RESUMEN
Correction for 'Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics' by Hyungkook Jeon et al., Lab Chip, 2020, 20, 3612-3624, https://doi.org/10.1039/D0LC00675K.
RESUMEN
A fully-automated and portable leukocyte separation platform was developed based on a new type of inertial microfluidic device, multi-dimensional double spiral (MDDS) device, as an alternative to centrifugation. By combining key innovations in inertial microfluidic device designs and check-valve-based recirculation processes, highly purified and concentrated WBCs (up to >99.99% RBC removal, â¼80% WBC recovery, >85% WBC purity, and â¼12-fold concentrated WBCs compared to the input sample) were achieved in less than 5 minutes, with high reliability and repeatability (coefficient of variation, CV < 5%). Using this, one can harvest up to 0.4 million of intact WBCs from 50 µL of human peripheral blood (50 µL), without any cell damage or phenotypic changes in a fully-automated operation. Alternatively, hand-powered operation is demonstrated with comparable separation efficiency and speed, which eliminates the need for electricity altogether for truly field-friendly sample preparation. The proposed platform is therefore highly deployable for various point-of-care applications, including bedside assessment of the host immune response and blood sample processing in resource-limited environments.
Asunto(s)
Leucocitos , Microfluídica , Separación Celular , Humanos , Dispositivos Laboratorio en un Chip , Reproducibilidad de los ResultadosRESUMEN
Stimulation of PC-12 cells with epidermal (EGF) versus nerve (NGF) growth factors (GFs) biases the distribution between transient and sustained single-cell ERK activity states, and between proliferation and differentiation fates within a cell population. We report that fibroblast GF (FGF2) evokes a distinct behavior that consists of a gradually changing population distribution of transient/sustained ERK signaling states in response to increasing inputs in a dose response. Temporally controlled GF perturbations of MAPK signaling dynamics applied using microfluidics reveal that this wider mix of ERK states emerges through the combination of an intracellular feedback, and competition of FGF2 binding to FGF receptors (FGFRs) and heparan sulfate proteoglycan (HSPG) co-receptors. We show that the latter experimental modality is instructive for model selection using a Bayesian parameter inference. Our results provide novel insights into how different receptor tyrosine kinase (RTK) systems differentially wire the MAPK network to fine-tune fate decisions at the cell population level.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Animales , Teorema de Bayes , Relación Dosis-Respuesta a Droga , Proteoglicanos de Heparán Sulfato/metabolismo , Técnicas Analíticas Microfluídicas , Células PC12 , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Dysregulated leukocyte responses underlie the pathobiology of sepsis, which is a leading cause of death. However, measures of leukocyte function are not routinely available in clinical care. Here we report the development and testing of an inertial microfluidic system for the label-free isolation and downstream functional assessment of leukocytes from 50 µl of peripheral blood. We used the system to assess leukocyte phenotype and function in serial samples from 18 hospitalized patients with sepsis and 10 healthy subjects. The sepsis samples had significantly higher levels of CD16dim and CD16- neutrophils and CD16+ 'intermediate' monocytes, as well as significantly lower levels of neutrophil-elastase release, O2- production and phagolysosome formation. Repeated sampling of sepsis patients over 7 days showed that leukocyte activation (measured by isodielectric separation) and leukocyte phenotype and function were significantly more predictive of the clinical course than complete-blood-count parameters. We conclude that the serial assessment of leukocyte function in microlitre blood volumes is feasible and that it provides significantly more prognostic information than leukocyte counting.
Asunto(s)
Leucocitos , Técnicas Analíticas Microfluídicas/métodos , Sepsis/sangre , Sepsis/diagnóstico , Índice de Severidad de la Enfermedad , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Femenino , Proteínas Ligadas a GPI , Humanos , Recuento de Leucocitos , Elastasa de Leucocito/sangre , Masculino , Técnicas Analíticas Microfluídicas/instrumentación , Persona de Mediana Edad , Monocitos , Neutrófilos , Fenotipo , Receptores de IgG , Adulto JovenRESUMEN
Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.
