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Two-dimensional (2D) tumor model has always poorly predicted drug response of animal model due to the lack of recapitulation of tumor microenvironment. Establishing a biomimetic, controllable, and cost-effective three-dimensional (3D) model and large-scale validation of its in vivo predictivity has shown promise in bridging the gap between the 2D tumor model and animal model. Here, we established a matrigel-based 3D micro-tumor model on an array chip for large-scale anticancer drug evaluation. Compared with the 2D tumor model, the 3D tumor model on the chip showed spheroid morphology, slower proliferation kinetics, and comparable reproducibility. Next, the results of the chemotherapeutic evaluation from 18 drugs against 27 cancer cell lines showed 17.6% of drug resistance on the 3D tumor model. Moreover, the evaluation results of targeted drugs showed expected sensitivity and higher specificity on the 3D tumor model compared with the 2D model. Finally, the evaluation results on the 3D tumor model were more consistent with the in vivo cell-derived xenograft model, and excluded 95% false-positive results from the 2D model. Overall, the matrigel-based 3D micro-tumor model on the array chip provides a promising tool to accelerate anticancer drug discovery.
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Nine undescribed steroidal alkaloid glycosides, unibrasolanosides A-F, unibraverazosides A-B, and unibratomatoside A, were isolated from the bulbs of Fritillaria unibracteata P. K. Hsiao & K. C. Hsia (Liliaceae). Their structures were elucidated by HRESIMS and 1D and 2D NMR data analyses as well as chemical methods and single-crystal X-ray diffraction analyses. Further investigation revealed that eight steroidal alkaloid glycosides displayed moderate anti-inflammatory activity in vivo in a CuSO4-induced transgenic zebrafish model.
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The development of an in vitro model for predicting drug permeability through the human blood-brain barrier (BBB) will greatly accelerate the development of neural therapy. Previously reported platforms for BBB model construction cannot meet the requirements of constant-rate and high-throughput flow, as well as compatibility with the commercial meter for real-time transendothelial electrical resistance (TEER) measurement. Herein, a constant-rate perfused array chip (cPAC) was developed to establish a brain endothelium model for screening drug permeability. The cPAC consisted of 24 units with four layers. Three reservoirs on the top had a 0.5 mm center-to-center spacing, enabling real-time detection of the TEER with the commercial volt-ohm meter. With the optimized chip design, the constant-rate and high-throughput flow by gravity was achieved. Compared with the static culture of the Transwell, the brain endothelium model on the cPAC exhibited superior performance in barrier function, efflux functionality of the transporters, and reversible osmotic opening of the brain endothelium. More importantly, the permeability of model drugs on the cPAC matched the in vivo results with the correlation coefficient reaching 0.994. Finally, the brain endothelium model was cocultured with 3D tumor cells for simultaneous evaluation of drug permeability and brain tumor therapy. The drug efficacy at the target cells on the coculture model was also consistent with clinical findings. These results demonstrated that this platform provides a promising tool for brain endothelium model establishment to predict drug permeability and brain therapy. We anticipate the cPAC to be widely accepted for establishing various barrier models.
