Due to their label-free and noninvasive nature,impedance measurements have attracted increasing interest in biological research.Advances in microfabrication and integrated-circuit technology have opened a route to usi...Due to their label-free and noninvasive nature,impedance measurements have attracted increasing interest in biological research.Advances in microfabrication and integrated-circuit technology have opened a route to using large-scale microelectrode arrays for real-time,high-spatiotemporal-resolution impedance measurements of biological samples.In this review,we discuss different methods and applications of measuring impedance for cell and tissue analysis with a focus on impedance imaging with microelectrode arrays in in vitro applications.We first introduce how electrode configurations and the frequency range of the impedance analysis determine the information that can be extracted.We then delve into relevant circuit topologies that can be used to implement impedance measurements and their characteristic features,such as resolution and data-acquisition time.Afterwards,we detail design considerations for the implementation of new impedance-imaging devices.We conclude by discussing future fields of application of impedance imaging in biomedical research,in particular applications where optical imaging is not possible,such as monitoring of ex vivo tissue slices or microelectrode-based brain implants.展开更多
Carious lesions are bacteria-caused destructions of the mineralised dental tissues,marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue.While major molec...Carious lesions are bacteria-caused destructions of the mineralised dental tissues,marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue.While major molecular players in tooth decay have been uncovered during the past years,a detailed map of the molecular and cellular landscape of the diseased pulp is still missing.In this study we used single-cell RNA sequencing analysis,supplemented with immunostaining,to generate a comprehensive single-cell atlas of the pulp of carious human teeth.Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth,such as immune cells,mesenchymal stem cells(MSC)and fibroblasts,when compared to the pulp of healthy human teeth.Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters.These changes include the upregulation of genes encoding extracellular matrix(ECM)components,including COL1A1 and Fibronectin(FN1),and the enrichment of the fibroblast cluster with myofibroblasts.The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses.Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues,compared to the healthy ones.The present data demonstrate molecular,cellular and biomechanical alterations in the pulp of human carious teeth,indicative of extensive ECM remodelling,reminiscent of fibrosis observed in other organs.This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.展开更多
AIM: To investigate the distribution of nestin-positive cells in pterygium, as well as the relationship between nestin-positive cells and proliferative cells in the pathogenesis of pterygium. METHODS: Nine pterygium s...AIM: To investigate the distribution of nestin-positive cells in pterygium, as well as the relationship between nestin-positive cells and proliferative cells in the pathogenesis of pterygium. METHODS: Nine pterygium specimens and 5 normal conjunctiva specimens were investigated. All explanted specimens were immediately immersed in 5-Ethynyl-2'-deoxyuridine, and were subjected to hematoxylin and eosin staining, as well as immunostaining to detect nestin. RESULTS: Small sub-populations of nestin-expressing cells in both normal and pterygial conjunctiva epithelium were found. These were located at the superficial layer of the epithelium, and were significantly increased (P=0.007) and spread out in the pterygial conjunctiva epithelium, even though these cells were mitotically quiescent. CONCLUSION: In pterygium, more nestin-positive cells were present at the superficial layer of the epithelium. With growing scientific evidence that nestin plays an important role in defining various specialized cell types, such as stem cells, cancer cells and angiogenic cells, further investigations on the roles of nestin-expressing cells in pterygium may help to uncover the mechanisms of initiation, development and the prognosis of this disease.展开更多
In vitro evaluation of novel therapeutic approaches often fails to reliably predict efficacy and toxicity,especially when recapitulating conditions involving recirculating cells.Current testing strategies are often ba...In vitro evaluation of novel therapeutic approaches often fails to reliably predict efficacy and toxicity,especially when recapitulating conditions involving recirculating cells.Current testing strategies are often based on static co-culturing of cells in suspension and 3D tissue models,where cell sedimentation on the target tissue can occur.The observed effects may then mostly be a consequence of sedimentation and of the corresponding forced cell-tissue interactions.The realization of continuous medium flow helps to better recapitulate physiological conditions and cell-tissue interactions.To tackle current limitations of perfused organ-on-chip approaches,we developed a microfluidic chip and operation concept,which prevents undesired sedimentation and accumulation of suspended cells during multiple