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1.
Methods Mol Biol ; 2429: 189-199, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507162

RESUMO

In order to use induced Pluripotent Stem Cells (iPSCs) to model neurodegenerative diseases, efficient and homogeneous generation of neurons in vitro represents a key step. Here we describe a method to obtain and characterize functional human spinal and cranial motoneurons using a combined approach of microfluidic chips and programs designed for scientific multidimensional imaging. We have used this approach to analyze axonal phenotypes. These tools are useful to investigate the cellular and molecular bases of neuromuscular diseases, including amyotrophic lateral sclerosis and spinal muscular atrophy.


Assuntos
Esclerose Amiotrófica Lateral , Células-Tronco Pluripotentes Induzidas , Axônios/fisiologia , Humanos , Dispositivos Lab-On-A-Chip , Neurônios Motores , Fenótipo
2.
Anal Chim Acta ; 1208: 339829, 2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35525588

RESUMO

In sample preparation, simultaneous extraction of analytes of very different polarity from biological matrixes represents a challenge. In this work, verapamil hydrochloride (VRP), amitriptyline (AMP), tyramine (TYR), atenolol (ATN), metopropol (MTP) and nortriptyline (NRP) were used as basic model analytes and simultaneously extracted from urine samples by liquid-phase microextraction (LPME) in a microfluidic device. The model analytes (target compounds) were pharmaceuticals with 0.4 < log P < 5. Different organic solvents and mixtures of them were investigated as supported liquid membrane (SLM), and a mixture of 2:1 (v/v) tributyl phosphate (TBP) and dihexyl ether (DHE) was found to be highly efficient for the simultaneous extraction of the non-polar and polar model analytes. TBP reduced the intrinsic hydrophobicity of the SLM and facilitated extraction of polar analytes, while DHE served to minimize trapping of non-polar analytes. Sample and acceptor phase composition were adjusted to pH 12 and pH 1.5, respectively. Urine samples were pumped into the microfluidic system at 1 µL min-1 and the extraction was completed in 7 min. Recoveries exceeded 78% for the target analytes, and the relative standard deviation (n = 4) was below 7% in all cases. Using five microliters of SLM, the microfluidic extraction system showed good long-term stability, and the same SLM was used for more than 18 consecutive extractions.


Assuntos
Microextração em Fase Líquida , Microfluídica , Éteres , Humanos , Dispositivos Lab-On-A-Chip , Membranas Artificiais , Preparações Farmacêuticas , Solventes
3.
Sci Rep ; 12(1): 7392, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513689

RESUMO

Circulating tumor cells (CTCs) survive in the bloodstream and then seed and invade to foster tumor metastasis. The arrest of cancer cells is favored by permissive flow forces and geometrical constraints. Through the use of high-throughput microfluidic devices designed to mimic capillary-sized vessels, we applied pressure differences to cancer cells (MCF-7 cell line) and recorded the cell traverse-vessel behaviors. Our results showed that cancer cells transform from a Newtonian droplet state to an adhesion/migration state when cancer cells traverse artificial vessels. To explain these phenomena, a modified Newtonian droplet model was also proposed. These phenomena and the modified model may reveal how CTCs in the blood seed and invade vessels under suitable conditions.


Assuntos
Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Humanos , Dispositivos Lab-On-A-Chip , Células MCF-7 , Células Neoplásicas Circulantes/patologia
4.
Biomaterials ; 285: 121531, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35533441

RESUMO

Recent advances in biomaterials, microfabrication, microfluidics, and cell biology have led to the development of organ-on-a-chip devices that can reproduce key functions of various organs. Such platforms promise to provide novel insights into various physiological events, including mechanisms of disease, and evaluate the effects of external interventions, such as drug administration. The neuroscience field is expected to benefit greatly from these innovative tools. Conventional ex vivo studies of the nervous system have been limited by the inability of cell culture to adequately mimic in vivo physiology. While animal models can be used, their relevance to human physiology is uncertain and their use is laborious and associated with ethical issues. To date, organ-on-a-chip systems have been developed to model different tissue components of the brain, including brain regions with specific functions and the blood brain barrier, both in normal and pathophysiological conditions. While the field is still in its infancy, it is expected to have major impact on studies of neurophysiology, pathology and neuropharmacology in future. Here, we review advances made and limitations faced in an effort to stimulate development of the next generation of brain-on-a-chip devices.


