Pingwei Xu, Junjie Chi, Xiaochen Wang, Meng Zhu, Kai Chen, Qihui Fan, Fangfu Ye, Changmin Shao
Liver cancer, characterized as a kind of malignant tumor within the digestive system, poses great health harm, and immune escape stands out as an important reason for its occurrence and development. Chemokines, pivotal in guiding immune cells' migration, is necessary to initiate and deliver an effective anti-tumor immune response. Therefore, understanding the chemotactic environment and identifying chemokines that regulate recruitment of immune cells to the tumor microenvironment (TME) are critical to improve current immunotherapy interventions. Herein, we report a well-defined inverse opal scaffold generated with a microfluidic emulsion template for the construction of a vascularized liver tumor model, offering insights into immune cells' recruitment. Due to the excellent 3D porous morphology of the inverse opal scaffold, human hepatocellular carcinoma cells can aggregate in the pores of the scaffold to form uniform multicellular tumor spheroids. More attractively, the vascularized liver tumor model can be achieved by constructing a 3D co-culture system involving endothelial cells and hepatocellular carcinoma cells. The results demonstrate that the 3D co-cultured tumor cells increase the neutrophil chemokines remarkably and recruit neutrophils to tumor tissues, then promote tumor progression. This approach opens a feasible avenue for realizing a vascularized liver tumor model with a reliable immune microenvironment close to that of a solid tumor of liver cancer.
{"title":"<i>In vitro</i> vascularized liver tumor model based on a microfluidic inverse opal scaffold for immune cell recruitment investigation.","authors":"Pingwei Xu, Junjie Chi, Xiaochen Wang, Meng Zhu, Kai Chen, Qihui Fan, Fangfu Ye, Changmin Shao","doi":"10.1039/d4lc00341a","DOIUrl":"https://doi.org/10.1039/d4lc00341a","url":null,"abstract":"<p><p>Liver cancer, characterized as a kind of malignant tumor within the digestive system, poses great health harm, and immune escape stands out as an important reason for its occurrence and development. Chemokines, pivotal in guiding immune cells' migration, is necessary to initiate and deliver an effective anti-tumor immune response. Therefore, understanding the chemotactic environment and identifying chemokines that regulate recruitment of immune cells to the tumor microenvironment (TME) are critical to improve current immunotherapy interventions. Herein, we report a well-defined inverse opal scaffold generated with a microfluidic emulsion template for the construction of a vascularized liver tumor model, offering insights into immune cells' recruitment. Due to the excellent 3D porous morphology of the inverse opal scaffold, human hepatocellular carcinoma cells can aggregate in the pores of the scaffold to form uniform multicellular tumor spheroids. More attractively, the vascularized liver tumor model can be achieved by constructing a 3D co-culture system involving endothelial cells and hepatocellular carcinoma cells. The results demonstrate that the 3D co-cultured tumor cells increase the neutrophil chemokines remarkably and recruit neutrophils to tumor tissues, then promote tumor progression. This approach opens a feasible avenue for realizing a vascularized liver tumor model with a reliable immune microenvironment close to that of a solid tumor of liver cancer.</p>","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jaehoon Kim, Taehee Yoon, Sungryeong Lee, Paul J Kim, YongTae Kim
Tissue barriers in a body, well known as tissue-to-tissue interfaces represented by endothelium of the blood vessels or epithelium of organs, are essential for maintaining physiological homeostasis by regulating molecular and cellular transports. It is crucial for predicting drug response to understand physiology of tissue barriers through which drugs are absorbed, distributed, metabolized and excreted. Since the FDA Modernization Act 2.0, which prompts the inception of alternative technologies for animal models, tissue barrier chips, one of the applications of organ-on-a-chip or microphysiological system (MPS), have only recently been utilized in the context of drug development. Recent advancements in stem cell technology have brightened the prospects for the application of tissue barrier chips in personalized medicine. In past decade, designing and engineering these microfluidic devices, and demonstrating the ability to reconstitute tissue functions were main focus of this field. However, the field is now advancing to the next level of challenges: validating their utility in drug evaluation and creating personalized models using patient-derived cells. In this review, we briefly introduce key design parameters to develop functional tissue barrier chip, explore the remarkable recent progress in the field of tissue barrier chips and discuss future perspectives on realizing personalized medicine through the utilization of tissue barrier chips.