days by relying on gravity-driven perfusion.Our platform,which we termed“human immune flow(hiFlow)chip”,enables to co-culture cells in suspension with up to 7 preformed microtissue models.Here,we present the design principle and operation of the platform,and we validate its performance by culturing cells and microtissues of a variety of different origins.Cells and tissues could be monitored on chip via high-resolution microscopy,while cell suspensions and microtissues could be easily retrieved for off-chip analysis.Our results demonstrate that primary immune cells and a range of different spheroid models of healthy and diseased tissues can be maintained for over 6 days on chip.As proof-of-concept cell-tissue interaction assay,we used an antibody treatment against diffuse midline glioma,a highly aggressive pediatric tumor.We are confident that our platform will help to increase the prediction power of in vitro preclinical testing of novel therapeutics that rely on the interaction of circulating cells with organ tissues.展开更多
Cell therapy approaches that employ engineered mam-malian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine.The therapeutic cel...Cell therapy approaches that employ engineered mam-malian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine.The therapeutic cells can be customized to sense user-defined signals,pro-cess them,and respond in a programmable and pre-dictable way.In this paper,we introduce the available tools and strategies employed to design therapeutic cells.Then,various approaches to control cell behav-iors,including open-loop and closed-loop systems,are discussed.We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental dis-ease models.Finally,we consider emerging technolo-gies such as digital devices and their potential for incorporation into future cell-based therapies.展开更多
Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities.Here we present the integration of enzyme-based multi-analyte biosensors into...Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities.Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for‘body-on-a-chip’applications.The microfluidic platform is based on the technology of hanging-drop networks,which is designed for the formation,cultivation,and analysis of fluidically interconnected organotypic spherical three-dimensional(3D)microtissues of multiple cell types.The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes,a platinum counter electrode,and an Ag/AgCl reference electrode.They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended.The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry.The biosensors featured high sensitivities of 322±41 nA mM^(−1) mm^(−2) for glucose and 443±37 nA mM^(−1) mm^(−2) for lactate;the corresponding limits of detection were below 10μM.The proposed technology enabled tissue-size-dependent,real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops.Furthermore,glucose consumption and lactate secretion were monitored in parallel,and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.展开更多
Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal.In the context of personalized cancer treatment,the profiling of individual tumour cell...Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal.In the context of personalized cancer treatment,the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs.Currently,genomic analysis at the single-cell level is available through highly sensitive sequencing approaches.However,the identification and quantification of intracellular or secreted proteins or metabolites remains challenging.Here,we introduce a microfluidic method that facilitates capture,automated data acquisition and the multiplexed quantification of proteins from individual cells.The microfluidic platform comprises 1026 chambers with a volume of 152 pL each,in which single cells and barcoded beads are co-immobilized.We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines,including two model breast cancer cell lines.We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase(GAPDH),galectin-3(Gal-3)and galectin-3 binding protein(Gal-3bp)with detection limits as low as 7.0×10^(4),2.3×10^(5)and 1.8×10^(3)molecules per cell,respectively.The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp,which are important factors that contribute to cancer metastasis.Because it employed commercially available barcoded beads for this study,our platform could be easily used for the single-cell protein profiling of several hundred different targets.Moreover,this versatile method is applicable to the analysis of bacteria,yeast and mammalian cells and nanometre-sized lipid vesicles.展开更多
Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns,often leading to ineffective treatment.Current clinical practice for treatment decision-making is co...Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns,often leading to ineffective treatment.Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies,yet when disease progression accelerates,tissue biopsies are not performed on a regular basis.It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities,providing valuable information about previously underappreciated treatment opportunities.Here,we present MyCTC chip,a novel microfluidic device enabling the isolation,culture and drug susceptibility testing of cancer cells derived from liquid biopsies.Cancer cell capture is achieved through a label-free,antigen-agnostic enrichment method,and it is followed by cultivation in dedicated conditions,allowing on-chip expansion of captured cells.Upon growth,cancer cells are then transferred to drug screen chambers located within the same device,where multiple compounds can be tested simultaneously.We demonstrate MyCTC chip performance by means of spike-in experiments with patientderived breast circulating tumour cells,enabling>95%capture rates,as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian,tubal and endometrial cancer,where sensitivity to specific chemotherapeutic agents was identified.Together,we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.展开更多
