Assessment of nanotoxicity in a human placenta-on-a-chip from trophoblast stem cells.

Maternal exposure to nanoparticles during gestation poses potential risks to fetal development. The placenta, serving as a vital interface for maternal-fetal interaction, plays a pivotal role in shielding the fetus from direct nanoparticle exposure. However, the impact of nanoparticles on placental function is still poorly understood, primarily due to the absence of proper human placental models. In this study, we established a placenta-on-a-chip model capable of recapitulating nanoparticle exposure to assess potential nanotoxicity. The model was assembled by coculturing human trophoblast stem cells (hTSCs) and endothelial cells within a dynamic microsystem. hTSCs exhibited progressive differentiation into syncytiotrophoblasts under continuous fluid flow, forming a bilayered trophoblastic epithelium that mimicking both structural and functional aspects of human placental villi. Copper oxide nanoparticles (CuO NPs) were introduced into the trophoblastic side to simulate maternal blood exposure. Our findings revealed that CuO NPs hindered hTSCs differentiation, leading to diminished hormone secretion and impaired glucose transport. Subsequent analysis indicated that CuO NPs disrupted the autophagic flux in trophoblasts and induced apoptosis. Furthermore, the placenta-on-a-chip model exhibited inflammatory responses to CuO NP exposure, including maternal macrophage activation, inflammatory cytokine secretion, and endothelial barrier disruption. Dysfunction of the placental barrier and the ensuing inflammatory cascades may contribute to aberrant fetal development. Overall, our placenta-on-a-chip model offers a promising platform for assessing nanoparticle exposure-related risks and conducting toxicology studies.

Microfluidic-Assisted Pneumatic Droplet Generators Designed for Multiscenario Biomanufacturing with Favorable Biocompatibility and Extendibility.

Droplets, tiny liquid compartments, are increasingly emerging in the biomedical and biomanufacturing fields due to their unique properties to serve as templates or independent reaction units. Currently, the straightforward and efficient generation of various functional droplets in a biofriendly manner remains challenging. Herein, a novel microfluidic-assisted pneumatic strategy is described for the customizable and high-throughput production of monodispersed droplets, and the droplet size can be precisely controlled via a simplified gas pressure regulation module. In particular, numerous uniform alginate microcarriers can be rapidly fabricated in an all-aqueous manner, wherein the encapsulated islet or liver cells exhibit favorable viability and biological functions. Furthermore, by changing the microchannel configuration, several fluid manipulation functions developed by microfluidic technology, such as mixing and laminar flow, can be successfully incorporated into this platform. The droplet generators with scalable functionality are demonstrated in many biomanufacturing scenarios, including on-demand distribution of cell-mimetic particles, continuous synthesis of biomedical metal-organic framework (MOF), controllable preparation of compartmental microgel, etc. These may provide sustainable inspiration for developing droplet generators and their applications in tissue and organ engineering, biomaterials design, bioprinting nozzles, and other fields.

A flexible strategy to fabricate trumpet-shaped porous PDMS membranes for organ-on-chip application.

Porous polydimethylsiloxane (PDMS) membrane is a crucial element in organs-on-chips fabrication, supplying a unique substrate that can be used for the generation of tissue-tissue interfaces, separate co-culture, biomimetic stretch application, etc. However, the existing methods of through-hole PDMS membrane production are largely limited by labor-consuming processes and/or expensive equipment. Here, we propose an accessible and low-cost strategy to fabricate through-hole PDMS membranes with good controllability, which is performed via combining wet-etching and spin-coating processes. The porous membrane is obtained by spin-coating OS-20 diluted PDMS on an etched glass template with a columnar array structure. The pore size and thickness of the PDMS membrane can be adjusted flexibly via optimizing the template structure and spinning speed. In particular, compared to the traditional vertical through-hole structure of porous membranes, the membranes prepared by this method feature a trumpet-shaped structure, which allows for the generation of some unique bionic structures on organs-on-chips. When the trumpet-shape faces upward, the endothelium spreads at the bottom of the porous membrane, and intestinal cells form a villous structure, achieving the same effect as traditional methods. Conversely, when the trumpet-shape faces downward, intestinal cells spontaneously form a crypt-like structure, which is challenging to achieve with other methods. The proposed approach is simple, flexible with good reproducibility, and low-cost, which provides a new way to facilitate the building of multifunctional organ-on-chip systems and accelerate their translational applications.