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Astrócitos , Ensaios de Triagem em Larga Escala , Humanos , Barreira Hematoencefálica , Encéfalo , Permeabilidade , Técnicas de Cocultura , Endotélio Vascular , Células CultivadasRESUMO
An in vitro human renal proximal tubule model that represents the proper transporter expression and pronounced epithelial polarization is necessary for the accurate prediction of nephrotoxicity. Here, we constructed a high-throughput human renal proximal tubule model based on an integrated biomimetic array chip (iBAC). Primary human renal proximal tubule epithelial cells (hRPTECs) cultured on this microfluidic platform were able to form a tighter barrier, better transporter function and more sensitive nephrotoxicity prediction than those on the static Transwell. Compared with the human immortalized HK2 model, the hRPTECs model on the chip gained improved apical-basolateral polarization, barrier function and transporter expression. Polymyxin B could induce nephrotoxicity not only from the apical of the hRPTECs, but also from the basolateral side on the iBAC. However, other chemotherapeutic agents, such as doxorubicin and sunitinib, only induced nephrotoxicity from the apical surface of the hRPTECs on the iBAC. In summary, our renal proximal tubule model on the chip exhibits improved epithelial polarization and membrane transporter activity, and can be implemented as an effective nephrotoxicity-screening toolkit.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Dispositivos Lab-On-A-Chip , Doxorrubicina , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/farmacologia , Polimixina B/metabolismo , Polimixina B/farmacologia , Sunitinibe/metabolismo , Sunitinibe/farmacologiaRESUMO
Drug-induced liver injury (DILI) is a leading cause of therapy failure in the clinic and also contributes much to acute liver failure cases. Investigations of predictive sensitivity in animal models have limitations due to interspecies differences. Previously reported in vitro models of liver injury based on primary human hepatocytes (PHHs) cannot meet the requirements of high physiological fidelity, low cost, simple operation, and high throughput with improved sensitivity. Herein, we developed an integrated biomimetic array chip (iBAC) for establishing extracellular matrix (ECM)-based models. A collagen-based 3D PHH model was constructed on the iBAC as a case for the prediction of clinical DILI at throughput. The iBAC has a three-layer structure with a core component of 3D implanting holes. At an initial cell seeding numbers of 5000-10,000, the collagen-based 3D PHH model was optimized with improved and stabilized liver functionality, including cell viability, albumin, and urea production. Moreover, basal activities of most metabolic enzymes on the iBAC were maintained for at least 12 days. Next, a small-scale hepatotoxicity screening indicated that the 3D PHH model on the iBAC was more sensitive for predicting hepatotoxicity than the 2D PHH model on the plate. Finally, a large-scale screening of liver toxicity using 122 clinical drugs further demonstrated that the collagen-based 3D PHH model on the iBAC had superior predictive sensitivity compared to all previously reported in vitro models. These results indicated the importance of 3D collagen for liver physiological functionality and hepatotoxicity prediction. We anticipant it being a promising tool for risk assessment of drug-induced hepatotoxicity with a widespread acceptance in drug industry.
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Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Hepatócitos , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Biomimética , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacosRESUMO
Cardiovascular diseases seriously endanger human health and life. The accompanying myocardial injury has been a focus of attention in society. Chinese medicine,serving as a natural and precious reservoir for the research and development of new drugs,is advantageous in resisting myocardial injury due to its multi-component,multi-pathway,and multi-target characteristics. In recent years,with the extensive application of culture method for isolated cardiomyocytes,a cost-effective,controllable in vitro model of cardiomyocyte injury with uniform samples is becoming a key tool for mechanism research on cardiomyocyte injury and drug development.A good in vitro model can reduce experimental and manpower cost,and also accurately stimulate clinical changes to reveal the mechanism. Therefore,the selection and establishment of in vitro model are crucial for the in-depth research. This study summarized the modeling principles,evaluation indicators,and application of more than ten models reflecting different clinical conditions,such as injuries induced by hypoxia-reoxygenation,hypertrophy,oxidative stress,inflammation,internal environmental disturbance,and toxicity. Furthermore,we analyzed advantages and technical difficulties,aiming to provide a reference for in-depth research on myocardial injury mechanism and drug development.