As 3D in vitro tissue models become more pervasive,their built-in nutrient,metabolite,compound,and waste gradients increase biological relevance at the cost of analysis simplicity.Investigating these gradients and the...As 3D in vitro tissue models become more pervasive,their built-in nutrient,metabolite,compound,and waste gradients increase biological relevance at the cost of analysis simplicity.Investigating these gradients and the resulting metabolic heterogeneity requires invasive and time-consuming methods.An alternative is using electrochemical biosensors and measuring concentrations around the tissue model to obtain size-dependent metabolism data.With our hanging-dropintegrated enzymatic glucose biosensors,we conducted current measurements within hanging-drop compartments hosting spheroids formed from the human colorectal carcinoma cell line HCT116.We developed a physics-based mathematical model of analyte consumption and transport,considering(1)diffusion and enzymatic conversion of glucose to form hydrogen peroxide(H_(2)O_(2))by the glucose-oxidase-based hydrogel functionalization of our biosensors at the microscale;(2)H_(2)O_(2)oxidation at the electrode surface,leading to amperometric H_(2)O_(2)readout;(3)glucose diffusion and glucose consumption by cancer cells in a spherical tissue model at the microscale;(4)glucose and H_(2)O_(2)transport in our hangingdrop compartments at the macroscale;and(5)solvent evaporation,leading to glucose and H_(2)O_(2)upconcentration.Our model relates the measured currents to the glucose concentrations generating the currents.The low limit of detection of our biosensors(0.4±0.1μM),combined with our current-fitting method,enabled us to reveal glucose dynamics within our system.By measuring glucose dynamics in hanging-drop compartments populated by cancer spheroids of various sizes,we could infer glucose distributions within the spheroid,which will help translate in vitro 3D tissue model results to in vivo.展开更多
Growth rate is a widely studied parameter for various cell-based biological studies.Growth rates of cell populations can be monitored in chemostats and micro-chemostats,where nutrients are continuously replenished.Her...Growth rate is a widely studied parameter for various cell-based biological studies.Growth rates of cell populations can be monitored in chemostats and micro-chemostats,where nutrients are continuously replenished.Here,we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of(i)growth rates of cells by means of impedance measurements and of(ii)specific other cellular events by means of high-resolution optical or fluorescence microscopy.Yeast colonies were grown in a monolayer under culturing pads,which enabled high-resolution microscopy,as all cells were in the same focal plane.Upon cell growth and division,cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements.The impedance data could then be used to directly determine the growth rates of the cells in the culturing area.The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel.As a demonstration,we modulated the growth rates of engineered yeast strains using calcium.The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells.展开更多
Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micromet...Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micrometer-sized channels, electric fields are efficient mechanisms for manipulating liquids, leading to deflection, injection, poration or electrochemical modification of cells and droplets. While PDMS-based microfluidic devices are used due to their inexpensive fabrication, they are limited in terms of electrode integration. Using silicon as the channel material, microfabrication techniques can be used to create nearby electrodes. Despite the advantages that silicon provides, its opacity has prevented its usage in most important microfluidic applications that need optical access. To overcome this barrier, silicon-on-insulator technology in microfluidics is introduced to create optical viewports and channel-interfacing electrodes. More specifically, the microfluidic channel walls are directly electrified via selective, nanoscale etching to introduce insulation segments inside the silicon device layer, thereby achieving the most homogeneous electric field distributions and lowest operation voltages feasible across microfluidic channels. These ideal electrostatic conditions enable a drastic energy reduction, as effectively shown via picoinjection and fluorescence-activated droplet sorting applications at voltages below 6 and 15 V, respectively, facilitating low-voltage electric field applications in next-generation microfluidics.展开更多
