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1.
EMBO J ; 42(24): e113761, 2023 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-38009333

RESUMEN

Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer-based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.


Asunto(s)
Actinas , Nanotubos , Actinas/metabolismo , Polimerizacion , Proteómica , Nanotubos/química , Citoesqueleto de Actina/metabolismo
2.
Analyst ; 149(13): 3537-3546, 2024 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-38758167

RESUMEN

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.


Asunto(s)
Reacción en Cadena de la Ligasa , Límite de Detección , Liposomas , Liposomas/química , Humanos , Reacción en Cadena de la Ligasa/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Polimorfismo de Nucleótido Simple , Biotina/química , Acústica , Avidina/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Oro/química , ADN/genética , ADN/química , Colesterol , Mutación Puntual
3.
Anal Chem ; 95(49): 17988-17996, 2023 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-38032406

RESUMEN

The extraction and separation of cellular compounds are crucial steps in numerous biological protocols, particularly in multiomics studies, where several cellular modalities are examined simultaneously. While magnetic particle extraction is commonly used, it may not be applicable for ultralow input samples. Microfluidics has made possible the analysis of rare or low-materiality samples such as circulating tumor cells or single cells through miniaturization of numerous protocols. In this study, a microfluidics workflow for separating different cellular modalities from ultralow input samples is presented. This approach is based on magnetic tweezers technology, allowing the extraction and resuspension of magnetic particles between consecutive nanoliter droplets to perform multistep assays on small volumes. The ability to separate and recover mRNA and gDNA in samples containing less than 10 cells is demonstrated, achieving separation efficiency comparable to the one obtained with conventional pipetting but with a significantly lower amount of starting material, typically 1-2 orders of magnitude less.


Asunto(s)
Técnicas Analíticas Microfluídicas , Técnicas Analíticas Microfluídicas/métodos , Multiómica , Microfluídica/métodos , Bioensayo/métodos , Flujo de Trabajo
4.
PLoS Comput Biol ; 17(3): e1008870, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33784299

RESUMEN

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.


Asunto(s)
Apoptosis/fisiología , Técnicas de Cocultivo/métodos , Linfocitos T Citotóxicos , Microambiente Tumoral/fisiología , Línea Celular Tumoral , Biología Computacional , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Cinética , Técnicas Analíticas Microfluídicas , Microscopía por Video , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/fisiología
5.
Electrophoresis ; 39(3): 526-533, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28868639

RESUMEN

The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (µMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the µMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.


Asunto(s)
Separación Inmunomagnética/métodos , Leche/química , Salmonella/aislamiento & purificación , Animales , Escherichia coli/aislamiento & purificación , Inmunoglobulina G/química , Separación Inmunomagnética/instrumentación , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microesferas , Salmonella/citología , Salmonella/inmunología
6.
Analyst ; 143(5): 1077-1086, 2018 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-29383369

RESUMEN

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.


Asunto(s)
Neuropatías Amiloides Familiares/diagnóstico , Técnicas Analíticas Microfluídicas/instrumentación , Prealbúmina/análisis , Humanos , Reproducibilidad de los Resultados
7.
Analyst ; 141(20): 5776-5783, 2016 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-27486596

RESUMEN

In this work, we have investigated Dyneon THV, a fluorinated material, as a new material to afford electrokinetic separations in microfluidic devices. To overcome protein adsorption, two poly(ethylene oxide) (PEO)-based coatings have been investigated: Pluronic F127 and PEO stearate 40. The best results were obtained with the PEO stearate 40 coating which allowed decreasing the surface contact angle from 91 ± 3 to 76°± 3. With this surface treatment, a 66% reduction of the electroosmotic mobility at pH 8.0 and a marked suppression of protein adsorption were observed compared to a native Dyneon THV microchip. Finally, a separation of fluorescently labeled proteins (bovine serum albumin and trypsin inhibitor), well-known for their strong tendency to adsorb on hydrophobic surfaces, was successfully achieved in an HEPES buffer with a PEO stearate 40 treated microchip by capillary zone electrophoresis. Furthermore, we demonstrated the possibility to perform non-aqueous capillary electrophoresis analysis of hydrophobic dyes using various solvents in untreated microchips. The overall results demonstrated not only the suitability of the Dyneon THV microchip for electrokinetic separations, but also its versatility allowing different separation modes to be implemented with the same microchip material.

