Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications.

In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21-26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21-26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Optimizing Microfluidic Channel Design with High-Performance Materials for Safe Neonatal Drug Delivery.

INTRODUCTION: Designing the microfluidic channel for neonatal drug delivery requires proper considerations to enhance the efficiency and safety of drug substances when used in neonates. Thus, this research aims to evaluate high-performance materials and optimize the channel design by modeling and simulation using COMSOL multiphysics in order to deliver an optimum flow rate between 0. 3 and 1 mL/hr. METHOD: Some of the materials used in the study included PDMS, glass, COC, PMMA, PC, TPE, and hydrogels, and the evaluation criterion involved biocompatibility, mechanical properties, chemical resistance, and ease of fabrication. The simulation was carried out in the COMSOL multiphysics platform and demonstrated the fog fluid behavior in different channel geometries, including laminar flow and turbulence. The study then used systematic changes in design parameters with the aim of establishing the best implementation models that can improve the efficiency and reliability of the drug delivery system. The comparison was based mostly on each material and its appropriateness in microfluidic usage, primarily in neonatal drug delivery. The biocompatibility of the developed materials was verified using the literature analysis and adherence to the ISO 10993 standard, thus providing safety for the use of neonatal devices. Tensile strength was included to check the strength of each material to withstand its operation conditions. Chemical resistance was also tested in order to determine the compatibility of the materials with various drugs, and the possibility of fabrication was also taken into consideration to identify appropriate materials that could be used in the rapid manufacturing of the product. RESULTS: The results we obtained show that PDMS, due to its flexibility and simplicity in simulation coupled with more efficient channel designs which have been extracted from COMSOL, present a feasible solution to neonatal drug delivery. CONCLUSION: The present comparative study serves as a guide on the choice of materials and design of microfluidic devices to help achieve safer and enhanced drug delivery systems suitable for the delicate reception of fragile neonates.

Enhanced capture system for mesenchymal­type circulating tumor cells using a polymeric microfluidic device 'CTC­Chip' incorporating cell­surface vimentin.

CellSearch, the only approved epithelial cell adhesion molecule (EpCAM)­dependent capture system approved for clinical use, overlooks circulating tumor cells (CTCs) undergoing epithelial­mesenchymal transition (EMT­CTCs), which is considered a crucial subtype responsible for metastasis. To address this limitation, a novel polymeric microfluidic device 'CTC­chip' designed for the easy introduction of any antibody was developed, enabling EpCAM­independent capture. In this study, antibodies against EpCAM and cell surface vimentin (CSV), identified as cancer­specific EMT markers, were conjugated onto the chip (EpCAM­chip and CSV­chip, respectively), and the capture efficiency was examined using lung cancer (PC9, H441 and A549) and colon cancer (DLD1) cell lines, classified into three types based on EMT markers: Epithelial (PC9), intermediate (H441 and DLD1) and mesenchymal (A549). PC9, H441 and DLD1 cells were effectively captured using the EpCAM­chip (average capture efficiencies: 99.4, 88.8 and 90.8%, respectively) when spiked into blood. However, A549 cells were scarcely captured (13.4%), indicating that EpCAM­dependent capture is not suitable for mesenchymal­type cells. The expression of CSV tended to be higher in cells exhibiting mesenchymal properties and A549 cells were effectively captured with the CSV­chip (72.4 and 88.4% at concentrations of 10 and 100 µg/ml, respectively) when spiked into PBS. When spiked into blood, the average capture efficiencies were 27.7 and 46.8% at concentrations of 10 and 100 µg/ml, respectively. These results suggest that the CSV­chip is useful for detecting mesenchymal­type cells and has potential applications in capturing EMT­CTCs.

Barrier-free, open-top microfluidic chip for generating two distinct, interconnected 3D microvascular networks.

