhancing the development and assembly, offering a research model and platform for neurological diseases.

Label-Free Multiple Reaction Monitoring-Mass Spectrometry for Quantifying Phosphopeptides from Extracellular Vesicles.

Increasing efforts have been made to develop proteins in circulating extracellular vesicles (EVs) as potential disease markers. It is in particular intriguing to measure post-translational modifications (PTMs) such as phosphorylation, preserved and stable in EVs. To facilitate the quantitative measurement of EV protein phosphorylation for potential clinical use, a label-free (LF) multiple reaction monitoring (MRM) strategy is introduced by utilizing a synthetic phosphopeptide set (phos-iRT) as the internal standards and a local normalization method. The quantitation method was investigated in terms of its linear dynamic range, sensitivity, accuracy, precision, and matrix effect, with a dynamic range spanning from 10 to 1000 ng/mL and an accuracy ranging from 82.4 to 116.8% for EV samples. Then, the LF-MRM-based local normalization method was utilized to evaluate and optimize our recently developed EVTOP method for the enrichment of phosphopeptides from EVs. Finally, we applied the optimized EV enrichment approach and the LF-MRM-based local normalization method to quantify phosphopeptides in urine EVs from patients with prostate cancer (PCa) and healthy individuals, showcasing the strategy's superiority in quantifying phosphopeptides without isotopic internal standards and validating that the method is generally applicable in MRM-based EV phosphopeptide quantification.

Visualizing and quantifying dynamic cellular forces with photonic crystal hydrogels.

Cellular forces play a crucial role in numerous biological processes, including tissue development, morphogenesis, and disease progression. However, existing methods for detecting cellular forces, such as traction force microscopy and atomic force microscopy, often face limitations in terms of high throughput, real-time monitoring, and applicability to complex biological systems. In this study, we utilized a novel Photonic Crystal Cellular Force Microscopy (PCCFM) system to visualize and quantify dynamic cellular forces. This system consists of a conventional optical microscope and a photonic crystal substrate formed by the periodic arrangement of silica nanoparticles within polyacrylamide hydrogels. Taking MDCK cells and BMSCs as examples, we found that PCCFM can capture dynamic cellular forces with high spatial and temporal resolution during the cell adhesion, spread, proliferation, and osteogenic differentiation. The application of this technique revealed distinct force patterns in different cellular stages, offering insights into the interplay between cellular forces and morphological changes. By investigating the migration of cells from MDCK cyst fragments, we could gain significant insights into tumour cell migration behaviours. The real-time, high-throughput analysis of cellular biomechanics from the PCCFM system offers valuable information on the mechanisms of tumour metastasis, potentially guiding therapeutic development and improving disease treatment strategies.

Magnetic Torque-Driven All-Terrain Microrobots.

All-terrain microrobots possess significant potential in modern medical applications due to their superior maneuverability in complex terrains and confined spaces. However, conventional microrobots often struggle with adaptability and operational difficulties in variable environments. This study introduces a magnetic torque-driven all-terrain multiped microrobot (MTMR) to address these challenges. By coupling the structure's multiple symmetries with different uniform magnetic fields, such as rotating and oscillating fields, the MTMR demonstrates various locomotion modes, including rolling, tumbling, walking, jumping, and their combinations. Experimental results indicate that the robot can navigate diverse terrains, including flat surfaces, steep slopes (up to 75°), and gaps over twice its body height. Additionally, the MTMR performs well in confined spaces, capable of passing through slits (0.1 body length) and low tunnels (0.25 body length). The robot shows potential for clinical applications like minimally invasive hemostasis in internal bleeding and thrombus removal from blood vessels through accurate cargo manipulation capability. Moreover, the MTMR can carry temperature sensors to monitor environmental temperature changes in real time while simultaneously manipulating objects, displaying its potential for in-situ sensing and parallel task implementation. This all-terrain microrobot technology demonstrates notable adaptability and versatility, providing a solid foundation for practical applications in interventional medicine.

Bioinspired Capillary Transistors.