8.
Analyst ; 140(17): 5891-900, 2015 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-26206107

RESUMEN

A new sample treatment approach for sensitive determination of three amyloid-ß peptides (Aß 1-42, Aß 1-40 and Aß 1-38) with capillary electrophoresis coupled with laser induced fluorescent detection is reported herein. These Aß peptides are considered an important family of biomarkers in the cerebrospinal fluid (CSF) for early diagnosis of Alzheimer's disease (AD). Due to their extremely low abundance in CSF (down to sub nM ranges), batch-wise preconcentration via magneto-immunocapture with enrichment factors up to 100 was implemented. The Aß peptides were first captured onto magnetic micro-beads. Then, on-beads fluorescent labeling of the captured Aß peptides were carried out to avoid the unwanted presence of extra fluorescent dye in the eluent as in the case of in-solution labeling. Finally thermal elution was performed and eluted labeled peptides were analyzed off line with CE-LIF. The Aß-capturing efficiencies of different commercially available antibodies grafted onto magnetic beads were tested. Aß peptides in CSF samples collected from AD's patients and healthy persons (used as controls) were measured and evaluated. As a proof of concept, the developed strategy was adapted into a miniaturized fluidized bed configuration that has the potential for coupling with a microchip separation system.


Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Técnicas Analíticas Microfluídicas/métodos , Fragmentos de Péptidos/líquido cefalorraquídeo , Espectrometría de Fluorescencia , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/aislamiento & purificación , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Electroforesis Capilar , Colorantes Fluorescentes/química , Humanos , Rayos Láser , Magnetismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Espectrometría de Fluorescencia/instrumentación
9.
Lab Chip ; 24(3): 584-593, 2024 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-38175160

RESUMEN

The manipulation of magnetic microparticles has always been pivotal in the development of microfluidic devices, as it encompasses a broad range of applications, such as drug delivery, bioanalysis, on-chip diagnostics, and more recently organ-on-chip development. However, predicting the behavior and trajectory of these particles remains a recurring and partly unresolved question. Magnetic particle-laden flows can display intricate collective behaviors, such as packed plugs, column-shaped aggregates, or fluidization, which are difficult to predict. In this study, we introduce a finite-element model to simulate highly dense flows of magnetic microparticles. Our method relies on an interpenetrating continuum approach, where both the liquid and particle phases are described by the Navier-Stokes equations, in which the magnetic force, interphase friction, and interparticle forces were included. We demonstrate its applicability across the entire range of particle packing densities and compare the results with experimental data from real microfluidic application cases. The model successfully replicates complex behaviors, such as particle aggregation, plug formation and fluidization. This approach has potential to accelerate microfluidic device development by reducing the need for costly and time-consuming experimental optimization.

10.
Methods Mol Biol ; 2804: 163-176, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38753147

RESUMEN

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis, and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecule bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, the extraction of 1 µm diameter magnetic particles was achieved at high throughput (20 droplets per second) with an efficiency close to 100% in 450 pL droplets. The first demonstration of its adaptability to single-cell analysis is demonstrated with the extraction of mRNA. Using a purified nucleic acid solution, this unique magnetic configuration was able to reach a RNA extraction rate of 72%. This is the first demonstration of a physical separation in droplets at high throughput at single-cell scale.