{"title":"Reconstitution of human tissue barrier function for precision and personalized medicine.","authors":"Jaehoon Kim, Taehee Yoon, Sungryeong Lee, Paul J Kim, YongTae Kim","doi":"10.1039/d4lc00104d","DOIUrl":"https://doi.org/10.1039/d4lc00104d","url":null,"abstract":"<p><p>Tissue barriers in a body, well known as tissue-to-tissue interfaces represented by endothelium of the blood vessels or epithelium of organs, are essential for maintaining physiological homeostasis by regulating molecular and cellular transports. It is crucial for predicting drug response to understand physiology of tissue barriers through which drugs are absorbed, distributed, metabolized and excreted. Since the FDA Modernization Act 2.0, which prompts the inception of alternative technologies for animal models, tissue barrier chips, one of the applications of organ-on-a-chip or microphysiological system (MPS), have only recently been utilized in the context of drug development. Recent advancements in stem cell technology have brightened the prospects for the application of tissue barrier chips in personalized medicine. In past decade, designing and engineering these microfluidic devices, and demonstrating the ability to reconstitute tissue functions were main focus of this field. However, the field is now advancing to the next level of challenges: validating their utility in drug evaluation and creating personalized models using patient-derived cells. In this review, we briefly introduce key design parameters to develop functional tissue barrier chip, explore the remarkable recent progress in the field of tissue barrier chips and discuss future perspectives on realizing personalized medicine through the utilization of tissue barrier chips.</p>","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Thomas Kellerer, Bettina Sailer, Patrick Byers, Rune Barnkob, Oliver Hayden, Thomas Hellerer
We combine two-photon-excited fluorescence microscopy and acoustofluidic trapping in a spherical microchamber to in vitro study cells and cell clusters three-dimensionally close to in vivo conditions. The two-photon microscopy provides the in-depth 3D analysis of the spherical microchamber dimensions as well as the positions of trapped samples therein with high spatial precision and high temporal resolution enabling even tracking of the fast moving particles. Furthermore, optical sectioning allows to gather information of individual cells in trapped cell clusters inside the chamber. We demonstrate real-time monitoring of osmosis in A549 lung cells and red blood cells as one possible biomedical application. The observed osmosis reduced the cell membrane diameter by approximately 4 μm in the A549 cells and by approximately 2 μm in the red blood cells. Our approach provides an important optical tool for future investigations of cell functions and cell-cell interactions avoiding wall-contact inside an acoustofluidic device.
{"title":"Two-photon microscopy of acoustofluidic trapping for highly sensitive cell analysis.","authors":"Thomas Kellerer, Bettina Sailer, Patrick Byers, Rune Barnkob, Oliver Hayden, Thomas Hellerer","doi":"10.1039/d4lc00144c","DOIUrl":"https://doi.org/10.1039/d4lc00144c","url":null,"abstract":"<p><p>We combine two-photon-excited fluorescence microscopy and acoustofluidic trapping in a spherical microchamber to <i>in vitro</i> study cells and cell clusters three-dimensionally close to <i>in vivo</i> conditions. The two-photon microscopy provides the in-depth 3D analysis of the spherical microchamber dimensions as well as the positions of trapped samples therein with high spatial precision and high temporal resolution enabling even tracking of the fast moving particles. Furthermore, optical sectioning allows to gather information of individual cells in trapped cell clusters inside the chamber. We demonstrate real-time monitoring of osmosis in A549 lung cells and red blood cells as one possible biomedical application. The observed osmosis reduced the cell membrane diameter by approximately 4 μm in the A549 cells and by approximately 2 μm in the red blood cells. Our approach provides an important optical tool for future investigations of cell functions and cell-cell interactions avoiding wall-contact inside an acoustofluidic device.</p>","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141416708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shi Feng, Chundong Xue, Cunliang Pan, Shengyang Tao
Droplets generated through microfluidics serve as a common platform for assembling artificial cells, which are feasibly tailored using microfluidic methodology. The ability of natural cells to undergo shape changes, such as phagocytosis, is a typical characteristic that researchers aim to mimic in artificial cells. However, simulating the deformation behavior of natural cells within droplets is exceptionally challenging. Here, this study reports a pinocytosis-like phenomenon observed in droplets, termed "droplet drinking." When droplets traverse a capillary with slits, the shear force from the continuous-phase fluid induces relative motion within the droplets, creating concave regions at the rear. These regions facilitate engulfing of the continuous-phase fluid, resulting in the formation of multiple emulsions. This behavior is influenced by the capillary number, and the size of the ingested droplets is governed by the interfacial tension between the two phases. The production of multicore or multi-shell emulsions can be easily accomplished by making slight adjustments to the slit structure. Furthermore, this method demonstrates the integration of reactants into pre-existing droplets, facilitating biochemical reactions. This study presents a convenient approach for generating complex emulsions and an innovative strategy for studying deformation behavior in droplet-based artificial cells.
