Cell maturation: Hallmarks, triggers, and manipulation.

How cells become specialized, or "mature," is important for cell and developmental biology. While maturity is usually deemed a terminal fate, it may be more helpful to consider maturation not as a switch but as a dynamic continuum of adaptive phenotypic states set by genetic and environment programing. The hallmarks of maturity comprise changes in anatomy (form, gene circuitry, and interconnectivity) and physiology (function, rhythms, and proliferation) that confer adaptive behavior. We discuss efforts to harness their chemical (nutrients, oxygen, and growth factors) and physical (mechanical, spatial, and electrical) triggers in vitro and in vivo and how maturation strategies may support disease research and regenerative medicine.

A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Dynamic in situ detection in iRhizo-Chip reveals diurnal fluctuations of Bacillus subtilis in the rhizosphere.

Effective colonization by microbe in the rhizosphere is critical for establishing a beneficial symbiotic relationship with the host plant. Bacillus subtilis, a soil-dwelling bacterium that is commonly found in association with plants and their rhizosphere, has garnered interest for its potential to enhance plant growth, suppress pathogens, and contribute to sustainable agricultural practices. However, research on the dynamic distribution of B. subtilis within the rhizosphere and its interaction mechanisms with plant roots remains insufficient due to limitations in existing in situ detection methodologies. To achieve dynamic in situ detection of the rhizosphere environment, we established iRhizo-Chip, a microfluidics-based platform. Using this device to investigate microbial behavior within the rhizosphere, we found obvious diurnal fluctuations in the growth of B. subtilis in the rhizosphere. Temporal dynamic analysis of rhizosphere dissolved oxygen (DO), pH, dissolved organic carbon, and reactive oxygen species showed that diurnal fluctuations in the growth of B. subtilis are potentially related to a variety of environmental factors. Spatial dynamic analysis also showed that the spatial distribution changes of B. subtilis and DO and pH were similar. Subsequently, through in vitro control experiments, we proved that rhizosphere DO and pH are the main driving forces for diurnal fluctuations in the growth of B. subtilis. Our results show that the growth of B. subtilis is driven by rhizosphere DO and pH, resulting in diurnal fluctuations, and iRhizo-Chip is a valuable tool for studying plant rhizosphere dynamics.

A vesicular Warburg effect: Aerobic glycolysis occurs on axonal vesicles for local NAD+ recycling and transport.

In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.

SPA, a Stigma-style-transmitting tract Physical microenvironment Assay for investigating mechano-signaling in pollen tubes.

Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it's naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.

Mimicking kidney flow shear efficiently induces aggregation of LECT2, a protein involved in renal amyloidosis.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma.

Extracellular vesicles (EVs) have recently received increasing attention as essential mediators of communication between tumor cells and their microenvironments. Tumor-associated macrophages (TAMs) play a proangiogenic role in various tumors, especially head and neck squamous cell carcinoma (HNSCC), and angiogenesis is closely related to tumor growth and metastasis. This research focused on exploring the mechanisms by which EVs derived from TAMs modulate tumor angiogenesis in HNSCC. Our results indicated that TAMs infiltration correlated positively with microvascular density in HNSCC. Then we collected and identified EVs from TAMs. In the microfluidic chip, TAMs derived EVs significantly enhanced the angiogenic potential of pHUVECs and successfully induced the formation of perfusable blood vessels. qPCR and immunofluorescence analyses revealed that EVs from TAMs transferred miR-21-5p to endothelial cells (ECs). And targeting miR-21-5p of TAMs could effectively inhibit TAM-EVs induced angiogenesis. Western blot and tube formation assays showed that miR-21-5p from TAM-EVs downregulated LATS1 and VHL levels but upregulated YAP1 and HIF-1α levels, and the inhibitors of YAP1 and HIF-1α could both reduce the miR-21-5p enhanced angiogenesis in HUVECs. The in vivo experiments further proved that miR-21-5p carried by TAM-EVs promoted the process of tumor angiogenesis via YAP1/HIF-1α axis in HNSCC. Conclusively, TAM-derived EVs transferred miR-21-5p to ECs to target the mRNA of LATS1 and VHL, which inhibited YAP1 phosphorylation and subsequently enhanced YAP1-mediated HIF-1α transcription and reduced VHL-mediated HIF-1α ubiquitination, contributing to angiogenesis in HNSCC. These findings present a novel regulatory mechanism of tumor angiogenesis, and miR-21-5p/YAP1/HIF-1α might be a potential therapeutic target for HNSCC.