Developing microphysiological cell culture platforms with a three-dimensional (3D) microenvironment has been a significant advancement from traditional monolayer cultures. Still, most of the current microphysiological platforms are limited in closed designs, i.e. are not accessible after 3D cell culture loading. Here, we report an open-top microfluidic chip which enables the generation of two sequentially loaded 3D cell cultures without physical barriers restricting the nurture, gas exchange and cellular communication. As a proof-of-concept, we demonstrated the formation of two 3D vasculatures, one in the upper and the other in the lower compartment, under three distinct flow conditions: asymmetric side-to-center, symmetric side-to-center and symmetric center-to-side. We used computational modelling to characterize initial flow pressures in cell culture compartments. We showed prominent vessel formation and branched vasculatures in upper and lower cell culture compartments with interconnecting, lumenized vessels with in vivo-relevant diameter in all flow conditions. With advanced image processing, we quantified and compared the overall vascular network volume and the total length formed in asymmetric side-to-center, symmetric side-to-center and symmetric center-to-side flow conditions. Our results indicate that the developed chip can house two distinct 3D cell cultures with merging vessels between compartments and by providing asymmetric side-to-center or symmetric center-to-side flow vascular morphogenesis is enhanced in terms of overall network length. The developed open-top microfluidic chip may find various applications in generation of tissue-specific 3D-3D co-cultures for studying cellular interactions in vascularized tissues and organs.

Inertial microfluidic mixer for biological CubeSat missions.

Nanosatellites of CubeSat type due to, i.a., minimized costs of space missions, as well as the potential large application area, have become a significant part of the space economy sector recently. The opportunity to apply miniaturized microsystem (MEMS) tools in satellite space missions further accelerates both the space and the MEMS markets, which in the coming years are considered to become inseparable. As a response to the aforementioned perspectives, this paper presents a microfluidic mixer system for biological research to be conducted onboard CubeSat nanosatellites. As a high complexity of the space systems is not desired due to the need for failure-free and remotely controlled operation, the principal concept of the work was to design an entirely passive micromixer, based on lab-on-chip technologies. For the first time, the microfluidic mixer that uses inertial force generated by rocket engines during launch to the orbit is proposed to provide an appropriate mixing of liquid samples. Such a solution not only saves the space occupied by standard pumping systems, but also reduces the energy requirements, ultimately minimizing the number of battery modules and the whole CubeSat size. The structures of the microfluidic mixers were fabricated entirely out of biocompatible resins using MultiJet 3D printing technology. To verify the functionality of the passive mixing system, optical detection consisting of the array of blue LEDs and phototransistors was applied successfully. The performance of the device was tested utilizing an experimental rocket, as a part of the Spaceport America Cup 2023 competition.

Electrochemical biosensors on microfluidic chips as promising tools to study microbial biofilms: a review.

Microbial biofilms play a pivotal role in microbial infections and antibiotic resistance due to their unique properties, driving the urgent need for advanced methodologies to study their behavior comprehensively across varied environmental contexts. While electrochemical biosensors have demonstrated success in understanding the dynamics of biofilms, scientists are now synergistically merging these biosensors with microfluidic technology. This combined approach offers heightened precision, sensitivity, and real-time monitoring capabilities, promising a more comprehensive understanding of biofilm behavior and its implications. Our review delves into recent advancements in electrochemical biosensors on microfluidic chips, specifically tailored for investigating biofilm dynamics, virulence, and properties. Through a critical examination of these advantages, properties and applications of these devices, the review highlights the transformative potential of this technology in advancing our understanding of microbial biofilms in different settings.

Lymph node metastasis diagnosis of postoperative OSCC patients by analyzing extracellular vesicles in drainage fluid based on microfluidic isolation.

Lymph node metastasis (LNM) is a typical marker in oral squamous cell carcinoma (OSCC) indicating poor prognosis. Pathological examination by artificial image acquisition and analysis, as the main diagnostic method for LNM, often takes a week or longer which may cause great anxiety of the patient and also retard timely treatment. However, there are few efficient fast LNM diagnosis methods in clinical applications currently. Our previous study profiled the proteomics of extracellular vesicles (EVs) derived from postoperative drainage fluid (PDF) and showed the potential of detecting specific EVs that expressed aspartate ß-hydroxylase (ASPH) for LNM diagnosis in OSCC patients. Considering that the analysis of ASPH+ PDF-EVs is challenging due to their low abundance (counting less than 10% of total EVs in PDF) and the complex EV isolation process of ultra-centrifugation, we developed a facile platform containing two microfluidic chips filled with antibody-modified microbeads to isolate ASPH+ PDF-EVs, with both the capture and retrieval rate reaching around 90%. Clinical sample analysis based on our method revealed that a mean of 6 × 106 /mL ASPH+ PDF-EVs could be isolated from LNM+ OSCC patients compared to 2.5 × 106 /mL in LNM- OSCC ones. When combined with enzyme-linked immunosorbent assay (ELISA) technique that was commonly used in clinical laboratories in hospitals, this microfluidic platform could precisely distinguish postoperative OSCC patients with LNM or not in several hours, which were validated by a double-blind test containing 6 OSCC patients. We believe this strategy has promise for early diagnosis of LNM in postoperative OSCC patients and finally helps guiding timely and reasonable treatment in clinic.