Assuntos
Dispositivos Lab-On-A-Chip , Microfluídica , Animais , Materiais Biocompatíveis , Barreira Hematoencefálica , Microfluídica/métodos , Microtecnologia
5.
Biosens Bioelectron ; 210: 114351, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35569269

RESUMO

Epidermal microfluidic devices with long microchannels have been developed for continuous sweat analysis, which are crucial to assess personal hydration status and underlying health conditions. However, the flow resistance in long channels and the ionic concentration variation significantly affect the accuracy of both the sweat rate and electrolyte concentration measurements. Herein, we present a novel fluidic-controlled wearable platform for synchronously dropwise-detecting the sweat rate and total electrolyte concentration. The unconventional platform consisting of a vertically shortened channel, a pair of embedded electrodes and an absorption layer, is designed to minimize the flow resistance and transform sweat fluidics into uniform micro-droplets for chronological and dropwise detection. Real-time sweat conductance is decoupled from a square-wave-like curve, where the sweat rate and electrolyte concentration can be derived from the interval time and peak value, respectively. Flexible and wearable band devices are demonstrated to show their potential application for hydration status assessment during exercises.


Assuntos
Técnicas Biossensoriais , Dispositivos Eletrônicos Vestíveis , Eletrólitos , Dispositivos Lab-On-A-Chip , Suor
6.
Methods Mol Biol ; 2502: 329-349, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412249

RESUMO

Cancer metastasis, that is, the spreading of tumor cells from the primary tumor to distant sites, requires cancer cells to travel through pores substantially smaller than their cross section . This "confined migration" requires substantial deformation by the relatively large and rigid nucleus, which can impact nuclear compartmentalization, trigger cellular mechanotransduction pathways, and increase genomic instability. To improve our understanding of how cells perform and respond to confined migration, we developed polydimethylsiloxane (PDMS) microfluidic devices in which cells migrate through a precisely controlled "field of pillars" that closely mimic the intermittent confinement of tumor microenvironments and interstitial spaces. The devices can be designed with various densities of pillars, ranging from a very low density that does not require nuclear deformation to high densities that present microenvironment conditions with severe confinement. The devices enable assessment of cellular fitness for confined migration based on the distance traveled through the constriction area over several days. In this protocol, we present two complementary techniques to generate silicon master molds for the device fabrication: (1) SU-8 soft lithography for rapid prototyping and for devices with relatively large features; and (2) reactive ion etching (RIE) to achieve finer features and more durable molds. In addition, we describe the production, use, and validation of the devices, along with the analysis pipeline for experiments using the devices with fluorescently labeled cells. Collectively, this protocol enables the study of confined migration and is readily amendable to investigate other aspects of confined migration mechanobiology, such as nuclear pore complex function in response to nuclear deformation.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Biofísica , Movimento Celular/fisiologia , Núcleo Celular , Mecanotransdução Celular
7.
Sci Rep ; 12(1): 6750, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468928

RESUMO

We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a "big chamber" microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent.


Assuntos
Neurospora crassa , Difusão , Dispositivos Lab-On-A-Chip , Percepção de Quorum
8.
Sci Rep ; 12(1): 6855, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477984

RESUMO

Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development.


Assuntos
Dispositivos Lab-On-A-Chip , Neutrófilos , Colágeno , Endotélio , Matriz Extracelular , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Neutrófilos/fisiologia
9.
J Vis Exp ; (181)2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35404350

RESUMO

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.


Assuntos
COVID-19 , Nanopartículas , Humanos , Dispositivos Lab-On-A-Chip , Lipídeos , Lipossomos , RNA Interferente Pequeno/metabolismo , Distribuição Tecidual
10.
Int J Mol Sci ; 23(8)2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35457056

RESUMO

Psoriasis is a chronic inflammatory disease with unmet medical needs. To clarify potential therapeutic targets, different animal models have been developed. In the current study, imiquimod-induced psoriasiform dermatitis was used for monitoring the changes in skin thickness, transepidermal water loss, body weight, blood perfusion and drug permeability for a topical cream formulation of caffeine, both in wild type and in knock out mice. Morphological characterization of control and diseased tissues was performed by scanning electron microscopy and two-photon microscopy. The chemically induced psoriatic group showed increased skin permeability for the model drug during disease progression. In wild type and TRPA1 KO mice, however, enhanced skin thickness and hyperkeratosis blocked further increase of drug penetration at the late phase (96 h). These results indicate that topical drug therapy can be more effective in early phases of plaque development, when skin thickness is lower. Although paracellular connections (tight junctions) are looser in the advanced phase, hyperkeratosis blocks drug delivery through the transappendageal routes. Novel drug formulations may have the potency for effective drug delivery across the epidermal barrier even in the advanced phase. For development of more effective topical drugs, further research is proposed to explore drug penetration both in healthy and diseased conditions.