As a stress hormone existing in the human body,cortisol can reflect the psychological stress and health status in daily life,and is a potential biomarker of the body’s stress response.To effectively collect sweat and...As a stress hormone existing in the human body,cortisol can reflect the psychological stress and health status in daily life,and is a potential biomarker of the body’s stress response.To effectively collect sweat and accurately identify the target,this paper reports a flexible wearable cortisol detection device with outstanding reliability and sensitivity.Molecular imprinted polymer(MIP)ensures cortisol specificity.And carbon nanotubes(CNT)on electrodes increase sensitivity,expanding the detection range to 10^(−3) to 10^(4) nM,with sensitivity at 189.2 nA/lg(nM).In addition,porous chitosan hydrogel(PCSH)collects sweat effectively,its adhesive properties and 80%swelling rate offer a low-cost alternative to microfluidics.Flexible printed circuit board(FPCB)and serpentine electrode(SE)ensure device durability.This non-invasive,highly sensitive device offers a novel method for mental stress monitoring and clinical diagnosis,advancing human physiological state monitoring.展开更多
基金supported by the European Research Council Advanced Grant 694829“neuroXscales”by the Swiss National Science Foundation under Contracts 205320_188910 and CR32I2_166329 and a Marie Heim-Vögtlin grant to R.B.by an ETH Postdoctoral Fellowship to F.C.
文摘Due to their label-free and noninvasive nature,impedance measurements have attracted increasing interest in biological research.Advances in microfabrication and integrated-circuit technology have opened a route to using large-scale microelectrode arrays for real-time,high-spatiotemporal-resolution impedance measurements of biological samples.In this review,we discuss different methods and applications of measuring impedance for cell and tissue analysis with a focus on impedance imaging with microelectrode arrays in in vitro applications.We first introduce how electrode configurations and the frequency range of the impedance analysis determine the information that can be extracted.We then delve into relevant circuit topologies that can be used to implement impedance measurements and their characteristic features,such as resolution and data-acquisition time.Afterwards,we detail design considerations for the implementation of new impedance-imaging devices.We conclude by discussing future fields of application of impedance imaging in biomedical research,in particular applications where optical imaging is not possible,such as monitoring of ex vivo tissue slices or microelectrode-based brain implants.
文摘Carious lesions are bacteria-caused destructions of the mineralised dental tissues,marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue.While major molecular players in tooth decay have been uncovered during the past years,a detailed map of the molecular and cellular landscape of the diseased pulp is still missing.In this study we used single-cell RNA sequencing analysis,supplemented with immunostaining,to generate a comprehensive single-cell atlas of the pulp of carious human teeth.Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth,such as immune cells,mesenchymal stem cells(MSC)and fibroblasts,when compared to the pulp of healthy human teeth.Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters.These changes include the upregulation of genes encoding extracellular matrix(ECM)components,including COL1A1 and Fibronectin(FN1),and the enrichment of the fibroblast cluster with myofibroblasts.The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses.Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues,compared to the healthy ones.The present data demonstrate molecular,cellular and biomechanical alterations in the pulp of human carious teeth,indicative of extensive ECM remodelling,reminiscent of fibrosis observed in other organs.This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.
基金National Natural Science Foundation of China (No. 81100691 No. 31200739)+1 种基金Doctoral Fund of Ministry of Education of China (No. 20120101120003)Independent Research Project, Zhejiang University, China(No. 5A7000*172210131/005)
文摘AIM: To investigate the distribution of nestin-positive cells in pterygium, as well as the relationship between nestin-positive cells and proliferative cells in the pathogenesis of pterygium. METHODS: Nine pterygium specimens and 5 normal conjunctiva specimens were investigated. All explanted specimens were immediately immersed in 5-Ethynyl-2'-deoxyuridine, and were subjected to hematoxylin and eosin staining, as well as immunostaining to detect nestin. RESULTS: Small sub-populations of nestin-expressing cells in both normal and pterygial conjunctiva epithelium were found. These were located at the superficial layer of the epithelium, and were significantly increased (P=0.007) and spread out in the pterygial conjunctiva epithelium, even though these cells were mitotically quiescent. CONCLUSION: In pterygium, more nestin-positive cells were present at the superficial layer of the epithelium. With growing scientific evidence that nestin plays an important role in defining various specialized cell types, such as stem cells, cancer cells and angiogenic cells, further investigations on the roles of nestin-expressing cells in pterygium may help to uncover the mechanisms of initiation, development and the prognosis of this disease.