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Bacterias/clasificación , Hibridación de Ácido Nucleico/métodos , ARN Ribosómico 16S/genética , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Mycobacterium/clasificación , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Mycobacterium/patogenicidad , Filogenia , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/patogenicidadRESUMEN
We used 3D cell printing to emulate an airway coupled with a naturally-derived blood vessel network in vitro. Decellularized extracellular matrix bioink derived from porcine tracheal mucosa (tmdECM) was used to encapsulate and print endothelial cells and fibroblasts within a designated polycarprolactone (PCL) frame. Providing a niche that emulates conditions in vivo, tmdECM gradually drives endothelial re-orientation, which leads to the formation of a lumen and blood vessel network. A fully-differentiated in vitro airway model was assembled with the printed vascular platform, and collectively reproduced a functional interface between the airway epithelium and the vascular network. The model presented respiratory symptoms including asthmatic airway inflammation and allergen-induced asthma exacerbation in physiological context. Because of the adaptable and automated nature of direct 3D cell printing, we expect that this will have relevance in vivo and high reproducibility for production of high-content platforms for preclinical trials in biomedical research.
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Bioimpresión/métodos , Células Endoteliales/citología , Fibroblastos/citología , Impresión Tridimensional , Ingeniería de Tejidos/normas , Animales , Diferenciación Celular , Proliferación Celular , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Porcinos , Tráquea/irrigación sanguínea , Tráquea/citologíaRESUMEN
Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal stimulation. Using this platform, we observed that static Epidermal growth factor (EGF) stimulation led to transient ERK activation with a significant cell-to-cell variation, while dynamic stimulation of 3' EGF pulse led to faster adaptation kinetics with no discrepancy. Multiple EGF pulses retriggered ERK activity with respect to frequency of stimulation. We also observed oscillation of ERK activity of each cell at basal state. Introducing of Mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, was not only dropping the average of basal activity for 7.5%, but also diminishing oscillatory behavior. Activity level raised up when inhibitor was removed, followed by transient peak of ERK kinetics. We expect this platform to probe Mitogen-associated protein kinase (MAPK) signaling network for systems biology research at single cellular level.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Análisis de la Célula Individual/métodos , Técnicas Biosensibles/métodos , Butadienos/farmacología , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Cinética , Modelos Teóricos , Nitrilos/farmacologíaRESUMEN
Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient's immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.
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Microfluídica/métodos , Neutrófilos/metabolismo , Sistema Respiratorio/metabolismo , HumanosRESUMEN
In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.
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Sangre/virología , Separación Celular , Técnicas Analíticas Microfluídicas , Virión/aislamiento & purificación , Virus/aislamiento & purificación , Células Sanguíneas , ADN Viral/análisis , ADN Viral/genética , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , ARN Viral/análisis , ARN Viral/genética , Virión/genética , Virus/genéticaRESUMEN
Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1-deficient (Thbs1-/-) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1-/- mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger.
Asunto(s)
Matriz Extracelular/metabolismo , Lesión Pulmonar/patología , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/patología , Trombospondina 1/metabolismo , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunología , Humanos , Elastasa de Leucocito/metabolismo , Pulmón/citología , Pulmón/inmunología , Pulmón/patología , Lesión Pulmonar/inmunología , Lesión Pulmonar/microbiología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Proteolisis , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Respiración Artificial/efectos adversos , Trombospondina 1/genética , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/metabolismoRESUMEN
Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device. This microfluidic device provides at least 12h of continuous stimulations that can be used to observe responses from mammalian cells. Combination of the microfluidic de-vice with live-cell imaging facilitates real-time observation of dynamic cellular response at single cell level. Using stable HEK cells with biosensors, ERK (Extracellular signal-Regulated Kinase) activities were observed un-der the pulsatile and ramping stimulations of EGF (Epidermal Growth Factor). We quantified ERK activation even at extremely low EGF concentration (0.0625µg/ml), which can not be observed using conventional techniques such as western blot. Cytoskeleton re-arrangement of the 3T3 fibroblast (stable transfection with Lifeact-GFP) was compared under abrupt and gradually changing gradient of PDGF.