Levonorgestrel protected Au10 cluster for hypochlorite sensing in living organisms.

BACKGROUND: Highly reactive oxygen species (ROS) could lead to serious damage in living cells and are associated with many diseases like cancers. Metal cluster with strong fluorescence has great potential in biosensing and many thiolate ligands-protected clusters have been applied in ROS sensing. RESULTS: In this work, we synthesized levonorgestrel protected Au10 cluster with specific sensing ability for highly ROS via crystal transformation from Au8 cluster, demonstrating the significance of inner core structure on detecting performance. The detection limit of Au10 cluster for ClO- could reach as low as 0.1 µM. This fluorescent probe not only achieving detection of exogenous ClO- in living cells and zebrafish, but also successful imaging of endogenous ClO- in HeLa and HepG2 cells. SIGNIFICANCE: In comparison to previously reported cluster-based sensors for ROS, this work proposes a different reaction mechanism of metal nanoclusters for ROS detection (breakage of gold-alkynyl bond and oxidation of alkynyl group). This provides new directions for designing specific ROS probes and broadens the applications of metal clusters in disease diagnostics.

In vitro evaluation of the toxicological effects of cooking oil fumes using a self-designed microfluidic chip.

Cooking oil fumes (COFs) are widely acknowledged as substantial contributors to indoor air pollution, having detrimental effects on human health. Despite the existence of commercialized in vitro aerosol exposure platforms, assessment risks of aerosol pollutants are primarily evaluated based on multiwell plate experiments by trapping and redissolving aerosols to conduct comprehensive in vitro immersion exposure manner. Therefore, an innovative real-time exposure system for COF aerosol was constructed, featuring a self-designed microfluidic chip as its focal component. The chip was used to assess toxicological effects of in vitro exposure to COF aerosol on cells cultured at the gas-liquid interface. Meanwhile, we used transcriptomics to analyze genes that exhibited differential expression in cells induced by COF aerosol. The findings indicated that the MAPK signaling pathway, known for its involvement in inflammatory response and oxidative stress, played a crucial role in the biological effects induced by COF aerosol. Biomarkers associated with inflammatory response and oxidative stress exhibited corresponding alterations. Furthermore, the concentration of COF aerosol exposure and post-exposure duration exert decisive effects on these biomarkers. Thus, the study suggests that COF can induce oxidative stress and inflammatory response in BEAS-2B cells, potentially exerting a discernible impact on human health.

In vitro assessment of ferroptosis of cells exposed to cigarette smoke aerosol using a self-designed on-chip evaluation system based on gas-liquid dual-dimensional exposure.

Aerosol pollutants significantly cause health concerns. Herein, we established an original real-time aerosol exposure system that used a self-designed bionic-lung microfluidic chip. The chip features a 4 × 4 intersecting array within gas and liquid layers, creating 16 distinct microenvironments. A membrane situated between the layers offers attachment for cells and establishes a gas-liquid interface. This design provides a reliable screening capacity for investigating the biological effects of aerosol exposure in vitro by manipulating the gas and/or liquid conditions. Using this system, we validated that cigarette smoke (CS) aerosol triggered a concentration- and time-dependent reduction in cell viability and intracellular glutathione levels, accompanied by an increase in intracellular reactive oxygen species and Fe2+. Furthermore, CS aerosol significantly downregulated the expression of GPX4, SLC7A11, and FTL mRNA while inducing a notable increase in that of ACSL4 mRNA. Additionally, CS aerosol markedly stimulated the release of proinflammatory cytokines. Crucially, the ferroptosis inhibitor deferoxamine mesylate reversed these biological indicators. These results demonstrate that our novel bionic-lung chip presents a suitably achievable approach to investigate the biological effects induced by aerosol exposure.

A Pump-Free Strategy for the Controllable Generation of Alginate Microgels as Cellular Microcarriers.