基金the support for flow cytometry and microscopy by the single cell facility(SCF)at the Department of Biosystems Science and Engineering at ETH Zurichfinancially supported by the Innosuisse grant 38880.1 IP-LS.by the“Personalized Health and Related Technologies(PHRT)”of the ETH Domain(Project#2021-351).
文摘In vitro evaluation of novel therapeutic approaches often fails to reliably predict efficacy and toxicity,especially when recapitulating conditions involving recirculating cells.Current testing strategies are often based on static co-culturing of cells in suspension and 3D tissue models,where cell sedimentation on the target tissue can occur.The observed effects may then mostly be a consequence of sedimentation and of the corresponding forced cell-tissue interactions.The realization of continuous medium flow helps to better recapitulate physiological conditions and cell-tissue interactions.To tackle current limitations of perfused organ-on-chip approaches,we developed a microfluidic chip and operation concept,which prevents undesired sedimentation and accumulation of suspended cells during multiple days by relying on gravity-driven perfusion.Our platform,which we termed“human immune flow(hiFlow)chip”,enables to co-culture cells in suspension with up to 7 preformed microtissue models.Here,we present the design principle and operation of the platform,and we validate its performance by culturing cells and microtissues of a variety of different origins.Cells and tissues could be monitored on chip via high-resolution microscopy,while cell suspensions and microtissues could be easily retrieved for off-chip analysis.Our results demonstrate that primary immune cells and a range of different spheroid models of healthy and diseased tissues can be maintained for over 6 days on chip.As proof-of-concept cell-tissue interaction assay,we used an antibody treatment against diffuse midline glioma,a highly aggressive pediatric tumor.We are confident that our platform will help to increase the prediction power of in vitro preclinical testing of novel therapeutics that rely on the interaction of circulating cells with organ tissues.
文摘Cell therapy approaches that employ engineered mam-malian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine.The therapeutic cells can be customized to sense user-defined signals,pro-cess them,and respond in a programmable and pre-dictable way.In this paper,we introduce the available tools and strategies employed to design therapeutic cells.Then,various approaches to control cell behav-iors,including open-loop and closed-loop systems,are discussed.We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental dis-ease models.Finally,we consider emerging technolo-gies such as digital devices and their potential for incorporation into future cell-based therapies.
基金This work was financially supported by FP7 of the EU through the project‘Body on a chip’,ICT-FET-296257the ERC Advanced Grant‘NeuroCMOS’(contract 267351)as well as by an individual Ambizione Grant 142440 from the Swiss National Science Foundation for Olivier Frey.
文摘Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities.Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for‘body-on-a-chip’applications.The microfluidic platform is based on the technology of hanging-drop networks,which is designed for the formation,cultivation,and analysis of fluidically interconnected organotypic spherical three-dimensional(3D)microtissues of multiple cell types.The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes,a platinum counter electrode,and an Ag/AgCl reference electrode.They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended.The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry.The biosensors featured high sensitivities of 322±41 nA mM^(−1) mm^(−2) for glucose and 443±37 nA mM^(−1) mm^(−2) for lactate;the corresponding limits of detection were below 10μM.The proposed technology enabled tissue-size-dependent,real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops.Furthermore,glucose consumption and lactate secretion were monitored in parallel,and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.
基金we acknowledge funding from the European Research Council(ERC Consolidator Grant No.681587“HybCell”to P.S.D.).