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Técnicas Biosensibles , Técnicas Analíticas Microfluídicas , Análisis de la Célula Individual , Células 3T3-L1 , Animales , Técnicas de Cultivo de Célula , Quimiotaxis/genética , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/aislamiento & purificación , Fibroblastos/ultraestructura , Humanos , Ratones , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/aislamiento & purificaciónRESUMEN
We introduce a novel microfluidic device to co-culture a blood vessel network and cell tissues in an in vivo-like niche. Our "open-top" microfluidic device is composed of microchannels with micropores in the ceiling, which provides direct fluid access from reservoir to microchannel. Fluid connections through micropores afford novel advantages, including: i) the long-term culture of large-scale microvessel network, ii) access of different fluids to inner and exterior sides of the microvessel, and iii) co-culturing of the microvessel network and small cell tissue. In this study, we have successfully assembled microvessels with 5 mm channel widths. We were also able to mimic capillary bed conditions by co-culturing microvessels with cancer spheroids. Intimate contact between the cancer spheroid and microvessel caused vessel recruitment and an increase in vessel formation, and affected vessel morphology. We expect this device to be used as a novel platform for vascularized tissue models.
Asunto(s)
Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Línea Celular , Técnicas de Cocultivo , Técnicas Citológicas/instrumentación , Técnicas Citológicas/métodos , Diseño de Equipo , Humanos , Microvasos/citología , Microvasos/metabolismo , Microvasos/fisiología , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Assessment of airway secretion cells, both for research and clinical purposes, is a highly desired goal in patients with acute and chronic pulmonary diseases. However, lack of proper cell isolation and enrichment techniques hinder downstream evaluation and characterization of cells found in airway secretions. Here, we demonstrate a novel enrichment method to capture immune-related cells from clinical airway secretions using closed-loop separation of spiral inertial microfluidics (C-sep). By recirculating the output focusing stream back to the input reservoir and running continuously with a high flow processing rate, one can achieve optimal concentration, recovery and purity of airway immune cells from a large volume of diluent, which was not readily possible in the single-pass operation. Our method reproducibly recovers 94.0% of polymorphonuclear leukocytes (PMNs), with up to 105 PMNs in clear diluted buffer from 50 µL of airway secretions obtained from mechanically ventilated patients. We show that C-sep isolated PMNs show higher neutrophil elastase (NE) release following activation by phorbol 12-myristate 13-acetate (PMA) than cells isolated by conventional mucolytic method. By capturing cells without chemically disrupting their potential function, our method is expected to expand the possibility of clinical in vitro cell based biological assays for various pulmonary diseases such as acute respiratory distress syndrome, pneumonia, cystic fibrosis, and bronchiectasis.
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Separación Celular/métodos , Microfluídica , Neutrófilos/citología , Esputo/citología , Separación Celular/instrumentación , Ditiotreitol/farmacología , Humanos , Elastasa de Leucocito/metabolismo , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/patología , Mucinas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
There are many proposed mechanisms by which single cells can be trapped; among them is the through-hole membrane for the characterization of individual microorganisms. Due to the small scale of the fabricated pores, the construction of through-hole membranes on a large scale and with relatively large areas faces many difficulties. This paper describes novel fabrication methods for a large-area, freestanding micro/nano through-hole membrane constructed from versatile membrane materials using through-hole membranes on a microfluidic chip (THMMC). This process can rapidly (<20 min) fabricate membranes with high fidelity multiscale hole size without residual layers. The through-hole site was easily customizable from the micro to the nanoscale, with a low or high aspect ratio giving rise to reliable membranes. Also, the rigidity and biocompatibility of the through-hole membrane are easily tunable by simple injection of versatile membrane materials to obtain a large area (up to 3600 mm2). Membranes produced in this manner were then applied as a proof of concept for the isolation, cultivation, and quantification of individual micro-algal cells for selection with respect to the growth rate, while controlling the quorum sensing mediated metabolic and proliferative changes.