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Apoptose , Miócitos Cardíacos , Hipóxia Celular , Humanos , Miocárdio , Estresse OxidativoRESUMO
Species differences in metabolism may produce failure prediction of drug efficacy/toxicity in humans. Integration of metabolic competence and cellular effect assays in vitro can provide insight into the species differences in metabolism; however, a co-culture platform with features of high throughput, operational simplicity, low sample consumption, and independent layouts is required for potential usage in industrial test settings. Herein, we developed an integrated array chip (IAC) to evaluate the species differences in metabolism through metabolism-induced anticancer bioactivity as a case. The IAC consisted of two functional parts: a micropillar chip for immobilization of liver microsomes and a microwell chip for three-dimensional (3D) tumor cell culture. First, optimized parameters of the micropillar chip for microsomal encapsulation were obtained by cross-shaped protrusions and a 2.5 µL volume of 3D agarose spots. Next, we examined factors influencing metabolism-induced anticancer bioactivity. Feasibility of the IAC was validated by four model prodrugs using image-based bioactivity detection and mass spectrometry (MS)-based metabolite analysis. Finally, a species-specific IAC was used for selection of animal species that best resembles metabolism-induced drug response to humans at throughputs. Overall, the IAC provides a promising co-culture platform for identifying species differences in metabolism and selection of animal models to accelerate drug discovery.
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Desenvolvimento de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Metabolismo/genética , Especificidade da Espécie , Animais , HumanosRESUMO
The integration of liver metabolism and hepatotoxicity evaluation for anticancer bioactivity assays in vitro is of fundamental importance to better predict the efficacy and safety of anticancer drugs. In particular, there is a lack of co-culture techniques that can fully mimic the physiological microenvironment at speeds consistent with high-throughput screening. Herein, an integrated Biomimetic Array Chip (iBAC) that enables co-culture of three-dimensional (3D) liver and tumor microtissues was developed for advanced anticancer bioactivity screening at throughputs. The iBAC consisted of two functional chips, a liver chip and a tumor chip containing a cross-shaped protrusion on the tip of a pillar array for co-culture. First, the 3D biomimetic liver microtissue on the liver chip was optimized to mimic superior liver function. Next, the constructed iBAC was evaluated for metabolism-induced anticancer bioactivity by using model prodrugs and for the effect of drug-drug interactions. Finally, the functionality of the iBAC for simultaneous evaluation of anticancer bioactivity and hepatotoxicity was verified. The iBAC exhibits superior performance in biomimetic and integrated functions as well as operationally simple and high-throughput co-culture that makes a good balance between functionality and throughput. Overall, the iBAC provides an integrated, biomimetic and high-throughput co-culture platform to complement the conventional bioactivity assay in tiered screening strategies and could be used as a secondary screening tool at the early phase of drug development.
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Biomimética , Neoplasias , Técnicas de Cocultura , Ensaios de Triagem em Larga Escala , Humanos , Fígado , Análise de Sequência com Séries de Oligonucleotídeos , Microambiente TumoralRESUMO
Transient receptor potential (TRP) channels are emerging drug targets, and TRP channel modulators possess therapeutic potential for many indications. However, there is a lack of intellectual and robust screening assays against TRP channels utilizing the least amount of compounds. Here, a precise microfluidic assay in single-cell profile is developed for the screening of TRP channel modulators. The geometrically optimized microchip is designed for both trapping single cells and utilizing passive pumping for sequential media replacement with low shear stress. The microfluidic chip exhibits superior performance in screening, repeatable compound administration, and improved reproducibility. Using this screening platform, the false-positive and negative rate of the commonly used Ca2+ imaging is reduced from 76.2% to 4.8% and four coumarin derivatives isolated from Murraya species that inhibit TRP channels are identified. One coumarin derivative B-304 reverses TRPA1-mediated inflammatory pain in vivo. Taken together, the data demonstrate that the established microfluidic assay in single-cell profile could be used for the screening of TRP channel modulators that may have therapeutic potential for the channelopathies.