Microgels are advanced scaffolds for tissue engineering due to their proper biodegradability, good biocompatibility, and high specific surface area for effective oxygen and nutrient transfer. However, most of the current monodispersed microgel fabrication systems rely heavily on various precision pumps, which highly increase the cost and complexity of their downstream application. In this work, we developed a simple and facile system for the controllable generation of uniform alginate microgels by integrating a gas-shearing strategy into a glass microfluidic device. Importantly, the cell-laden microgels can be rapidly prepared in a pump-free manner under an all-aqueous environment. The three-dimensional cultured green fluorescent protein-human A549 cells in alginate microgels exhibited enhanced stemness and drug resistance compared to those under two-dimensional conditions. The pancreatic cancer organoids in alginate microgels exhibited some of the key features of pancreatic cancer. The proposed microgels showed decent monodispersity, biocompatibility, and versatility, providing great opportunities in various biomedical applications such as microcarrier fabricating, organoid engineering, and high-throughput drug screening.

Investigation of the in vitro toxic effects induced by real-time aerosol of electronic cigarette solvents using microfluidic chips.

The safety of propylene glycol (PG) and vegetable glycerin (VG) as solvents in electronic cigarette liquid has received increasing attention and discussion. However, the conclusions derived from toxicity assessments conducted through animal experiments and traditional in vitro methodologies have consistently been contentious. This study constructed an original real-time aerosol exposure system, centered around a self-designed microfluidic bionic-lung chip, to assess the biological effects following exposure to aerosols from different solvents (PG, PG/VG mixture alone and PG/VG mixture in combination with nicotine) on BEAS-2B cells. The study aimed to investigate the impact of aerosols from different solvents on gene expression profiles, intracellular biomarkers (i.e., reactive oxygen species content, nitric oxide content, and caspase-3/7 activity), and extracellular biomarkers (i.e., IL-6, IL-8, TNF-α, and malondialdehyde) of BEAS-2B cells on-chip. Transcriptome analyses suggest that ribosomal function could serve as a potential target for the impact of aerosols derived from various solvents on the biological responses of BEAS-2B cells on-chip. And the results showed that aerosols of PG/VG mixtures had significantly less effect on intracellular and extracellular biomarkers in BEAS-2B cells than aerosols of PG, whereas increasing nicotine levels might elevate these effects of aerosol from PG/VG mixture.

The application of a self-designed microfluidic lung chip in the assessment of different inhalable aerosols.

Microfluidic-based assessment platforms have recently attracted considerable attention and have been widely used for evaluating in vitro toxic effects. In the present study, we developed an original real-time aerosol exposure system, which focused on a self-designed microfluidic chip, in order to evaluate the toxicological effects following exposure to inhalable aerosols. The three-layer structured microfluidic chip enables real-time aerosol exposure at the gas-liquid interface. The comprehensive detection of toxic effect biomarkers based on this assessment platform encompasses transcriptomics, in situ fluorescence detection, and the identification of extracellular secretagogues. Correspondingly, the effects of selected inhalable aerosols such as cigarette smoke (CS), heated tobacco product smoke (HS), and electronic cigarette smoke (ES) on gene expression profiles, cell viability, intracellular biomarkers (reactive oxygen species and nitric oxide), apoptosis (caspase-3/7 activity), and extracellular biomarkers (IL-8, IL-1ß, TNF-α, and malondialdehyde) in the BEAS-2B cells present on the chip were investigated. Following exposure to aerosols derived from CS, HS, and ES, the transcriptome analysis revealed differential expression in these cells. In addition, the overlapping DEGs from the different treatment groups were found to be primarily associated with stimuli and inflammatory responses. Correspondingly, each of the three categories of selected inhalable aerosols was confirmed to induce significant changes in biomarkers that were associated with toxic effects. These results suggest that the original real-time aerosol exposure system centered around a self-designed chip can be applied to the toxic effect evaluation of inhalable aerosol exposure.

Analysis of the response to cigarette smoke exposure in cell coculture and monoculture based on bionic-lung microfluidic chips.