Osteochondral Tissue-On-a-Chip: A Novel Model for Osteoarthritis Research.

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

Looping Flexible Fluoropolymer Microcapillary Film Extends Analysis Times for Vertical Microfluidic Blood Testing.

The microfluidic measurement of capillary flow can be used to evaluate the response of biological samples to stimulation, where distance and velocity are altered. Melt-extruded multi-bored microfluidic capillaries allow for high-throughput testing with low device cost, but simple devices may limit control over sample flow when compared to the more complex "lab-on-a-chip" devices produced using advanced microfluidic fabrication methods. Previously, we measured the dynamics of global haemostasis stimulated by thrombin by dipping straight vertical microcapillaries into blood, but only the most rapid response could be monitored, as flow slowed significantly within 30 s. Here, we show an innovative method to extend both the stimulation process and flow measurement time without increasing the cost of the device by adding simple loops to the flexible extruded device. The loops enable longer time-scale measurements by increasing resistance to flow, thereby reducing the dependence on high stimulus concentrations for rapid reactions. The instantaneous velocity and equilibrium heights of straight and looped vertical microcapillary films were assessed with water, plasma and whole blood, showing that the loops create additional frictional resistances, reduce flow velocity and prolong residence times for increased time scales of the stimulation process. A modified pressure balance model was used to capture flow dynamics with the added loop. Looped devices loaded with thrombin and collagen showed an improved detection of blood stimulation responses even with lower stimulus concentrations, compared to straight vertical capillaries. Thrombin-activated blood samples in straight capillaries provided a maximum measurement zone of only 4 mm, while the looped design significantly increased this to 11 mm for much longer time scale measurements. Our results suggest that extending stimulation times can be achieved without complex microfluidic fabrication methods, potentially improving concentration-response blood stimulation assays, and may enhance the accuracy and reliability. We conclude adding a loop to low-cost extruded microfluidic devices may bring microfluidic devices closer to delivering on their promise of widespread, decentralized low-cost evaluation of blood response to stimulation in both research and clinical settings.

A Synergistic Overview between Microfluidics and Numerical Research for Vascular Flow and Pathological Investigations.

Vascular diseases are widespread, and sometimes such life-threatening medical disorders cause abnormal blood flow, blood particle damage, changes to flow dynamics, restricted blood flow, and other adverse effects. The study of vascular flow is crucial in clinical practice because it can shed light on the causes of stenosis, aneurysm, blood cancer, and many other such diseases, and guide the development of novel treatments and interventions. Microfluidics and computational fluid dynamics (CFDs) are two of the most promising new tools for investigating these phenomena. When compared to conventional experimental methods, microfluidics offers many benefits, including lower costs, smaller sample quantities, and increased control over fluid flow and parameters. In this paper, we address the strengths and weaknesses of computational and experimental approaches utilizing microfluidic devices to investigate the rheological properties of blood, the forces of action causing diseases related to cardiology, provide an overview of the models and methodologies of experiments, and the fabrication of devices utilized in these types of research, and portray the results achieved and their applications. We also discuss how these results can inform clinical practice and where future research should go. Overall, it provides insights into why a combination of both CFDs, and experimental methods can give even more detailed information on disease mechanisms recreated on a microfluidic platform, replicating the original biological system and aiding in developing the device or chip itself.

A Novel Size-Based Centrifugal Microfluidic Design to Enrich and Magnetically Isolate Circulating Tumor Cells from Blood Cells through Biocompatible Magnetite-Arginine Nanoparticles.

This paper presents a novel centrifugal microfluidic approach (so-called lab-on-a-CD) for magnetic circulating tumor cell (CTC) separation from the other healthy cells according to their physical and acquired chemical properties. This study enhances the efficiency of CTC isolation, crucial for cancer diagnosis, prognosis, and therapy. CTCs are cells that break away from primary tumors and travel through the bloodstream; however, isolating CTCs from blood cells is difficult due to their low numbers and diverse characteristics. The proposed microfluidic device consists of two sections: a passive section that uses inertial force and bifurcation law to sort CTCs into different streamlines based on size and shape and an active section that uses magnetic forces along with Dean drag, inertial, and centrifugal forces to capture magnetized CTCs at the downstream of the microchannel. The authors designed, simulated, fabricated, and tested the device with cultured cancer cells and human cells. We also proposed a cost-effective method to mitigate the surface roughness and smooth surfaces created by micromachines and a unique pulsatile technique for flow control to improve separation efficiency. The possibility of a device with fewer layers to improve the leaks and alignment concerns was also demonstrated. The fabricated device could quickly handle a large volume of samples and achieve a high separation efficiency (93%) of CTCs at an optimal angular velocity. The paper shows the feasibility and potential of the proposed centrifugal microfluidic approach to satisfy the pumping, cell sorting, and separating functions for CTC separation.