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Traditional Chinese medicine(TCM) is a research area with highly original innovation features,and is also a Chinese name card to the world. However,TCM owns a unique theoretical system which is quite different from western modern medicine,leading to an awkward situation of deficient modern social identity as well as poor international spread. Therefore,how to establish a research strategy in line with the characteristics of TCM itself to systematically interpret the unique scientific connotation of TCM is always a public hot topic. Based on persistent practical exploration and scientific consideration in TCM,our group firstly promoted the concept of traditional Chinese medicine chemical biology(TCM chemical biology,TCMCB). The major idea of TCMCB is to clarify the nature of TCM regulating life progress to link TCM to modern medicine by using TCM components as chemical tools. Notably,TCMCB mainly focuses on TCM target identification and TCM-guided disease molecular mechanism exploration,further to clarify the basic law of TCM mediating disease process. Finally,TCMCB-guided scientific studies can help explain TCM theory and promote the developmentof modern innovative drugs based on identified targets using TCM active components. Moreover,TCMCB is of vital importance for investigating the scientific nature of biological progress and the pattern of disease occurrence and development,indicating a key significance for modern life science and medicine. This review introduces the definition of TCMCB as well as its academic thought,research method,technology system and scientific significance,for providing new research ideas and scientific thoughts for TCM development.
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Pesquisa Interdisciplinar , Medicina Tradicional Chinesa , Biologia , Química , Medicina , Projetos de PesquisaRESUMO
Emerging awareness of cardiac macrophages' role in inflammation after myocardial infarction indicates that overabundant proinflammatory macrophages induce accentuated myocardial injury. The investigation of the macrophages-cardiomyocytes interaction and inflammation-induced dynamic damage in myocardial infarction, especially in a spatiotemporally controlled manner, remains a huge challenge. Here, we developed an in vitro model using a microfluidic coculture system to mimic inflammatory cardiac injury. To our knowledge, on-chip pathological models focused on inflammation-induced myocardial injury have not been reported. The device consists of two sets of thin interconnecting grooves that isolate heterogeneous cells spatially but maintain their soluble factors communication. The mass transportation is visually characterized, and the complete diffusion reaches equilibrium within 100 s. We investigate the dynamic interaction between the macrophages and the cardiomyocytes in the spatiotemporal controlled microenvironment, mimicking a key aspect of the in vivo pathophysiological process. The results show that the activated macrophages induce time-lapsed apoptotic responses of the cardiac cells and damage mitochondria membrane integrity. The anti-inflammatory and cardio-protective effects of quercetin were explored on the chip. The extent of caspase-3 activation is asynchronous in the individual cardiac cells, suggesting the different apoptosis dynamics. We further demonstrate that the mechanism of activated inflammation is associated with the upregulation of several inflammatory cytokines and NF-κB pathway. Thus, the developed microfluidic coculture device provides a useful tool for real-time monitoring of inflammatory response for myocardial disease and holds potential for anti-inflammatory drug screening.
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Técnicas de Cocultura , Inflamação/fisiopatologia , Técnicas Analíticas Microfluídicas , Infarto do Miocárdio/fisiopatologia , Animais , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular , Células Cultivadas , Inflamação/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica , Células RAW 264.7 , RatosRESUMO
Caenorhabditis elegans is a widely used model organism to study development, aging and behavior. Many of these biological studies require staging a large number of worms to assay a synchronized population of animals. Conventional synchronization techniques such as manual picking, gravity stratification and chemical bleaching are labor-intensive and could perturb animals' physiology. Thus, there is a need for a simple inexpensive technology to sort a mixed population of worms based on their developmental stages with minimal perturbation. Here we demonstrate a simple but accurate and high-throughput technique to sort based on animal size, which correlates well with developmental stages. The device consists of an array of geometrically optimized pillars that act as a sieve to allow worms of specific sizes to rapidly move through. With optimized chamber heights, pillar spacing and driving pressures, these binary separation devices are capable of independently separating a mixture of worms at two different stages at average efficiency of around 95%, and throughput of hundreds of worms per minute. In addition, when four devices are used sequentially, we demonstrate the ability to stratify a mixture of worms of all developmental stages with >85% overall efficiency.