Assuntos
Dispositivos Lab-On-A-Chip , Psoríase , Animais , Modelos Animais de Doenças , Epiderme , Camundongos , Imagem Óptica , Permeabilidade , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Pele
12.
Lab Chip ; 22(9): 1650-1679, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35403636

RESUMO

Cilia are microscopic hair-like external cell organelles that are ubiquitously present in nature, also within the human body. They fulfill crucial biological functions: motile cilia provide transportation of fluids and cells, and immotile cilia sense shear stress and concentrations of chemical species. Inspired by nature, scientists have developed artificial cilia mimicking the functions of biological cilia, aiming at application in microfluidic devices like lab-on-chip or organ-on-chip. By actuating the artificial cilia, for example by a magnetic field, an electric field, or pneumatics, microfluidic flow can be generated and particles can be transported. Other functions that have been explored are anti-biofouling and flow sensing. We provide a critical review of the progress in artificial cilia research and development as well as an evaluation of its future potential. We cover all aspects from fabrication approaches, actuation principles, artificial cilia functions - flow generation, particle transport and flow sensing - to applications. In addition to in-depth analyses of the current state of knowledge, we provide classifications of the different approaches and quantitative comparisons of the results obtained. We conclude that artificial cilia research is very much alive, with some concepts close to industrial implementation, and other developments just starting to open novel scientific opportunities.


Assuntos
Incrustação Biológica , Cílios , Humanos , Dispositivos Lab-On-A-Chip , Campos Magnéticos , Microfluídica/métodos
13.
Lab Chip ; 22(9): 1680-1689, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35417508

RESUMO

Microfluidics offers tremendous opportunities to understand the underlying biology of fertilization at the single-cell level and improve infertility management, however, its true clinical impact is yet to be realized. Lab-on-a-chip devices have generally failed to diffuse into clinical practice due to issues associated with their translation or their practicality and performance in clinical settings. In this perspective, I reflect on how the full potential of microfluidic technologies for fertility can be realized by considering regulatory and manufacturing considerations at the development stage and by redefining our developmental goals to directly target the ultimate clinical needs. I also challenge the common rationale around developing technologies for infertility treatment based on reducing cost and complexity in operation as the ultimate outcome is invaluable, human life.


Assuntos
Infertilidade , Dispositivos Lab-On-A-Chip , Fertilidade , Humanos , Microfluídica , Tecnologia
14.
Sci Rep ; 12(1): 6380, 2022 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-35430583

RESUMO

Precise manipulation of microparticles have fundamental applications in the fields of lab-on-a-chip and biomedical engineering. Here, for the first time, we propose a fully operational microfluidic chip equipped with thin magnetic films composed of straight tracks and bends which precisely transports numerous single-particles in the size range of ~ 2.8-20 µm simultaneously, to certain points, synced with the general external three-axial magnetic field. The uniqueness of this design arises from the introduced vertical bias field that provides a repulsion force between the particles and prevents unwanted particle cluster formation, which is a challenge in devices operating in two-dimensional fields. Furthermore, the chip operates as an accurate sensor and detects low levels of proteins and DNA fragments, being captured by the ligand-functionalized magnetic beads, while lowering the background noise by excluding the unwanted bead pairs seen in the previous works. The image-processing detection method in this work allows detection at the single-pair resolution, increasing the sensitivity. The proposed device offers high-throughput particle transport and ultra-sensitive bio-detection in a highly parallel manner at single-particle resolution. It can also operate as a robust single-cell analysis platform for manipulating magnetized single-cells and assembling them in large arrays, with important applications in biology.


Assuntos
Campos Magnéticos , Técnicas Analíticas Microfluídicas , Separação Imunomagnética , Dispositivos Lab-On-A-Chip , Magnetismo , Microfluídica
15.
Mikrochim Acta ; 189(5): 177, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35381890

RESUMO

At present, analytical lab-on-chip devices find their usage in different facets of chemical analysis, biological analysis, point of care analysis, biosensors, etc. In addition, graphene has already established itself as an essential component of advanced lab-on-chip devices. Graphene-based lab-on-chip devices have achieved appreciable admiration because of their peerless performance in comparison to others. However, to accomplish a sustainable future, a device must undergo "green screening" to check its environmental compatibility. Thus, extensive research is carried out globally to make the graphene-based lab-on-chip green, though it is yet to be achieved. Nevertheless, as a ray of hope, there are few existing strategies that can be stitched together for feasible fabrication of environment-friendly green graphene-based analytical lab-on-chip, and those prospective pathways are reviewed in this paper.