BACKGROUND: In vitro toxicity assessment studies with various experimental models and exposure modalities frequently generate diverse outcomes. In the prevalent experimental, aerosol pollutants are dissolved in culture medium through capture for exposure to two-dimensional planar cellular models in multiwell plates via immersion. However, this approach can generate restricted and inconclusive experimental data, significantly constraining the applicability of risk assessment outcomes. Herein, the in vitro cocultivation of lung epithelial and/or vascular endothelial cells was performed using self-designed bionic-lung microfluidic chip housing a gas-concentration gradient generator (GCGG) unit. Exposure experiments involving a concentration gradient of cigarette smoke (CS) aerosol were then conducted through an original assembled real-time aerosol exposure system. RESULTS: Transcriptomic analysis revealed a potential involvement of the cGMP-signaling pathway following online CS aerosol exposure on different cell culture models. Furthermore, distinct responses to different concentrations of CS aerosol exposure on different culture models were highlighted by detecting inflammation- and oxidative stress-related biomarkers (i.e., cell viability, reactive oxygen species, nitric oxide, IL-6, IL-8, TNF-α, GM-CSF, malondialdehyde, and superoxide dismutase). SIGNIFICANT: The results underscore the importance of improving chip biomimicry while addressing multi-throughput demands, given the substantial influence of the coculture model on cellular responses triggered by CS. Furthermore, the coculture model exhibited a mutually beneficial protective effect on cells at low CS concentrations within the GCGG unit, yet revealed a mutually amplified damaging effect at higher CS concentrations in contrast to the monoculture model.

Combining a lung microfluidic chip exposure model with transcriptomic analysis to evaluate the inflammation in BEAS-2B cells exposed to cigarette smoke.

BACKGROUND: Typically, in vitro studies on the exposure of complex gaseous substances are performed in multi-well plate experiments by trapping and redissolving them, which could introduce potential bias into the results due to the use of inadequate trapping methods. Therefore, a more effective method is to expose complex gaseous substances in gaseous form online, such as using microfluidic chips in experiments. To address these challenges, we introduce a methodology that integrates a self-designed bionic-lung chip with transcriptome analysis to assess the impact of cigarette smoke (CS) exposure on changes in BEAS-2B cells cultured on-chip. RESULTS: After the microfluidic chip underwent online gas exposure, total RNA was extracted via in situ cell lysis, and RNA-Seq transcriptome analysis was conducted. And the RNA-Seq findings revealed the significant involvement of the MAPK signaling pathway associated with the inflammatory response in the cellular effects induced by CS exposure. Moreover, the validation of inflammatory response-related biomarkers through in situ fluorescence corroborated the outcomes of the transcriptome analysis. Besides, the experiment involving the inhibition of inflammation by DEX on the microfluidic chip provided additional confirmation of the previous experimental findings. SIGNIFICANT: In this study, we present an analytical strategy that combines microfluidic-based CS in situ exposure method with RNA-Seq technology. This strategy offers an experimental scheme for in situ exposure to complex gases, transcriptome analysis, and in situ fluorescence detection. Through the integration of the comprehensiveness of transcriptome analysis with the chip's direct and intuitive in situ fluorescence detection with the stability and reliability of RT-PCR and Western blot experiments, we have successfully addressed the challenges associated with in vitro risk assessment for online exposure to complex gaseous substances.

Association of Fine Particulate Matter Constituents with the Predicted 10-Year Atherosclerotic Cardiovascular Disease Risk: Evidence from a Large-Scale Cross-Sectional Study.

