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Although the dendritic cell (DC)-based modulation of immune responses has emerged as a promising therapeutic strategy for tumors, infections, and autoimmune diseases, basic research and therapeutic applications of DCs are hampered by expensive growth factors and sophisticated culture procedures. Furthermore, the platform to drive the differentiation of a certain DC subset without any additional biochemical manipulations has not yet been developed. Here, five types of polymer films with different hydrophobicity via an initiated chemical vapor deposition (iCVD) process to modulate the interactions related to cell-substrate adhesion are introduced. Especially, poly(cyclohexyl methacrylate) (pCHMA) substantially enhances the expansion and differentiation of conventional type 1 DCs (cDC1s), the prime DC subset for antigen cross-presentation, and CD8+ T cell activation, by 4.8-fold compared to the conventional protocol. The cDC1s generated from the pCHMA-coated plates retain the bona fide DC functions including the expression of co-stimulatory molecules, cytokine secretion, antigen uptake and processing, T cell activation, and induction of antitumor immune responses. To the authors' knowledge, this is the first report highlighting that the modulation of surface hydrophobicity of the culture plate can be an incisive approach to construct an advanced DC culture platform with high efficiency, which potentially facilitates basic research and the development of immunotherapy employing DCs.
Assuntos
Células Dendríticas , Polímeros , Apresentação de Antígeno , Técnicas de Cultura de Células/métodos , Células Dendríticas/metabolismo , Ativação Linfocitária , Polímeros/metabolismoRESUMO
For efficient therapeutic use of human mesenchymal stem cells (hMSCs), maximizing their self-renewal performance and multipotency must be fully retained. However, conventional trypsin-based cell passaging methods are known to damage the attached cells to be detached because of the inherent corrosive nature of trypsin, and continuous passaging substantially degrades the self-renewal and differentiation capacity of hMSCs. Therefore, it is imperative to secure a damage-free passaging method that supports cell growth as well as their stem cell function. Here, an enzyme-free cell detachment method using a poly(ethylene glycol dimethacrylate) (pEGDMA)-coated surface is developed, which allows for reduced integrin-dependent cell adhesion. Cell detachment can be facilitated simply by treating the plated cells on the pEGDMA surface with Ca2+ and Mg2+-depleted DPBS. Spontaneous cell detachment occurs within 10 min with the full retention of the cell viability and proliferation ability of hMSCs. Especially, the detachment method can minimize the surface protein damage of hMSCs compared to the conventional trypsin treatment and preserve the self-renewal property and differentiation capacity even with an increased passage number over 10. The developed enzyme-free detachment method using the pEGDMA-coated surface is highly promising for a culture platform to broaden its application to the field of tissue engineering and regenerative medicine.
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The aggregation of mesenchymal stem cells (MSCs) into three-dimensional (3D) spheroids has emerged as a promising therapeutic candidate for the treatment of a variety of diseases. In spite of the numerous 3D culture methods suggested recently for MSC spheroid generation, it is still elusive to fully reflect real stem cell niches; this effort majorly suffers from a lack of cell-extracellular matrix (ECM) interactions within the 3D spheroids. In this study, we develop a simple but versatile method for generating human MSC (hMSC) spheroids by culturing the cells on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). Interestingly, the pV4D4-coated surface allows a dynamic cell adhesion to the polymer surface while developing the formation of 3D spheroids. The corresponding mechanotransduction promotes the expression of the endogenous ECM and, in turn, results in a remarkable improvement in self-renewal abilities, pro-angiogenic potency, and multilineage differentiation capabilities. This observation highlights the significance of our method compared to the conventional spheroid-generating methods in terms of recreating the ECM-rich microenvironment. We believe the developed surface can serve as a versatile but reliable method for stem cell-based tissue engineering and regenerative medicine.