Asunto(s)
Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Magnetismo/métodos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Humanos , Microfluídica/métodos , Microfluídica/instrumentación
11.
bioRxiv ; 2024 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-38948811

RESUMEN

Kidney tubular cells are submitted to two distinct mechanical forces generated by the urine flow: shear stress and hydrostatic pressure. In addition, the mechanical properties of the surrounding extracellular matrix modulate tubule deformation under constraints. These mechanical factors likely play a role in the pathophysiology of kidney diseases as exemplified by autosomal dominant polycystic kidney disease, in which pressure, flow and matrix stiffness have been proposed to modulate the cystic dilation of tubules with PKD1 mutations. The lack of in vitro systems recapitulating the mechanical environment of kidney tubules impedes our ability to dissect the role of these mechanical factors. Here we describe a perfused kidney-on-chip with tunable extracellular matrix mechanical properties and hydrodynamic constraints, that allows a decoupling of shear stress and flow. We used this system to dissect how these mechanical cues affect Pkd1 -/- tubule dilation. Our results show two distinct mechanisms leading to tubular dilation. For PCT cells (proximal tubule), overproliferation mechanically leads to tubular dilation, regardless of the mechanical context. For mIMCD-3 cells (collecting duct), tube dilation is associated with a squamous cell morphology but not with overproliferation and is highly sensitive to extracellular matrix properties and hydrodynamic constraints. Surprisingly, flow alone suppressed Pkd1 -/- mIMCD-3 tubule dilation observed in static conditions, while the addition of luminal pressure restored it. Our in vitro model emulating nephron geometrical and mechanical organization sheds light on the roles of mechanical constraints in ADPKD and demonstrates the importance of controlling intraluminal pressure in kidney tubule models.

12.
Methods Mol Biol ; 2804: 65-75, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38753140

RESUMEN

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).


Asunto(s)
Ácidos Nucleicos Libres de Células , Dispositivos Laboratorio en un Chip , Humanos , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Reacción en Cadena de la Polimerasa/métodos , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Magnetismo/métodos , Neoplasias/sangre , Neoplasias/genética , Neoplasias/diagnóstico
13.
Cell Rep Med ; 5(5): 101549, 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38703767

RESUMEN

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.


Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares , Medicina de Precisión , Receptor de Muerte Celular Programada 1 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología , Medicina de Precisión/métodos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Dispositivos Laboratorio en un Chip , Inmunoterapia/métodos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Línea Celular Tumoral
14.
Anal Biochem ; 435(2): 150-2, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23333271

RESUMEN

Aptamer-conjugated nanoparticles (Apt-NPs) are increasingly being developed for biomedical purposes and especially for diagnosis and therapy. However, there is no quantitative study of the targeting functionality of such grafted aptamers compared with free aptamers. Thus, we report the first determination of binding parameters for Apt-NP/target complexes, thanks to a continuous frontal analysis in a microchip electrophoresis format (named FACMCE), based on a methodology previously developed by our group. As a model system, the targeting ability of a lysozyme-binding aptamer conjugated to fluorescent maghemite nanoparticles was evaluated and showed evidence that the conjugation does not alter the affinity of this aptamer.


Asunto(s)
Aptámeros de Nucleótidos/química , Electroforesis por Microchip , Nanopartículas del Metal/química , Secuencia de Bases , Sitios de Unión , Compuestos Férricos/química
15.
Methods ; 57(3): 297-307, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22796377

RESUMEN

At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.


Asunto(s)
Separación Celular/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/patología , Antígenos de Superficie/análisis , Células Sanguíneas/citología , Adhesión Celular , Movimiento Celular , Separación Celular/instrumentación , Centrifugación , Electroforesis , Células Endoteliales/citología , Filtración , Fluorescencia , Humanos , Magnetismo , Técnicas Analíticas Microfluídicas , Microfluídica/instrumentación
16.
Methods Mol Biol ; 2664: 85-106, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37423984

RESUMEN

The organ-on-chip model offers versatility and modularity of in vitro models while approaching the biological fidelity of in vivo models. We propose a method to build a perfusable kidney-on-chip aiming at reproducing key features of the densely packed segments of nephrons in vitro; such as their geometry, their extracellular matrix, and their mechanical properties. The core of the chip is made of parallel tubular channels molded into collagen I that are as small as 80 µm in diameter and as close as 100 µm apart. These channels can further be coated with basement membrane components and seeded by perfusion of a suspension of cells originating from a given segment of the nephron. We optimized the design of our microfluidic device to achieve high reproducibility regarding the seeding density of the channels and high fluidic control of the channels. This chip was designed as a versatile tool to study nephropathies in general, contributing to building ever better in vitro models. It could be particularly interesting for pathologies such as polycystic kidney diseases where mechanotransduction of the cells and their interaction with adjacent extracellular matrix and nephrons may play a key role.