[Advances of gut-on-a-chip for exploring host-microbe interactions].

The human gut is a complex ecosystem harboring rich microbes that play a key role in the nutrient absorption, drug metabolism, and immune responses. With the continuous development of microfluidics and organ-on-a-chip, gut-on-a-chip has become a powerful tool for modeling host-microbe interactions. The chip is able to mimic the complex physiological environment of the human gut in vitro, providing a unique platform for studying host-microbe interactions. Firstly, we introduce the physiological characteristics of the human gut. Secondly, we comprehensively summarize the advantages of the microfluidic chip in vitro recapitulating the intestinal system by integrating microenvironmental factors, such as complex cell components, dynamic fluids, oxygen gradients, and mechanical mechanics. Thirdly, we expound the key performance indicators for evaluating the construction performance of gut-on-a-chip. In addition, we review the progress of gut-on-a-chip models in the research on gut microecology, disease modeling, and drug evaluation. Finally, we highlight the challenges and prospects in the applications of the emerging technology. The above is summarized with a view to informing the application of gut-on-a-chip for indepth studies of gut microbe-host interactions.

Overview of research on additive manufacturing of hydrogel-assisted lab-on-chip platforms for cell engineering applications in photodynamic therapy research.

Lab-on-chips supported by hydrogel matrices are excellent solutions for cell culture; thus, this literature review presents examples of scientific research in this area. Several works are presenting the properties of biocompatible hydrogels that mimic the cellular environment published recently. Hydrogels can also be treated as cell transporters or as a structural component of microfluidic devices. The rapidly growing scientific sector of hydrogel additive manufacturing is also described herein, with attention paid to the appropriate mechanical and biological properties of the inks used to extrude the material, specifically for biomedical purposes. The paper focuses on protocols employed for additive manufacturing, e.g., 3D printing parameters, calibration, ink preparation, crosslinking processes, etc. The authors also mention potential problems concerning manufacturing processes and offer example solutions. As the novel trend for hydrogels enriched with several biocompatible additives has recently risen, the article presents examples of the use of high-quality carbon nanotubes in hydrogel research enhancing biocompatibility, mechanical stability, and cell viability. Moving forward, the article points out the high applicability of the hydrogel-assisted microfluidic platforms used for cancer research, especially for photodynamic therapy (PDT). This innovative treatment strategy can be investigated directly on the chip, which was first proposed by Jedrych E. et al. in 2011. Summarizing, this literature review highlights recent developments in the additive manufacturing of microfluidic devices supported by hydrogels, toward reliable cell culture experiments with a view to PDT research. This paper gathers the current knowledge in these intriguing and fast-growing research paths.

Brain-on-a-chip: an emerging platform for studying the nanotechnology-biology interface for neurodegenerative disorders.

Neurological disorders have for a long time been a global challenge dismissed by drug companies, especially due to the low efficiency of most therapeutic compounds to cross the brain capillary wall, that forms the blood-brain barrier (BBB) and reach the brain. This has boosted an incessant search for novel carriers and methodologies to drive these compounds throughout the BBB. However, it remains a challenge to artificially mimic the physiology and function of the human BBB, allowing a reliable, reproducible and throughput screening of these rapidly growing technologies and nanoformulations (NFs). To surpass these challenges, brain-on-a-chip (BoC) - advanced microphysiological platforms that emulate key features of the brain composition and functionality, with the potential to emulate pathophysiological signatures of neurological disorders, are emerging as a microfluidic tool to screen new brain-targeting drugs, investigate neuropathogenesis and reach personalized medicine. In this review, the advance of BoC as a bioengineered screening tool of new brain-targeting drugs and NFs, enabling to decipher the intricate nanotechnology-biology interface is discussed. Firstly, the main challenges to model the brain are outlined, then, examples of BoC platforms to recapitulate the neurodegenerative diseases and screen NFs are summarized, emphasizing the current most promising nanotechnological-based drug delivery strategies and lastly, the integration of high-throughput screening biosensing systems as possible cutting-edge technologies for an end-use perspective is discussed as future perspective.