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier-penetrant nanoparticles.

The blood­brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood­brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood­brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood­brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

A Scalable Microfluidic Platform for Nanoparticle Formulation: For Exploratory- and Industrial-Level Scales.

Nanoparticle synthesis on microfluidic platforms provides excellent reproducibility and control over bulk synthesis. While there have been plenty of platforms for producing nanoparticles (NPs) with controlled physicochemical properties, such platforms often operate in a narrow range of predefined flow rates. The flow rate limitation restricts either up-scalability for industrial production or down-scalability for exploratory research use. Here, we present a universal flow rate platform that operates over a wide range of flow rates (0.1-75 mL/min) for small-scale exploratory research and industrial-level synthesis of NPs without compromising the mixing capabilities. The wide range of flow rate is obtained by using a coaxial flow with a triangular microstructure to create a vortex regardless of the flow regime (Reynolds number). The chip synthesizes several types of NPs for gene and protein delivery, including polyplex, lipid NPs, and solid polymer NPs via self-assembly and precipitation, and successfully expresses GFP plasmid DNA in human T cells.

Microfluidics-Derived Microparticles with Prebiotics and Probiotics for Enhanced In Situ Colonization and Immunoregulation of Colitis.

Oral administration of probiotics orchestrates the balance between intestinal microbes and the immune response. However, effective delivery and in situ colonization are limited by the harsh environment of the gastrointestinal tract. Herein, we provide a microfluidics-derived encapsulation strategy to address this problem. A novel synergistic delivery system composed of EcN Nissle 1917 and prebiotics, including alginate sodium and inulin gel, for treating inflammatory bowel disease and colitis-associated colorectal cancer is proposed. We demonstrated that EcN@AN microparticles yielded promising gastrointestinal resistance for on-demand probiotic delivery and colon-retentive capability. EcN@AN microparticles efficiently ameliorated intestinal inflammation and modulated the gut microbiome in experimental colitis. Moreover, the prebiotic composition of EcN@AN enhanced the fermentation of relative short-chain fatty acid metabolites, a kind of postbiotics, to exert anti-inflammatory and tumor-suppressive effects in murine models. This microfluidcis-based approach for the coordinated delivery of probiotics and prebiotics may have broad implications for gastrointestinal bacteriotherapy applications.

Cavitation-on-a-Chip Enabled Size-Specific Liposomal Drugs for Selective Pharmacokinetics and Pharmacodynamics.

The size of liposomal drugs has been demonstrated to strongly correlate with their pharmacokinetics and pharmacodynamics. While the microfluidic method successfully achieves the production of liposomes with well-controlled sizes across various buffer/lipid flow rate ratio (FRR) settings, any adjustments to the FRR inevitably influence the concentration, encapsulation efficiency (EE), and stability of liposomal drugs. Here we describe a controllable cavitation-on-a-chip (CCC) strategy that facilitates the precise regulation of liposomal drug size at any desired FRR. The CCC-enabled size-specific liposomes exhibited striking differences in uptake and biodistribution behaviors, thereby demonstrating distinct antitumor efficacy in both tumor-bearing animal and melanoma patient-derived organoid (PDO) models. Intriguingly, as the liposome size decreased to approximately 80 nm, the preferential accumulation of liposomal drugs in the liver transitioned to a predominant enrichment in the kidneys. These findings underscore the considerable potential of our CCC approach in influencing the pharmacokinetics and pharmacodynamics of liposomal nanomedicines.