文摘Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal.In the context of personalized cancer treatment,the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs.Currently,genomic analysis at the single-cell level is available through highly sensitive sequencing approaches.However,the identification and quantification of intracellular or secreted proteins or metabolites remains challenging.Here,we introduce a microfluidic method that facilitates capture,automated data acquisition and the multiplexed quantification of proteins from individual cells.The microfluidic platform comprises 1026 chambers with a volume of 152 pL each,in which single cells and barcoded beads are co-immobilized.We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines,including two model breast cancer cell lines.We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase(GAPDH),galectin-3(Gal-3)and galectin-3 binding protein(Gal-3bp)with detection limits as low as 7.0×10^(4),2.3×10^(5)and 1.8×10^(3)molecules per cell,respectively.The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp,which are important factors that contribute to cancer metastasis.Because it employed commercially available barcoded beads for this study,our platform could be easily used for the single-cell protein profiling of several hundred different targets.Moreover,this versatile method is applicable to the analysis of bacteria,yeast and mammalian cells and nanometre-sized lipid vesicles.
基金supported by the European Research Council(101001652)the strategic focus area of Personalized Health and Related Technologies at ETH Zurich(PHRT-541)+5 种基金the Future and Emerging Technologies programme of the European Commission(801159-B2B)the Swiss National Science Foundation(310030_212183)the Swiss Cancer League(KLS-4834-08-2019)the Basel Cancer League(KLbB-4763-02-2019)and ETH Zürichthe strategic focus area of Personalized Health and Related Technologies at ETH Zurich(PHRT-541)the European Research Council(681587).
文摘Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns,often leading to ineffective treatment.Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies,yet when disease progression accelerates,tissue biopsies are not performed on a regular basis.It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities,providing valuable information about previously underappreciated treatment opportunities.Here,we present MyCTC chip,a novel microfluidic device enabling the isolation,culture and drug susceptibility testing of cancer cells derived from liquid biopsies.Cancer cell capture is achieved through a label-free,antigen-agnostic enrichment method,and it is followed by cultivation in dedicated conditions,allowing on-chip expansion of captured cells.Upon growth,cancer cells are then transferred to drug screen chambers located within the same device,where multiple compounds can be tested simultaneously.We demonstrate MyCTC chip performance by means of spike-in experiments with patientderived breast circulating tumour cells,enabling>95%capture rates,as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian,tubal and endometrial cancer,where sensitivity to specific chemotherapeutic agents was identified.Together,we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.
基金N.R.acknowledges financial support from a doctoral scholarship(FRQNT 199851)from the Fonds de Recherche du Québec—Nature et Technologies.R.L.S.acknowledges financial support from a scholarship(LCF/BQ/EU19/11710046)from the“La Caixa”FoundationWe acknowledge the help of Christian Lohasz,ETH Zürich,with the cell culture,maintenance,and characterization of HCT116 spheroids.We acknowledge the help of Flavio Bonanini,ETH Zürich,with the determination of cell seeding densities and control of HCT116 growth dynamicsWe acknowledge the help of Stefan Strebel,University of Basel,and Felix Franke,ETH Zürich,with developing the mathematical models.
文摘As 3D in vitro tissue models become more pervasive,their built-in nutrient,metabolite,compound,and waste gradients increase biological relevance at the cost of analysis simplicity.Investigating these gradients and the resulting metabolic heterogeneity requires invasive and time-consuming methods.An alternative is using electrochemical biosensors and measuring concentrations around the tissue model to obtain size-dependent metabolism data.With our hanging-dropintegrated enzymatic glucose biosensors,we conducted current measurements within hanging-drop compartments hosting spheroids formed from the human colorectal carcinoma cell line HCT116.We developed a physics-based mathematical model of analyte consumption and transport,considering(1)diffusion and enzymatic conversion of glucose to form hydrogen peroxide(H_(2)O_(2))by the glucose-oxidase-based hydrogel functionalization of our biosensors at the microscale;(2)H_(2)O_(2)oxidation at the electrode surface,leading to amperometric H_(2)O_(2)readout;(3)glucose diffusion and glucose consumption by cancer cells in a spherical tissue model at the microscale;(4)glucose and H_(2)O_(2)transport in our hangingdrop compartments at the macroscale;and(5)solvent evaporation,leading to glucose and H_(2)O_(2)upconcentration.Our model relates the measured currents to the glucose concentrations generating the currents.The low limit of detection of our biosensors(0.4±0.1μM),combined with our current-fitting method,enabled us to reveal glucose dynamics within our system.By measuring glucose dynamics in hanging-drop compartments populated by cancer spheroids of various sizes,we could infer glucose distributions within the spheroid,which will help translate in vitro 3D tissue model results to in vivo.