Assuntos
Polímeros , Esferoides Celulares , Células-Tronco , Matriz Extracelular , Humanos , Mecanotransdução CelularRESUMO
3D spheroids are considered as the improved in vitro model to mimic the distinct arrangements of the cells in vivo. To date, low-attachment surfaces have been most widely used to induce the spontaneous aggregation of cells in suspension by simply tuning the relative strength of the cell-cell adhesion over cell-substrate adhesion. However, aggregating cancer cells into 3D clusters should mean more than just adjoining the cells in the physical proximity. The tumor cell functionality is strongly affected by the adhesion networks between cancer cells and extracellular matrix (ECM). Here, we performed an in-depth analysis of how the nonmetastatic breast cancer cells (MCF7) can be transformed to gain invasive phenotypes through compact aggregation into 3D spheroids on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). By comparing the adhesion networks and invasion dynamics between 3D spheroids cultured on the pV4D4 surface with those cultured on conventional ultra-low-attachment (ULA) dishes, we report that only spheroids on the pV4D4 display active and sporadic cell-surface binding activities via dynamic protrusions, which correlates strongly with an increase in integrin ß1. Moreover, localized laminin expression at the core of the pV4D4-cultured spheroids confirms the prominence of the intimate integrin-laminin interactions prompted by the exposure to pV4D4. This study suggests that structurally and functionally dissimilar 3D spheroids can be generated from the same type of cells on the surfaces of different physicochemical properties without any chemical treatment or genetic manipulation.
Assuntos
Neoplasias , Esferoides Celulares , Adesão Celular , Comunicação Celular , Matriz Extracelular , PolímerosRESUMO
Cell sheet engineering, a technique utilizing a monolayer cell sheet, has recently emerged as a promising technology for scaffold-free tissue engineering. In contrast to conventional tissue-engineering approaches, the cell sheet technology allows cell harvest as a continuous cell sheet with intact extracellular matrix proteins and cell-cell junction, which facilitates cell transplantation without any other artificial biomaterials. A facile, non-thermoresponsive method is demonstrated for a rapid but highly reliable platform for cell-sheet engineering. The developed method exploits the precise modulation of cell-substrate interactions by controlling the surface energy of the substrate via a series of functional polymer coatings to enable prompt cell sheet harvesting within 100 s. The engineered surface can trigger an intrinsic cellular response upon the depletion of divalent cations, leading to spontaneous cell sheet detachment under physiological conditions (pH 7.4 and 37 °C) in a non-thermoresponsive manner. Additionally, the therapeutic potential of the cell sheet is successfully demonstrated by the transplantation of multilayered cell sheets into mouse models of diabetic wounds and ischemia. These findings highlight the ability of the developed surface for non-thermoresponsive cell sheet engineering to serve as a robust platform for regenerative medicine and provide significant breakthroughs in cell sheet technology.
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Polímeros/química , Engenharia Tecidual/métodos , Adsorção , Fibronectinas/química , Propriedades de Superfície , Temperatura , Fatores de TempoRESUMO
Postsurgical intraocular lens (IOL) infection caused by pathogenic bacteria can result in blindness and often requires a secondary operation to replace the contaminated lens. The incorporation of an antibacterial property onto the IOL surface can prevent bacterial infection and postoperative endophthalmitis. This study describes a polymeric nanopillar array (NPA) integrated onto an IOL, which captures and eradicates the bacteria by rupturing the bacterial membrane. This is accomplished by changing the behavior of the elastic nanopillars using bending, restoration, and antibacterial surface modification. The combination of the polymer coating and NPA dimensions can decrease the adhesivity of corneal endothelial cells and posterior capsule opacification without causing cytotoxicity. An ionic antibacterial polymer layer is introduced onto an NPA using an initiated chemical vapor deposition process. This improves bacterial membrane rupture efficiency by increasing the interactions between the bacteria and nanopillars and damages the bacterial membrane using quaternary ammonium compounds. The newly developed ionic polymer-coated NPA exceeds 99% antibacterial efficiency against Staphylococcus aureus, which is achieved through topological and physicochemical surface modification. Thus, this paper provides a novel, efficient strategy to prevent postoperative complications related to bacteria contamination of IOL after cataract surgery.