Dynamics of axonal ß-actin mRNA in live hippocampal neurons.

Localization of mRNA facilitates spatiotemporally controlled protein expression in neurons. In axons, mRNA transport followed by local protein synthesis plays a critical role in axonal growth and guidance. However, it is not yet clearly understood how mRNA is transported to axonal subcellular sites and what regulates axonal mRNA localization. Using a transgenic mouse model in which endogenous ß-actin mRNA is fluorescently labeled, we investigated ß-actin mRNA movement in axons of hippocampal neurons. We cultured neurons in microfluidic devices to separate axons from dendrites and performed single-particle tracking of axonal ß-actin mRNA. Compared with dendritic ß-actin mRNA, axonal ß-actin mRNA showed less directed motion and exhibited mostly subdiffusive motion, especially near filopodia and boutons in mature dissociated hippocampal neurons. We found that axonal ß-actin mRNA was likely to colocalize with actin patches (APs), regions that have a high density of filamentous actin (F-actin) and are known to have a role in branch initiation. Moreover, simultaneous imaging of F-actin and axonal ß-actin mRNA in live neurons revealed that moving ß-actin mRNA tended to be docked in the APs. Our findings reveal that axonal ß-actin mRNA localization is facilitated by actin networks and suggest that localized ß-actin mRNA plays a potential role in axon branch formation.

Molecular Profiling of Circulating Tumour Cells and Circulating Tumour DNA: Complementary Insights from a Single Blood Sample Utilising the Parsortix® System.

The study of molecular drivers of cancer is an area of rapid growth and has led to the development of targeted treatments, significantly improving patient outcomes in many cancer types. The identification of actionable mutations informing targeted treatment strategies are now considered essential to the management of cancer. Traditionally, this information has been obtained through biomarker assessment of a tissue biopsy which is costly and can be associated with clinical complications and adverse events. In the last decade, blood-based liquid biopsy has emerged as a minimally invasive, fast, and cost-effective alternative, which is better suited to the requirement for longitudinal monitoring. Liquid biopsies allow for the concurrent study of multiple analytes, such as circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), from a single blood sample. Although ctDNA assays are commercially more advanced, there is an increasing awareness of the clinical significance of the transcriptome and proteome which can be analysed using CTCs. Herein, we review the literature in which the microfluidic, label-free Parsortix® system is utilised for CTC capture, harvest and analysis, alongside the analysis of ctDNA from a single blood sample. This detailed summary of the literature demonstrates how these two analytes can provide complementary disease information.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Enhanced responses to inflammatory cytokine interleukin-6 in micropatterned networks of cultured cortical neurons.

Cortical neurons in dissociated cultures are an indispensable model system for pharmacological research that provides insights into chemical responses in well-defined environments. However, cortical neurons plated on homogeneous substrates develop an unstructured network that exhibits excessively synchronized activity, which occasionally masks the consequences induced by external substances. Here, we show that hyperactivity and excessive synchrony in cultured cortical networks can be effectively suppressed by growing neurons in microfluidic devices. These devices feature a hierarchically modular design that resembles the in vivo network. We focused on interleukin-6, a pro-inflammatory cytokine, and assessed its acute and chronic effects. Fluorescence calcium imaging of spontaneous neural activity for up to 20 days of culture showed detectable modulation of collective activity events and neural correlation in micropatterned neurons, which was not apparent in neurons cultured on homogeneous substrates. Our results indicate that engineered neuronal networks provide a unique platform for detecting and understanding the fundamental effects of biochemical compounds on neuronal networks.

A machine learning based method for tracking of simultaneously imaged neural activity and body posture of freely moving maggot.