Construction of a pumpless gravity-driven vascularized Skin-on-a-Chip for the study of hepatocytotoxicity in percutaneous exposure to exogenous chemicals.

The utilization of existing Skin-on-a-Chip (SoC) is constrained by the complex structures, the multiplicity of auxiliary devices, and the inability to evaluate exogenous chemicals that are hepatotoxic after percutaneous metabolism. In this study, a gravity-driven SoC without any auxiliary devices was constructed for the hepatocytotoxicity study of exogenous chemicals. The SoC possesses 3 layers of culture chambers, from top to bottom, for human skin equivalent (HSE), Human Umbilical Vein Endothelial Cells (HUVEC) and hepatocytes (HepG2), and the maintenance and expression capacity of the corresponding cells on the SoC were verified by specificity parameters. The reactivity of the SoC to exogenous chemicals was verified by 2-aminofluorene (2-AF). The SoC can realistically simulate the in vivo exposure process of exogenous chemicals that are percutaneously exposed and metabolized into the bloodstream and then to the liver to produce toxicity, and it can achieve the same effects on transcriptome as those of animal tests at lower exposure levels while examining multiple toxicological targets of the skin, vascular endothelial cells, and hepatocytes. Both in terms of species similarity, the principles of reduction, replacement and refinement (3R), or the level of exposure suggest that the present SoC has a degree of replacement for animal models in assessing exogenous chemicals, especially those that are hepatotoxic after percutaneous metabolism.

Microfluidic Device with an Oxygen Gradient Generator for Investigating Effects of Specific Hypoxia Conditions on Responses of Tumor Cells.

The oxygen level in the tumor microenvironment (TME) plays a critical role in regulating cell fates such as proliferation, migration, apoptosis, and so forth. To better elucidate how hypoxia affects tumor cell behaviors, a series of microfluidic strategies have been utilized to generate an oxygen gradient covering both hypoxia and normoxia conditions. However, in most studies, some chemicals are introduced into microfluidic chips, causing the potential of their poor biocompatibility. The common oxygen gradient with linear variation does not allow the effects of specific oxygen concentrations on tumor cells to be analyzed accurately. In this paper, based on the physical method of gas diffusion, a microfluidic device integrated with an oxygen gradient generator is proposed for investigating effects of different hypoxia levels on responses of tumor cells. This device consists of three layers, i.e., upper layer, thin film layer, and bottom layer. The upper layer is used for introducing the initial gas and generating an oxygen gradient in the form of gas. The bottom layer is used for introducing cells and culture medium. The thin film layer separates the former two layers, allowing the gas to diffuse from the top to the bottom through it. The oxygen gradient in the bottom layer is finally generated in the form of dissolved oxygen. The device is fabricated using microfabrication technology. The effects of structural and working parameters of the device on the oxygen gradient are evaluated by finite element simulation. The oxygen gradient in cell culture channels is characterized by using oxygen-sensitive fluorescence materials. The proliferation and morphology of HeLa cells under specific oxygen levels are compared after culturing for 48 h. The oxygen gradient with a ladder-like distribution demonstrates that this microfluidic device can provide a prospective experimental platform for in vitro cell studies and revelation of the mechanism of tumor metastasis associated with a specific hypoxic microenvironment.

Polarization of Macrophages in Tumor Microenvironment Using High-Throughput Single-Cell Metabolomics.

Macrophages consist of a heterogeneous population of functionally distinct cells that participate in many physiological and pathological processes. They exhibit prominent plasticity by changing their different functional phenotypes represented by proinflammatory (M1) and anti-inflammatory (M2) in response to different environmental stimuli. Emerging evidence illustrates the importance of intracellular metabolic pathways in macrophage polarizations and functions. In the tumor microenvironment (TME), macrophages tend to M2 polarization, which promotes tumor growth and leads to adverse physiological effects. Due to the lack of highly specific antigens in M1 and M2 macrophages, significant challenges present in isolating these subtypes from clinical samples or in vitro coculture models of tumor-immune cells. In reverse, the single-cell technique provides the possibility to investigate the factors influencing macrophage polarization in the TME. In this research, we employed inertial microfluidic chip-mass spectrometry (IMC-MS) to conduct single-cell metabolomics analysis of macrophages polarized into the two major phenotypes, respectively, and 213 metabolites were identified in total. Subsequently, differential metabolites between macrophage phenotypes were analyzed using volcano plots and binary logistic regression models. Glutamine was pinpointed as a key metabolite for the M1 and M2 phenotypes. Experimental results from both monoculture and coculture cell models demonstrated that M1 polarization is more reliant on the presence of glutamine in the culture environment than M2 polarization. Glutamine deficiency resulted in failed M1 polarization, while its absence had a less pronounced effect on M2 polarization. Replenishing an appropriate amount of glutamine during the intermediate stages of coculture models significantly enhanced the proportion of M1 polarization and suppressed the growth of tumor cells. This research elucidated glutamine as a key factor influencing macrophage polarization in the TME via single-cell metabolomics based on IMC-MS, offering promising insights and targets for tumor therapies.