{"title":"Droplet Drinking in Constrictions","authors":"Shi Feng, Chundong Xue, Cunliang Pan, Shengyang Tao","doi":"10.1039/d4lc00381k","DOIUrl":"https://doi.org/10.1039/d4lc00381k","url":null,"abstract":"Droplets generated through microfluidics serve as a common platform for assembling artificial cells, which are feasibly tailored using microfluidic methodology. The ability of natural cells to undergo shape changes, such as phagocytosis, is a typical characteristic that researchers aim to mimic in artificial cells. However, simulating the deformation behavior of natural cells within droplets is exceptionally challenging. Here, this study reports a pinocytosis-like phenomenon observed in droplets, termed \"droplet drinking.\" When droplets traverse a capillary with slits, the shear force from the continuous-phase fluid induces relative motion within the droplets, creating concave regions at the rear. These regions facilitate engulfing of the continuous-phase fluid, resulting in the formation of multiple emulsions. This behavior is influenced by the capillary number, and the size of the ingested droplets is governed by the interfacial tension between the two phases. The production of multicore or multi-shell emulsions can be easily accomplished by making slight adjustments to the slit structure. Furthermore, this method demonstrates the integration of reactants into pre-existing droplets, facilitating biochemical reactions. This study presents a convenient approach for generating complex emulsions and an innovative strategy for studying deformation behavior in droplet-based artificial cells.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jie Hong, Hao He, Yingjia Xu, Shujing Wang, Chunxiong Luo
Cells can respond and adapt to complex forms of environmental change. Budding yeast is wildly used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a micro-heater(using liquid metal) and thermocouple(liquid metal vs conductive PDMS) on a chip. Three forms of temperature changes: step, gradient, and periodical oscillations were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37°C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated.
{"title":"An integrative temperature-controlled microfluidic system for budding yeast heat shock response analysis in single cell level","authors":"Jie Hong, Hao He, Yingjia Xu, Shujing Wang, Chunxiong Luo","doi":"10.1039/d4lc00313f","DOIUrl":"https://doi.org/10.1039/d4lc00313f","url":null,"abstract":"Cells can respond and adapt to complex forms of environmental change. Budding yeast is wildly used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a micro-heater(using liquid metal) and thermocouple(liquid metal vs conductive PDMS) on a chip. Three forms of temperature changes: step, gradient, and periodical oscillations were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37°C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141425408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Niels Klement, Elia Savino, Wesley R. Browne, Elisabeth Verpoorte
The control over fluid flow achievable in microfluidic devices creates opportunities for applications in many fields. In simple microchannels flow is purely laminar when one solvent is used, and hence, achieving reliable mixing is an important design consideration. Integration of structures, such as grooves, into the channels to act as static mixers is a commonly used approach. The mixing induced by these structures can be validated by determining concentration profiles in microfluidic channels following convergence of solvent streams from separate inlets. Spatially resolved characterisation is therefore necessary and requires in-line analysis methods. Here we report a line-focused illumination approach to provide operando, spatially resolved Raman spectra across the width of channels in the analysis of single- and multi-phase liquid systems and chemical reactions. A scientific complementary metal oxide semiconductor (sCMOS) sensor is used to overcome smearing encountered during spectral readout of images with CCD sensors. Isotopically labelled probes, in otherwise identical flow streams, show that z-confocality limits the spatial resolution and certainty as to the extent of mixing that can be achieved. These limitations are overcome using fast chemical reactions between reagents entering a microchannel in separate solvent streams. We show here that the progression of a chemical reaction, for which only the product is observable, is a powerful approach to determine the extent of mixing in a microchannel. Specifically resonance enhancement of Raman scattering from a product formed allows for determination of the true efficiency of mixing over the length and width of microchannels. Raman spectral images obtained by line-focused illumination show onset of mixing by observing the product of reagents entering from the separate inlets. Mixing is initially off-centre and immediately before the apex of the first groove of the static mixer, and then evolves along the entire width of the channel after a full cycle of grooves.