To understand neural basis of animal behavior, it is necessary to monitor neural activity and behavior in freely moving animal before building relationship between them. Here we use light sheet fluorescence microscope (LSFM) combined with microfluidic chip to simultaneously capture neural activity and body movement in small freely behaving Drosophila larva. We develop a transfer learning based method to simultaneously track the continuously changing body posture and activity of neurons that move together using a sub-region tracking network with a precise landmark estimation network for the inference of target landmark trajectory. Based on the tracking of each labelled neuron, the activity of the neuron indicated by fluorescent intensity is calculated. For each video, annotation of only 20 frames in a video is sufficient to yield human-level accuracy for all other frames. The validity of this method is further confirmed by reproducing the activity pattern of PMSIs (period-positive median segmental interneurons) and larval movement as previously reported. Using this method, we disclosed the correlation between larval movement and left-right asymmetry in activity of a group of unidentified neurons labelled by R52H01-Gal4 and further confirmed the roles of these neurons in bilateral balance of body contraction during larval crawling by genetic inhibition of these neurons. Our method provides a new tool for accurate extraction of neural activities and movement of freely behaving small-size transparent animals.

Janus-Structured Microgel Barrier with Tissue Adhesive and Hemostatic Characteristics for Efficient Prevention of Postoperative Adhesion.

Postoperative adhesion (POA) is a common and serious complication following various types of surgery. Current physical barriers either have a short residence time at the surgical site with a low tissue attachment capacity or are prone to undesired adhesion formation owing to the double-sided adhesive property, which limits the POA prevention efficacy of the barriers. In this study, Janus-structured microgels (Janus-MGs) with asymmetric tissue adhesion capabilities are fabricated using a novel bio-friendly gas-shearing microfluidic platform. The anti-adhesive side of Janus-MGs, which consists of alginate, hyaluronic acid, and derivatives, endows the material with separation, lubrication, and adhesion prevention properties. The adhesive side provided Janus-MGs with tissue attachment and retention capability through catechol-based adhesion, thereby enhancing the in situ adhesion prevention effect. In addition, Janus-MGs significantly reduced blood loss and shortened the hemostatic time in rats, further reducing adhesion formation. Three commonly used rat POA models with different tissue structures and motion patterns are established in this study, namely peritoneal adhesion, intrauterine adhesion, and peritendinous adhesion models, and the results showed that Janus-MGs effectively prevented the occurrence of POA in all the models. The fabrication of Janus-MGs offers a reliable strategy and a promising paradigm for preventing POA following diverse surgical procedures.

Hybrid Printing of Fully Integrated Microfluidic Devices for Biosensing.

The advent of 3D printing has facilitated the rapid fabrication of microfluidic devices that are accessible and cost-effective. However, it remains a challenge to fabricate sophisticated microfluidic devices with integrated structural and functional components due to limited material options of existing printing methods and their stringent requirement on feedstock material properties. Here, a multi-materials multi-scale hybrid printing method that enables seamless integration of a broad range of structural and functional materials into complex devices is reported. A fully printed and assembly-free microfluidic biosensor with embedded fluidic channels and functionalized electrodes at sub-100 µm spatial resolution for the amperometric sensing of lactate in sweat is demonstrated. The sensors present a sensitive response with a limit of detection of 442 nm and a linear dynamic range of 1-10 mm, which are performance characteristics relevant to physiological levels of lactate in sweat. The versatile hybrid printing method offers a new pathway toward facile fabrication of next-generation integrated devices for broad applications in point-of-care health monitoring and sensing.

Kinetically Controlled Nucleation Enabled by Tunable Microfluidic Mixing for the Synthesis of Dendritic Au@Pt Core/Shell Nanomaterials.

The nucleation stage plays a decisive role in determining nanocrystal morphology and properties; hence, the ability to regulate nucleation is critical for achieving high-level control. Herein, glass microfluidic chips with S-shaped mixing units are designed for the synthesis of Au@Pt core/shell materials. The use of hydrodynamics to tune the nucleation kinetics is explored by varying the number of mixing units. Dendritic Au@Pt core/shell nanomaterials are controllably synthesized and a formation mechanism is proposed. As-synthesized Au@Pt exhibited excellent ethanol oxidation activity under alkaline conditions (8.4 times that of commercial Pt/C). This approach is also successfully applied to the synthesize of Au@Pd core/shell nanomaterials, thus demonstrating its generality.