Predictive Analysis in Oral Cancer Immunotherapy: Profiling Dual PD-L1-Positive Extracellular Vesicle Subtypes with Step-Wedge Microfluidic Chips.

PD-L1-positive extracellular vesicles (PD-L1+ EVs) play a pivotal role as predictive biomarkers in cancer immunotherapy. These vesicles, originating from immune cells (I-PD-L1+ EVs) and tumor cells (T-PD-L1+ EVs), hold distinct clinical predictive values, emphasizing the importance of deeply differentiating the PD-L1+ EV subtypes for effective liquid biopsy analyses. However, current methods such as ELISA lack the ability to differentiate their cellular sources. In this study, a novel step-wedge microfluidic chip that combines magnetic microsphere separation with single-layer fluorescence counting is developed. This chip integrates magnetic microspheres modified with anti-PD-L1 antibodies and fluorescent nanoparticles targeting EpCAM (tumor cell marker) or CD45 (immunocyte marker), enabling simultaneous quantification and sensitive analysis of PD-L1+ EV subpopulations in oral squamous cell carcinoma (OSCC) patients' saliva without background interference. Analysis results indicate reduced levels of I-PD-L1+ EVs in OSCC patients compared to those in healthy individuals, with varying levels of heterogeneous PD-L1+ EVs observed among different patient groups. During immunotherapy, responders exhibit decreased levels of total PD-L1+ EVs and T-PD-L1+ EVs, accompanied by reduced levels of I-PD-L1+ EVs. Conversely, nonresponders show increased levels of I-PD-L1+ EVs. Utilizing the step-wedge microfluidic chip allows for simultaneous detection of PD-L1+ EV subtypes, facilitating the precise prediction of oral cancer immunotherapy outcomes.

Functionalization of microbubbles in a microfluidic chip for biosensing application.

Microbubbles are widely used for biomedical applications, ranging from imagery to therapy. In these applications, microbubbles can be functionalized to allow targeted drug delivery or imaging of the human body. However, functionalization of the microbubbles is quite difficult, due to the unstable nature of the gas/liquid interface. In this paper, we describe a simple protocol for rapid functionalization of microbubbles and show how to use them inside a microfluidic chip to develop a novel type of biosensor. The microbubbles are functionalized with biochemical ligand directly at their generation inside the microfluidic chip using a DSPE-PEG-Biotin phospholipid. The microbubbles are then organized inside a chamber before injecting the fluid with the bioanalyte of interest through the static bubbles network. In this proof-of-concept demonstration, we use streptavidin as the bioanalyte of interest. Both functionalization and capture are assessed using fluorescent microscopy thanks to fluorescent labeled chemicals. The main advantages of the proposed technique compared to classical ligand based biosensor using solid surface is its ability to rapidly regenerate the functionalized surface, with the complete functionalization/capture/measurement cycle taking less than 10 min.

A physiological perspective of the relevance of sweat biomarkers and their detection by wearable microfluidic technology: A review.

The great majority of published microfluidic wearable platforms for sweat sensing focus on the development of the technology to fabricate the device, the integration of sensing materials and actuators and the fluidics of sweat within the device. However, very few papers have discussed the physiological relevance of the metabolites measured using these novel approaches. In fact, some of the analytes present in sweat, which serve as biomarkers in blood, do not show a correlation with blood levels. This discrepancy can be attributed to factors such as contamination during measurements, the metabolism of sweat glands, or challenges in obtaining significant samples. The objective of this review is to present a critical and meaningful insight into the real applicability and potential use of wearable technology for improving health and sport performance. It also discusses the current limitations and future challenges of microfluidics, aiming to provide accurate information about the actual needs in this field. This work is expected to contribute to the future development of more suitable wearable microfluidic technology for health and sports science monitoring, using sweat as the biofluid for analysis.