{"title":"In-line Raman imaging of mixing by herringbone grooves in microfluidic channels","authors":"Niels Klement, Elia Savino, Wesley R. Browne, Elisabeth Verpoorte","doi":"10.1039/d4lc00115j","DOIUrl":"https://doi.org/10.1039/d4lc00115j","url":null,"abstract":"The control over fluid flow achievable in microfluidic devices creates opportunities for applications in many fields. In simple microchannels flow is purely laminar when one solvent is used, and hence, achieving reliable mixing is an important design consideration. Integration of structures, such as grooves, into the channels to act as static mixers is a commonly used approach. The mixing induced by these structures can be validated by determining concentration profiles in microfluidic channels following convergence of solvent streams from separate inlets. Spatially resolved characterisation is therefore necessary and requires in-line analysis methods. Here we report a line-focused illumination approach to provide operando, spatially resolved Raman spectra across the width of channels in the analysis of single- and multi-phase liquid systems and chemical reactions. A scientific complementary metal oxide semiconductor (sCMOS) sensor is used to overcome smearing encountered during spectral readout of images with CCD sensors. Isotopically labelled probes, in otherwise identical flow streams, show that z-confocality limits the spatial resolution and certainty as to the extent of mixing that can be achieved. These limitations are overcome using fast chemical reactions between reagents entering a microchannel in separate solvent streams. We show here that the progression of a chemical reaction, for which only the product is observable, is a powerful approach to determine the extent of mixing in a microchannel. Specifically resonance enhancement of Raman scattering from a product formed allows for determination of the true efficiency of mixing over the length and width of microchannels. Raman spectral images obtained by line-focused illumination show onset of mixing by observing the product of reagents entering from the separate inlets. Mixing is initially off-centre and immediately before the apex of the first groove of the static mixer, and then evolves along the entire width of the channel after a full cycle of grooves.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141333638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cardiac muscle, a subtype of striated muscle composing our heart, has garnered attention as a source of autonomously driving actuators due to its inherent capability for spontaneous contraction. However, conventional cardiac biohybrid robots have utilized planar (2D) cardiac tissue consisting of a thin monolayer of cardiac myotubes with a thickness of 3-5 μm, which can generate a limited contraction force per unit footprint. In this study, we propose 3D cardiac muscle rings as robotic actuator units, which not only exhibit higher contractile force per unit footprint due to gaining their height than their 2D counterparts but also can be integrated into desired 3D configurations. We fabricated cardiac muscle rings from human iPS cell-derived cardiomyocytes, evaluated their driving characteristics, and verified the actuation effects by integrating them with artificial components. We confirmed that the cardiac muscle rings exhibited rhythmic spontaneous contraction and increased contractile force in response to stretching stimuli. Furthermore, after constructing a centimeter-sized biohybrid self-beating actuator with an antagonistic pair structure of cardiac muscle rings, we confirmed the periodic antagonistic beating motion at its tail portion. We believe that 3D cardiac muscle rings, possessing high contractile force and capable of being positioned within limited 3D space, can be used as potent biohybrid robotic actuators.
{"title":"Human induced pluripotent stem cell-derived cardiac muscle rings for biohybrid self-beating actuator","authors":"Tomohiro Morita, Minghao Nie, Shoji Takeuchi","doi":"10.1039/d4lc00276h","DOIUrl":"https://doi.org/10.1039/d4lc00276h","url":null,"abstract":"Cardiac muscle, a subtype of striated muscle composing our heart, has garnered attention as a source of autonomously driving actuators due to its inherent capability for spontaneous contraction. However, conventional cardiac biohybrid robots have utilized planar (2D) cardiac tissue consisting of a thin monolayer of cardiac myotubes with a thickness of 3-5 μm, which can generate a limited contraction force per unit footprint. In this study, we propose 3D cardiac muscle rings as robotic actuator units, which not only exhibit higher contractile force per unit footprint due to gaining their height than their 2D counterparts but also can be integrated into desired 3D configurations. We fabricated cardiac muscle rings from human iPS cell-derived cardiomyocytes, evaluated their driving characteristics, and verified the actuation effects by integrating them with artificial components. We confirmed that the cardiac muscle rings exhibited rhythmic spontaneous contraction and increased contractile force in response to stretching stimuli. Furthermore, after constructing a centimeter-sized biohybrid self-beating actuator with an antagonistic pair structure of cardiac muscle rings, we confirmed the periodic antagonistic beating motion at its tail portion. We believe that 3D cardiac muscle rings, possessing high contractile force and capable of being positioned within limited 3D space, can be used as potent biohybrid robotic actuators.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141326829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hanfei Shen, Yasemin Atiyas, Zijian Yang, Andrew Lin, Jingbo Yang, Diao Liu, Juhwan Park, Wei Guo, David Issadore
The expression of Programmed Death-Ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput Droplet-based Extracellular Vesicle Analysis (DEVA) assay for ultrasensitive, quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360x enhancement in the limit of detection (LOD) and a 750x improvement in the limit of quantification (LOQ) compared to conventional plate enzyme linked immunoassay (ELISA). Underlying this performance is DEVA’s high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs versus background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per experiment, ~100x greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.