Assuntos
Técnicas Biossensoriais , Grafite , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Estudos Prospectivos
16.
Methods Mol Biol ; 2475: 239-257, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35451763

RESUMO

Relevant human in vitro models of the retinal microvasculature can be used to study the role of disease mediators on retinal barrier dysfunction and assess the efficacy of early drug candidates. This chapter describes an organ-on-a-chip model of the retinal microvasculature that allows for facile quantification of barrier permeability in response to leakage mediators, such as Vascular Endothelial Growth Factor (VEGF), and enables screening of VEGF-induced permeability inhibitors. This chapter also presents an automated confocal imaging method for the visualization of endothelial tube morphology as an additional measure of barrier integrity.


Assuntos
Barreira Hematorretiniana , Fator A de Crescimento do Endotélio Vascular , Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar/fisiologia , Humanos , Dispositivos Lab-On-A-Chip , Microvasos/metabolismo , Permeabilidade , Vasos Retinianos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Kidney360 ; 3(2): 217-231, 2022 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-35373131

RESUMO

Background: Renal ischemia/reperfusion injury (rIRI) is one of the major causes of AKI. Although animal models are suitable for investigating systemic symptoms of AKI, they are limited in translatability. Human in vitro models are crucial in giving mechanistic insights into rIRI; however, they miss out on crucial aspects such as reperfusion injury and the multitissue aspect of AKI. Methods: We advanced the current renal proximal tubule-on-a-chip model to a coculture model with a perfused endothelial vessel separated by an extracellular matrix. The coculture was characterized for its three-dimensional structure, protein expression, and response to nephrotoxins. Then, rIRI was captured through control of oxygen levels, nutrient availability, and perfusion flow settings. Injury was quantified through morphologic assessment, caspase-3/7 activation, and cell viability. Results: The combination of low oxygen, reduced glucose, and interrupted flow was potent to disturb the proximal tubules. This effect was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia-reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase-3/7 activation. Conclusions: A human in vitro rIRI model was developed using a coculture of a proximal tubule and blood vessel on-a-chip, which was used to characterize the renoprotective effect of adenosine. The robustness of the model and assays in combination with the throughput of the platform make it ideal to advance pathophysiological research and enable the development of novel therapeutic modalities.


Assuntos
Injúria Renal Aguda , Dispositivos Lab-On-A-Chip , Injúria Renal Aguda/prevenção & controle , Animais , Humanos , Isquemia/complicações , Túbulos Renais/metabolismo , Reperfusão/efeitos adversos
19.
Biosens Bioelectron ; 208: 114189, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35366427

RESUMO

Therapeutic drug monitoring (TDM) of adalimumab (ADM) at the point-of-care (POC) is key to prevent loss of response but has not been accomplished to date because true POC testing solutions are still lacking. Here, we present a novel "whole blood in - result out" self-powered microfluidic chip for detecting ADM within 30 min to enable TDM at POC. Hereto, we first demonstrated on-chip plasma separation from whole blood, followed by downscaling an ADM ELISA with maintained specificity and sensitivity in plasma. This assay was then performed on a robust and easy-to-use microfluidic chip we designed based on (i)SIMPLE technology, allowing autonomous function upon single finger press activation, which was successfully validated with patient samples. Herein, we prove the potential of our technology to detect targets starting from whole blood introduced directly on-chip and to integrate various immunoassays, both for TDM and other in vitro diagnostics applications, like infectious diseases.


Assuntos
Doenças Autoimunes , Técnicas Biossensoriais , Adalimumab/uso terapêutico , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/tratamento farmacológico , Monitoramento de Medicamentos , Humanos , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos
20.
Biochem Soc Trans ; 50(2): 665-673, 2022 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-35437569

RESUMO

As an emerging hot topic of the last decade, Organ on Chip (OoC) is a new technology that is attracting interest from both basic and translational scientists. The Biochemical Society, with its mission of supporting the advancement of science, with addressing grand challenges that have societal impact, has included OoC into their agenda to review the current state of the art, bottlenecks and future directions. This conference brought together representatives of the main stakeholders in the OoC field including academics, end-users, regulators and technology developers to discuss and identify requirements for this new technology to deliver on par with the expectations and the key challenges and gaps that still need to be addressed to achieve robust human-relevant tools, able to positively impact decision making in the pharmaceutical industry and reduce overreliance on poorly predictive animal models.


Assuntos
Dispositivos Lab-On-A-Chip , Tecnologia , Animais , Modelos Animais , Análise de Sequência com Séries de Oligonucleotídeos
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