Asunto(s)
Enfermedades Renales , Mecanotransducción Celular , Humanos , Reproducibilidad de los Resultados , Riñón , Nefronas , Dispositivos Laboratorio en un Chip
17.
Microsyst Nanoeng ; 9: 109, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37680311

RESUMEN

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

18.
Anal Chim Acta ; 1255: 341141, 2023 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-37032056

RESUMEN

This study reports on the development of a new concept of on-line dual preconcentration stages for capillary electrophoresis (CE), in which two completely different preconcentration approaches can be realized in the same capillary. In the first stage, a dynamic magneto-extraction of target analytes on circulating magnetic beads is implemented within the capillary. In the second one, electrokinetic preconcentration of eluted analytes via large volume sample stacking is carried out to focus them into a nano band, prior to CE separation of enriched analytes. To implement the dual-stage preconcentration operation, a purpose-made instrument was designed, combining electrophoretic and microfluidic modules to allow precise control of the movement of magnetic beads and analyte's flow. The potential of this new enrichment principle and its associated instrument was demonstrated for CE separation with light-emitting-diode-induced fluorescent (LEDIF) detection of target double-stranded DNA (ds-DNA). The workflow consists of purification and preconcentration of a target DNA fragment (300 bp) on negatively charged magnetic beads, followed by in-capillary elution and fluorescent labelling of the enriched DNA. Large volume sample stacking of the DNA eluent was then triggered to further preconcentrate the labelled DNA before its analysis by CE-LEDIF. An enrichment factor of 125 was achieved for the target DNA fragment. With our new approach, dual-stage sample pretreatment and CE separation can now be performed in-capillary without any mismatch of working volumes, nor any waste of pretreated samples.


Asunto(s)
Colorantes , Electroforesis Capilar , Electroforesis Capilar/métodos , Separación Inmunomagnética , Campos Magnéticos , Microfluídica
19.
Lab Chip ; 23(6): 1713, 2023 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-36852524

RESUMEN

Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.

20.
Lab Chip ; 23(18): 3906-3935, 2023 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-37592893

RESUMEN

Over the past 15 years, the field of oncology research has witnessed significant progress in the development of new cell culture models, such as tumor-on-chip (ToC) systems. In this comprehensive overview, we present a multidisciplinary perspective by bringing together physicists, biologists, clinicians, and experts from pharmaceutical companies to highlight the current state of ToC research, its unique features, and the challenges it faces. To offer readers a clear and quantitative understanding of the ToC field, we conducted an extensive systematic analysis of more than 300 publications related to ToC from 2005 to 2022. ToC offer key advantages over other in vitro models by enabling precise control over various parameters. These parameters include the properties of the extracellular matrix, mechanical forces exerted on cells, the physico-chemical environment, cell composition, and the architecture of the tumor microenvironment. Such fine control allows ToC to closely replicate the complex microenvironment and interactions within tumors, facilitating the study of cancer progression and therapeutic responses in a highly representative manner. Importantly, by incorporating patient-derived cells or tumor xenografts, ToC models have demonstrated promising results in terms of clinical validation. We also examined the potential of ToC for pharmaceutical industries in which ToC adoption is expected to occur gradually. Looking ahead, given the high failure rate of clinical trials and the increasing emphasis on the 3Rs principles (replacement, reduction, refinement of animal experimentation), ToC models hold immense potential for cancer research. In the next decade, data generated from ToC models could potentially be employed for discovering new therapeutic targets, contributing to regulatory purposes, refining preclinical drug testing and reducing reliance on animal models.


Asunto(s)
Técnicas de Cultivo de Célula , Neoplasias , Humanos , Animales , Industria Farmacéutica , Matriz Extracelular , Microambiente Tumoral , Neoplasias/tratamiento farmacológico
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