细胞外囊泡 (EVs) 上表达的程序性死亡配体 1 (PD-L1) 是一种新兴的癌症生物标记物，它在免疫疗法中的介导作用尤其引人关注。然而，临床样本中 PD-L1+ EVs 的精确定量仍然具有挑战性，因为它们的浓度稀少，而且人体血浆中的背景 EVs 数量巨大，限制了传统方法的适用性。在这项研究中，我们开发了一种基于液滴的高通量细胞外囊泡分析（DEVA）测定法，用于超灵敏地定量检测血浆中PD-L1和四泛素（CD81）双重阳性的EVs。与传统的平板酶联免疫测定 (ELISA) 相比，我们的性能大大超过了传统方法，检测限 (LOD) 提高了 360 倍，定量限 (LOQ) 提高了 750 倍。这种性能的基础是 DEVA 一次对单个 EV 的高通量分析，以及对目标 EV 相对于背景的高特异性。我们利用 EV 表面具有多个目标配体拷贝所产生的亲和力效应，使每个液滴的假阳性率仅为 0.006%。我们使用并行化光流体技术，每次实验可快速处理 1000 万个液滴，是传统方法的 100 倍。对 14 名黑色素瘤患者进行的验证研究证实，DEVA 能够在减少样本量的情况下与传统的 ELISA 测量相匹配，而且无需事先进行 EV 纯化。这项概念验证研究证明了 DEVA 在临床上的应用潜力，可用于加强预后判断和指导癌症治疗。
{"title":"Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay","authors":"Hanfei Shen, Yasemin Atiyas, Zijian Yang, Andrew Lin, Jingbo Yang, Diao Liu, Juhwan Park, Wei Guo, David Issadore","doi":"10.1039/d4lc00331d","DOIUrl":"https://doi.org/10.1039/d4lc00331d","url":null,"abstract":"The expression of Programmed Death-Ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput Droplet-based Extracellular Vesicle Analysis (DEVA) assay for ultrasensitive, quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360x enhancement in the limit of detection (LOD) and a 750x improvement in the limit of quantification (LOQ) compared to conventional plate enzyme linked immunoassay (ELISA). Underlying this performance is DEVA’s high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs versus background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per experiment, ~100x greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141315764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Optofluidic regulating of blood microflow in vivo represents a significant method for investigating illnesses linked to abnormal changes in blood circulation. Currently, non-invasive strategies are limited to regulation within capillaries of approximately 10 μm in diameter because the adaption to blood pressure levels in the order of several hundred pascals poses a significant challenge in larger microvessels. In this study, using laser-induced microbubble formation within microvessels of the mouse auricle, we regulate blood microflow in small vessels with diameters in the tens of micrometers. By controlling the laser power, we can control the growth and stability of microbubbles in vivo. This controlled approach enables the achievement of prolonged ischemia and subsequent reperfusion of blood flow, and it can also regulate the microbubbles to function as micro-pumps for reverse blood pumping. Furthermore, by controlling the microbubble, narrow microflow channels can be formed between the microbubbles and microvessels for assessing the apparent viscosity of leukocytes, which is 76.9 ± 11.8 Pa·s in the in vivo blood environment. The proposed design of in vivo microbubble valves opens new avenues for constructing real-time blood regulation and exploring cellular mechanics within living organisms.