Inspired by the unidirectional liquid spreading on Nepenthes peristome, Araucaria leaf, butterfly wings, etc., various microfluidic devices have been developed for water collection, irrigation, physical/chemical reaction, and oil-water separation. Despite extensive progress, most natural and artificial structures fail to enhance the Laplace pressure difference or capillary force, thus suffering from a low unidirectional capillary height (<30 mm). In this work, asymmetric re-entrant structures with long overhangs and connected forward/lateral microchannels are fabricated by 3D printing, resulting in a significantly increased unidirectional capillary height of 102.3 mm for water, which approximately corresponds to the theoretical limit. The overhangs can partially overlap the forward microchannels of the front structures without direct contact, thus enhancing the Laplace pressure difference and capillary force simultaneously. Based on asymmetric and symmetric re-entrant structures, capillary transistors are proposed and realized to programmably adjust the capillary direction, height, and width, which are envisioned to function as switches/valves and amplifiers/attenuators for highly efficient liquid patterning, desalination, and biochemical microreaction in 3D space.

3D Printing Photonic Crystals: A Review.

Living organisms in nature possess diverse and vibrant structural colors generated from their intrinsic surface micro/nanostructures. These intricate micro/nanostructures can be harnessed to develop a new generation of colorful materials for various fields such as photonics, information storage, display, and sensing. Recent advancements in the fabrication of photonic crystals have enabled the preparation of structurally colored materials with customized geometries using 3D printing technologies. Here, a comprehensive review of the historical development of fabrication methods for photonic crystals is provided. Diverse 3D printing approaches along with the underlying mechanisms, as well as the regulation methods adopted to generate photonic crystals with structural color, are discussed. This review aims to offer the readers an overview of the state-of-the-art 3D printing techniques for photonic crystals, present a guide and considerations to fabricate photonic crystals leveraging different 3D printing methods.

The mechano-chemical circuit in fibroblasts and dendritic cells drives basal cell proliferation in psoriasis.

Psoriasis is an intractable immune-mediated disorder that disrupts the skin barrier. While studies have dissected the mechanism by which immune cells directly regulate epidermal cell proliferation, the involvement of dermal fibroblasts in the progression of psoriasis remains unclear. Here, we identified that signals from dendritic cells (DCs) that migrate to the dermal-epidermal junction region enhance dermal stiffness by increasing extracellular matrix (ECM) expression, which further promotes basal epidermal cell hyperproliferation. We analyzed cell-cell interactions and observed stronger interactions between DCs and fibroblasts than between DCs and epidermal cells. Using single-cell RNA (scRNA) sequencing, spatial transcriptomics, immunostaining, and stiffness measurement, we found that DC-secreted LGALS9 can be received by CD44+ dermal fibroblasts, leading to increased ECM expression that creates a stiffer dermal environment. By employing mouse psoriasis and skin organoid models, we discovered a mechano-chemical signaling pathway that originates from DCs, extends to dermal fibroblasts, and ultimately enhances basal cell proliferation in psoriatic skin.

Beyond Frequency Bands: Complementary-Ensemble-Empirical-Mode-Decomposition-Enhanced Microstate Sequence Non-Randomness Analysis for Aiding Diagnosis and Cognitive Prediction of Dementia.

Exploring the spatiotemporal dynamic patterns of multi-channel electroencephalography (EEG) is crucial for interpreting dementia and related cognitive decline. Spatiotemporal patterns of EEG can be described through microstate analysis, which provides a discrete approximation of the continuous electric field patterns generated by the brain cortex. Here, we propose a novel microstate spatiotemporal dynamic indicator, termed the microstate sequence non-randomness index (MSNRI). The essence of the method lies in initially generating a sequence of microstate transition patterns through state space compression of EEG data using microstate analysis. Following this, we assess the non-randomness of these microstate patterns using information-based similarity analysis. The results suggest that this MSNRI metric is a potential marker for distinguishing between health control (HC) and frontotemporal dementia (FTD) (HC vs. FTD: 6.958 vs. 5.756, p < 0.01), as well as between HC and populations with Alzheimer's disease (AD) (HC vs. AD: 6.958 vs. 5.462, p < 0.001). Healthy individuals exhibit more complex macroscopic structures and non-random spatiotemporal patterns of microstates, whereas dementia disorders lead to more random spatiotemporal patterns. Additionally, we extend the proposed method by integrating the Complementary Ensemble Empirical Mode Decomposition (CEEMD) method to explore spatiotemporal dynamic patterns of microstates at specific frequency scales. Moreover, we assessed the effectiveness of this innovative method in predicting cognitive scores. The results demonstrate that the incorporation of CEEMD-enhanced microstate dynamic indicators significantly improved the prediction accuracy of Mini-Mental State Examination (MMSE) scores (R2 = 0.940). The CEEMD-enhanced MSNRI method not only aids in the exploration of large-scale neural changes in populations with dementia but also offers a robust tool for characterizing the dynamics of EEG microstate transitions and their impact on cognitive function.

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.

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.

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.