基金The work was financially supported by the Swiss SystemsX.ch IPhD program,by the FP7 of the EU through the MTN ISOLATE,Contract Number 289995the Ambizione Grant 142440 of the Swiss National Science Foundation for Olivier Frey.
文摘Growth rate is a widely studied parameter for various cell-based biological studies.Growth rates of cell populations can be monitored in chemostats and micro-chemostats,where nutrients are continuously replenished.Here,we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of(i)growth rates of cells by means of impedance measurements and of(ii)specific other cellular events by means of high-resolution optical or fluorescence microscopy.Yeast colonies were grown in a monolayer under culturing pads,which enabled high-resolution microscopy,as all cells were in the same focal plane.Upon cell growth and division,cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements.The impedance data could then be used to directly determine the growth rates of the cells in the culturing area.The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel.As a demonstration,we modulated the growth rates of engineered yeast strains using calcium.The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells.
基金We would like to thank the cleanroom operations team of the Binnig and Rohrer Nanotechnology Center(BRNC)for their help and support and A.Zulji for technical assistance.We would further like to thank B.Michel,T.Brunschwiler,H.Riel,and A.Curioni for their continuous support.The funding from the Swiss National Science Foundation–NCCR Molecular Systems Engineering(grant no.51NF40-205608)is gratefully acknowledged.
文摘Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micrometer-sized channels, electric fields are efficient mechanisms for manipulating liquids, leading to deflection, injection, poration or electrochemical modification of cells and droplets. While PDMS-based microfluidic devices are used due to their inexpensive fabrication, they are limited in terms of electrode integration. Using silicon as the channel material, microfabrication techniques can be used to create nearby electrodes. Despite the advantages that silicon provides, its opacity has prevented its usage in most important microfluidic applications that need optical access. To overcome this barrier, silicon-on-insulator technology in microfluidics is introduced to create optical viewports and channel-interfacing electrodes. More specifically, the microfluidic channel walls are directly electrified via selective, nanoscale etching to introduce insulation segments inside the silicon device layer, thereby achieving the most homogeneous electric field distributions and lowest operation voltages feasible across microfluidic channels. These ideal electrostatic conditions enable a drastic energy reduction, as effectively shown via picoinjection and fluorescence-activated droplet sorting applications at voltages below 6 and 15 V, respectively, facilitating low-voltage electric field applications in next-generation microfluidics.
基金supported by the Natural Science Foundation of Shandong Province(No.ZR2022QF120)Qingdao Postdoctoral Fund(No.QDBSH20230101005)Shandong Province Youth Innovation and Technology Support Plan for Higher Education Institutions(No.2023KJ362).
文摘As a stress hormone existing in the human body,cortisol can reflect the psychological stress and health status in daily life,and is a potential biomarker of the body’s stress response.To effectively collect sweat and accurately identify the target,this paper reports a flexible wearable cortisol detection device with outstanding reliability and sensitivity.Molecular imprinted polymer(MIP)ensures cortisol specificity.And carbon nanotubes(CNT)on electrodes increase sensitivity,expanding the detection range to 10^(−3) to 10^(4) nM,with sensitivity at 189.2 nA/lg(nM).In addition,porous chitosan hydrogel(PCSH)collects sweat effectively,its adhesive properties and 80%swelling rate offer a low-cost alternative to microfluidics.Flexible printed circuit board(FPCB)and serpentine electrode(SE)ensure device durability.This non-invasive,highly sensitive device offers a novel method for mental stress monitoring and clinical diagnosis,advancing human physiological state monitoring.