{"title":"Laser-induced Microbubble as an In Vivo Valve for Optofluidic Manipulation in Living Mice’s Microvessels","authors":"Meng Shao, Changxu Li, Chun Meng, Rui Liu, Panpan Yu, Fengya Lu, Zhensheng Zhong, Xunbin Wei, Jinhua Zhou, Min-Cheng Zhong","doi":"10.1039/d4lc00095a","DOIUrl":"https://doi.org/10.1039/d4lc00095a","url":null,"abstract":"Optofluidic regulating of blood microflow in vivo represents a significant method for investigating illnesses linked to abnormal changes in blood circulation. Currently, non-invasive strategies are limited to regulation within capillaries of approximately 10 μm in diameter because the adaption to blood pressure levels in the order of several hundred pascals poses a significant challenge in larger microvessels. In this study, using laser-induced microbubble formation within microvessels of the mouse auricle, we regulate blood microflow in small vessels with diameters in the tens of micrometers. By controlling the laser power, we can control the growth and stability of microbubbles in vivo. This controlled approach enables the achievement of prolonged ischemia and subsequent reperfusion of blood flow, and it can also regulate the microbubbles to function as micro-pumps for reverse blood pumping. Furthermore, by controlling the microbubble, narrow microflow channels can be formed between the microbubbles and microvessels for assessing the apparent viscosity of leukocytes, which is 76.9 ± 11.8 Pa·s in the in vivo blood environment. The proposed design of in vivo microbubble valves opens new avenues for constructing real-time blood regulation and exploring cellular mechanics within living organisms.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141315556","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Urinalysis is a heavily used diagnostic test in clinical laboratories, while it is chronically holden back by urine sediment microscopic examination. Current instruments are bulky and expensive to be widely adopted, making microscopic examination a procedure that still relies on manual operations and requires large time and labor costs. To improve the efficacy and automation of urinalysis, this study develops an acoustofluidics-based microscopic examination system. The system utilizes the combination of acoustofluidic manipulation and passive hydrodynamic mechanism thus achieves high throughput (1000 Lmin-1) and high concentration factor (95.2±2.1 fold) simultaneously, fulfilling the demands for urine examination. The concentrated urine sample is automatically dispensed into a hemocytometer chamber and the images are then analyzed by machine learning algorithm. The whole process is completed within 3 minutes with detection accuracies of erythrocytes and leukocytes at 94.6±3.5% and 95.1±1.8% respectively. The examination outcome of urine samples from 50 volunteers by this device shows a correlation coefficient of 0.96 compared to manual microscopic examination. Our system offers a promising tool for automated urine microscopic examination, thus it is of potential to save large amount of time and labor in clinical laboratories, as well as to promote point-of-care urine testing applications in and beyond hospitals.
{"title":"Acoustofluidics-based microscopic examination for automated and point-of-care urinalysis","authors":"Xin He, Feng Ren, Yangyang Wang, Zhiyuan Zhang, Jiming Zhou, Jian Huang, Shuye Cao, Jinying Dong, Renxin Wang, Mengxi Wu, Junshan Liu","doi":"10.1039/d4lc00408f","DOIUrl":"https://doi.org/10.1039/d4lc00408f","url":null,"abstract":"Urinalysis is a heavily used diagnostic test in clinical laboratories, while it is chronically holden back by urine sediment microscopic examination. Current instruments are bulky and expensive to be widely adopted, making microscopic examination a procedure that still relies on manual operations and requires large time and labor costs. To improve the efficacy and automation of urinalysis, this study develops an acoustofluidics-based microscopic examination system. The system utilizes the combination of acoustofluidic manipulation and passive hydrodynamic mechanism thus achieves high throughput (1000 Lmin-1) and high concentration factor (95.2±2.1 fold) simultaneously, fulfilling the demands for urine examination. The concentrated urine sample is automatically dispensed into a hemocytometer chamber and the images are then analyzed by machine learning algorithm. The whole process is completed within 3 minutes with detection accuracies of erythrocytes and leukocytes at 94.6±3.5% and 95.1±1.8% respectively. The examination outcome of urine samples from 50 volunteers by this device shows a correlation coefficient of 0.96 compared to manual microscopic examination. Our system offers a promising tool for automated urine microscopic examination, thus it is of potential to save large amount of time and labor in clinical laboratories, as well as to promote point-of-care urine testing applications in and beyond hospitals.","PeriodicalId":85,"journal":{"name":"Lab on a Chip","volume":null,"pages":null},"PeriodicalIF":6.1,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141315558","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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