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Membranas Artificiales , Técnicas Analíticas Microfluídicas/instrumentación , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos , Técnicas de Cultivo de Célula , Diseño de Equipo , Microalgas/citologíaRESUMEN
Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.
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Técnicas Biosensibles , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Análisis de la Célula Individual , Animales , Técnicas Biosensibles/métodos , Línea Celular , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia/métodos , Expresión Génica , Genes Reporteros , Vectores Genéticos/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Lentivirus/genética , Ratones , Microscopía Fluorescente , Reproducibilidad de los Resultados , Transducción de Señal , Análisis de la Célula Individual/métodos , Transducción GenéticaRESUMEN
The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.
Asunto(s)
Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Actinas/metabolismo , Amidas/farmacología , Animales , Técnicas Biosensibles , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Técnicas Analíticas Microfluídicas , Microscopía Fluorescente , Factor de Crecimiento Derivado de Plaquetas/farmacología , Piridinas/farmacología , Ratas , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.
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Diferenciación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Biología de Sistemas/métodos , Animales , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Células PC12 , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
The blood circulatory system links all organs from one to another to support and maintain each organ's functions consistently. Therefore, blood vessels have been considered as a vital unit. Engineering perfusable functional blood vessels in vitro has been challenging due to difficulties in designing the connection between rigid macroscale tubes and fragile microscale ones. Here, we propose a generalizable method to engineer a "long" perfusable blood vessel network. To form millimeter-scale vessels, fibroblasts were co-cultured with human umbilical vein endothelial cells (HUVECs) in close proximity. In contrast to previous works, in which all cells were permanently placed within the device, we developed a novel method to culture paracrine factor secreting fibroblasts on an O-ring-shaped guide that can be transferred in and out. This approach affords flexibility in co-culture, where the effects of secreted factors can be decoupled. Using this, blood vessels with length up to 2 mm were successfully produced in a reproducible manner (>90%). Because the vessels form a perfusable network within the channel, simple links to inlets and outlets of the device allowed connections to the outside world. The robust and reproducible formation of in vitro engineered vessels can be used as a module to link various organ components as parts of future body-on-a-chip applications.
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Vasos Sanguíneos/citología , Fibroblastos/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Comunicación Paracrina , Ingeniería de Tejidos/métodos , Circulación Sanguínea , Técnicas de Cultivo de Célula , Células Cultivadas , Técnicas de Cocultivo , Humanos , Dispositivos Laboratorio en un Chip/estadística & datos numéricos , Técnicas Analíticas Microfluídicas/instrumentación , Neovascularización Fisiológica , PerfusiónRESUMEN
Understanding the mechanism behind cancer metastasis is a major challenge in cancer biology. Several in vitro models have been developed to mimic a cancer microenvironment by engineering cancer-endothelial cell (EC) and cancer-stromal cell interactions. It has been challenging to realistically mimic angiogenesis, intravasation, and extravasation using macro-scale approaches but recent progress in microfluidics technology has begun to yield promising results. We present a metastasis chip that produce microvessels, where EC and stromal cells can be patterned in close proximity to tumor cells. The vessels are formed following a natural morphogenic process and have smooth boundaries with proper cell-cell junctions. The engineered microvessels are perfusable and have well-defined openings toward inlet and outlet channels. The ability to introduce cancer cells into different locations bordering to the microvessel wall allowed generation and maintenance of appropriate spatial gradients of growth factors and attractants. Cancer angiogenesis and its inhibition by anti-vascular endothelial growth factor (bevacizumab) treatment were successfully reproduced in the metastasis chip. Cancer intravasation and its modulation by treatment of tumor necrosis factor-α were also modeled. Compared to other models, the unique design of the metastasis chip that engineers a clear EC-cancer interface allows precise imaging and quantification of angiogenic response as well as tumor cell trans-endothelial migration. The metastasis chip presented here has potential applications in the investigation of fundamental cancer biology as well as in drug screening.