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Caenorhabditis elegans/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Tamanho Corporal/fisiologia , Caenorhabditis elegans/crescimento & desenvolvimento , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Modelos Animais , Óvulo/fisiologia , PressãoRESUMO
Chlamydomonas reinhardtii is widely used for studying cilia/flagella, organelles important for human health and disease. In situ monitoring of flagellar assembly/disassembly kinetics in single living cells has been difficult with conventional methods because of time-consuming media exchange and the requirement of whole cell fixation. Here, we develop a PDMS/glass hybrid microfluidic device for real-time tracking of flagellar length in single living cells of Chlamydomonas. Media exchange is precisely controlled by sequential gas-liquid plugs and complete medium replacement occurs within seconds. Rapid medium exchange allows the capture of transient flagellar dynamics. We show that Chlamydomonas cells respond to acidic medium exchange and deflagellate. However, the two flagella may shed asynchronously. After subsequent medium exchange, cells regenerate full-length flagella. Cells are also induced to shorten their flagella after being exposed to extracellular stimuli. The long-term kinetics of flagellar regeneration and disassembly for the whole cell population on the chip are comparable to those from conventional methods; however, individual cells display non-uniform response kinetics. We also find that flagellar growth rate is dependent on flagellar length. This device provides a potential platform to continuously monitor molecular activities associated with changes in flagellar length and to capture transient molecular changes upon flagellar loss, and initiation of flagellar assembly/disassembly.
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Chlamydomonas reinhardtii/citologia , Flagelos/ultraestrutura , Técnicas Analíticas Microfluídicas/métodos , Dimetilpolisiloxanos/química , Vidro/química , Cinética , Técnicas Analíticas Microfluídicas/instrumentação , Fatores de TempoRESUMO
A facile method is presented for achieving comprehensive two-dimensional droplet manipulations in closed microstructures consisting of microwell arrays and a straight microchannel. In this method, picoliter/nanoliter droplets with controllable sizes and numbers are sampled from nanoliter samples/reagents with almost 100% efficiency. Droplet motions are precisely controlled in the ±X-direction and ±Y-direction by managing hydrostatic pressure and magnetic repulsion, respectively. As a demonstration, a fluorescein-labeled droplet and a deionized droplet are successively generated and trapped in adjacent microwells. Then their positions are quickly exchanged without cross-contamination and fusion is implemented on-demand. After operations, hydrophobic ferrofluid can be completely replaced by mineral oil and droplets still remain in microwells safely. A typical fluorescence intensity-based assay is demonstrated: droplet arrays containing copper ion are diluted disproportionately first and then detected by addition of droplet arrays containing Calcein. With the ability of comprehensive two-dimensional droplet manipulations, this method could be used in various miniaturized biochemical analyses including requirements of multistep procedures and in situ monitoring.
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Técnicas Analíticas Microfluídicas , Cobre/análise , Dimetilpolisiloxanos/química , Fluoresceína/química , Fluoresceínas/química , Íons/química , Análise em Microsséries , Óleo Mineral/química , NanotecnologiaRESUMO
Multiple essential microdroplet operation units, including splitting, dispensing, oil-phase exchange, trapping, release and demulsification, were successfully implemented by combining hydrophobic ferrofluid with microfluidic chips.
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Interações Hidrofóbicas e Hidrofílicas , Técnicas Analíticas Microfluídicas/métodos , Magnetismo , Água/químicaRESUMO
Capillary electrophoresis has become a widely useful analytical technology. Amperometric detection is extensively employed in capillary electrophoresis for its many inherent virtues, such as rapid response, remarkable sensitivity, and low cost of both detectors and instrumentations. Analysis of inorganic and small organic ions by capillary electrophoresis is an important research field. This review focuses on the recent developments of capillary electrophoresis coupled with amperometric detection for analysis of inorganic and small organic ions. Advancements in electrophoresis separation modes, amperometric detection modes, working electrodes, and applications of inorganic ions, amino acids, phenols, and amines are discussed.