Little is known concerning the associations of fine particulate matter (PM2.5) and its constituents with atherosclerotic cardiovascular disease (ASCVD). A total of 31,162 participants enrolled from the Henan Rural Cohort were used to specify associations of PM2.5 and its constituents with ASCVD. Hybrid machine learning was utilized to estimate the 3-year average concentration of PM2.5 and its constituents (black carbon [BC], nitrate [NO3-], ammonium [NH4+], inorganic sulfate [SO42-], organic matter [OM], and soil particles [SOIL]). Constituent concentration, proportion, and residual models were utilized to examine the associations of PM2.5 constituents with 10-year ASCVD risk and to identify the most hazardous constituent. The isochronous substitution model (ISM) was employed to analyze the substitution effect between PM2.5 constituents. We found that each 1 µg/m3 increase in PM2.5, BC, NH4+, NO3-, OM, SO42-, and SOIL was associated with a 3.5%, 49.3%, 19.4%, 10.5%, 21.4%, 14%, and 28.5% higher 10-year ASCVD risk, respectively (all p < 0.05). Comparable results were observed in proportion and residual models. The ISM found that replacing BC with other constituents will generate the greatest health benefits. The results indicated that long-term exposure to PM2.5 and its constituents were associated with increased risks of ASCVD, with BC being the most attributable constituent.

Environmental Applications of Electromembrane Extraction: A Review.

Electromembrane extraction (EME) is a miniaturized extraction technique that has been widely used in recent years for the analysis and removal of pollutants in the environment. It is based on electrokinetic migration across a supported liquid membrane (SLM) under the influence of an external electrical field between two aqueous compartments. Based on the features of the SLM and the electrical field, EME offers quick extraction, effective sample clean-up, and good selectivity, and limits the amount of organic solvent used per sample to a few microliters. In this paper, the basic devices (membrane materials and types of organic solvents) and influencing factors of EME are first introduced, and the applications of EME in the analysis and removal of environmental inorganic ions and organic pollutants are systematically reviewed. An outlook on the future development of EME for environmental applications is also given.

Development of Au8 nanocluster-based fluorescent strip immunosensor for sensitive detection of aflatoxin B1.

Gold clusters with intriguing chemical/physical properties have great promise in applications such as sensing and bio-imaging due to their fascinating photoluminescence character. In this study, an immunofluorescence sensor based on levonorgestrel protected atomically precise Au8 nanocluster (Au8NC) for aflatoxin B1 (AFB1) detection was fabricated due to its strong carcinogenic and mutagenic effect on humans. The prepared polymer-Au8NC nanospheres displayed bright luminescence and good stability in aqueous solution. The obtained AFB1 fluorescent strip immunosensor achieved quantitative point-of-care detection of AFB1 in less than 15 min, with high selectivity and detection limits down to 0.27 ng/mL. In addition, the recovery rates of AFB1 from tea soup ranged from 96% to 105% with relative standard deviations less than 10%. This work not only realized high-sensitively fluorescent sensing for AFB1, but also expanded the bio-applications of atomic-precise metal clusters.

Atomic-precise Pt2Cu4 cluster-based fluorescent sensor for rapid interleukin-6 detection.

Levonorgestrel protected Pt2Cu4 clusters were assembled with a polymer to prepare nanobeads (NBs) with intense red fluorescence. An immunofluorescence sensor based on Pt2Cu4NBs was established for the rapid and sensitive detection of interleukin-6 (IL-6) owing to its significance in inflammatory diseases, with a limit of detection of 42.66 pg mL-1. IL-6 spiked in serum was also accurately detected.

Evaluation of cigarette smoke-induced oxidative stress and inflammation in BEAS-2B cells based on a lung microfluidic chip.

Oxidative stress and inflammation induced by cigarette smoking are associated with the pathology process of various chronic respiratory diseases, including asthma, emphysema, chronic obstructive pulmonary disease and cancer. Compared with conventional cell culture techniques, microfluidic chips can provide a continuous nutrient supply, mimic the in vivo physiological microenvironment of the cells, and conduct an integrated and flexible analysis of cell status and functions. Here, we designed and fabricated a bionic-lung chip, which was applied to perform cigarette smoke exposure of BEAS-2B cells cultured at the gas-liquid interface. The oxidative stress and inflammation in the cells exposed to cigarette smoke were investigated on chip. The results showed that cellular damage, oxidative stress and inflammatory response induced by cigarette smoke in the chip were dependent on smoke concentration and time after smoke exposure. N-Acetylcysteine (NAC) significantly inhibited these effects of cigarette smoke exposure on the cells at the gas-liquid interface within the chip.

Water-Soluble Au25 Clusters with Single-Crystal Structure for Mitochondria-Targeting Radioimmunotherapy.

Atomically precise gold clusters play an important role in the development of high-Z-element-based radiosensitizers, due to their intriguing structural diversity and advantages in correlating structures and properties. However, the synthesis of gold clusters with both water-solubility and single-crystal structure remains a challenge. In this study, atomically precise Au25(S-TPP)18 clusters (TPP-SNa = sodium 3-(triphenylphosphonio)propane-1-thiolate bromide) showing both mitochondria-targeting ability and water-solubility were obtained via ligand design for enhanced radioimmunotherapy. Compared with Au25(SG)18 clusters (SG = glutathione), Au25(S-TPP)18 exhibited higher radiosensitization efficiency due to its mitochondria-targeting ability, higher ROS production capacity, and obvious inhibition upon thioredoxin reductase (TrxR). In addition, the enhanced radiotherapy-triggered abscopal effect in combination with checkpoint blockade displayed effective growth inhibition of distant tumors. This work reveals the ligand-regulated organelle targeting ability of metal clusters by which feasible strategies to promote their application in precise theranostics could be realized.

Design of a microfluidic lung chip and its application in assessing the toxicity of formaldehyde.

In this work, a microfluidic lung chip with membrane supporting cell growth that can produce multiple concentration gradients of gas and liquid is introduced. The chip is composed of a gas gradient layer in the upper part, a porous membrane supporting cell growth in the middle and a liquid gradient layer in the lower part. The gas-liquid interface environment of the cells on the membrane can expose the cells to the gas in the upper layer and the liquid in the lower layer at the same time. Then, the chip is applied to the toxicity testing of formaldehyde in A549 cells. The results showed that at 6 × 10-5 mol/L formaldehyde, the survival rate of the cells in four channels were 90, 87, 81, and 71%, which shows a dose-response trend under the influence of different concentrations of formaldehyde. ROS staining results also showed that formaldehyde exposure at 6 × 10-5 mol/L lead to the increase of ROS level in the cells. These results suggest that the chip based on cell growth on membrane could be used for toxicological evaluation of environmental polluting gases.

Development and Validation of a Modified QuEChERS Method for Simultaneous Analysis of 250 Flavor Constituents in Tobacco by Gas Chromatography Tandem Mass Spectrometry.

BACKGROUND: Flavor constituents play an important role in the flavor characteristics of tobacco leaves and cigarettes. Sensitive, selective, and high-throughput multi-analyte analytical methods are needed to satisfy the demand for analyzing trace-level flavor constituents in tobacco. However, trace analysis of multi-targets in a complex tobacco matrix is significantly challenging. OBJECTIVE: This study was undertaken to develop and validate a fast, selective, sensitive, and accurate GC-tandem mass spectrometry (GC-MS/MS) method for the simultaneous analysis of 250 flavor constituents in tobacco using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure and backflushing technique. METHODS: The samples were extracted with a mixture of acetonitrile and phosphate buffer. GC-MS/MS served as a reliable tool to quantify the flavor constituents due to its high sensitivity, selectivity, and good repeatability. RESULTS: Our evaluation showed that 243 flavor constituents presented good linearity. Average recoveries of 216 target compounds in tobacco ranged from 70 to 120% with RSDs less than 20% at three fortification levels. The limits of quantitation of 225 and 25 compounds were in the range of 2-50 and 51-112 µg/kg, respectively. This method was then successfully applied to the analysis of 15 commercial cigarette samples with different style characteristics. CONCLUSION: The modified QuEChERS method worked very well for a wide range of flavor constituents that have not been reported by QuEChERS pretreatment previously, and the use of concurrent backflushing offered significant increase in system robustness and sample throughput. The method greatly improved the detection performance and the range of the flavor constituents, and proved to be more accurate, sensitive, selective, convenient, and practical than the reported method, and thus, can be applied in routine analysis. HIGHLIGHTS: A validated QuEChERS-based GC-MS/MS method for multiple flavor constituents analysis in tobacco was reported for the first time. The use of concurrent backflushing markedly improved the instrument robustness and sample throughput.