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Cristalino , Lentes Intraoculares , Antibacterianos/farmacologia , Células Endoteliais , PolímerosRESUMO
Physicochemical and biological gradients are desirable features for hydrogels to enhance their relevance to biological environments for three-dimensional (3D) cell culture. Therefore, simple and efficient techniques to generate chemical, physical and biological gradients within hydrogels are highly desirable. This work demonstrates a technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels by stacking and crosslinking prehydrogel solution in a layer by layer manner. Partial crosslinking of the hydrogel allows mixing of prehydrogel solution with the previous hydrogel layer, which makes a smooth gradient profile, rather than discrete layers. This technique enables the generation of concentration gradients of bovine serum albumin in both gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate hydrogels, as well as mechanical gradients across a hydrogel containing varying gel concentrations. Fluorescence microscopy, mechanical testing, and scanning electron microscopy show that the gradient profiles can be controlled by changing both the volume and concentration of each layer as well as intensity of UV exposure. GelMA hydrogel gradients with different Young's moduli were successfully used to culture human fibroblasts. The fibroblasts migrated along the gradient axis and showed different morphologies. In general, the proposed technique provides a rapid and simple approach to design and fabricate 3D hydrogel gradients for in vitro biological studies and potentially for in vivo tissue engineering applications.
Assuntos
Reagentes de Ligações Cruzadas/química , Hidrogéis/química , Luz , Fenômenos Mecânicos , Animais , Humanos , Metacrilatos/química , Camundongos , Células NIH 3T3 , Polímeros/química , Soroalbumina Bovina/metabolismo , Suínos , Raios UltravioletaRESUMO
Fabrication of new antibacterial surfaces has become a primary strategy for preventing device-associated infections (DAIs). Although considerable progress has recently been made in reducing DAIs, current antibacterial coating methods are technically complex and do not allow selective bacterial killing. Here, we propose novel anti-infective surfaces made of a cross-linked ionic polymer film that achieve selective bacteria killing while simultaneously favoring the survival of mammalian cells. A one-step polymerization process known as initiated chemical vapor deposition was used to generate a cross-linked ionic polymer film from 4-vinylbenzyl chloride and 2-(dimethylamino) ethyl methacrylate monomers in the vapor phase. In particular, the deposition process produced a polymer network with quaternary ammonium cross-linking sites, which provided the surface with an ionic moiety with an excellent antibacterial contact-killing property. This method confers substrate compatibility, which enables various materials to be coated with ionic polymer films for use in medical implants. Moreover, the ionic polymer-deposited surfaces supported the healthy growth of mammalian cells while selectively inhibiting bacterial growth in coculture models without any detectable cytotoxicity. Thus, the cross-linked ionic polymer-based antibacterial surface developed in this study can serve as an ideal platform for biomedical applications that require a highly sterile environment.
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Point-of-care (POC) molecular diagnostics plays a pivotal role for the prevention and treatment of infectious diseases. In spite of recent advancement in microfluidic based POC devices, there are still rooms for development to realize rapid, automatic and cost-effective sample-to-result genetic analysis. In this study, we propose an integrated rotary microfluidic system that is capable of performing glass microbead based DNA extraction, loop mediated isothermal amplification (LAMP), and colorimetric lateral flow strip based detection in a sequential manner with an optimized microfluidic design and a rotational speed control. Rotation direction-dependent coriolis force and siphon valving structures enable us to perform the fluidic control and metering, and the use of the lateral flow strip as a detection method renders all the analytical processes for nucleic acid test simplified and integrated without the need of expensive instruments or human intervention. As a proof of concept for point-of-care DNA diagnostics, we identified the food-borne bacterial pathogen which was contaminated in water or milk. Not only monoplex Salmonella Typhimurium but also multiplex Salmonella Typhimurium and Vibrio parahaemolyticus were analysed on the integrated rotary genetic analysis microsystem with a limit of detection of 50 CFU in 80min. In addition, three multiple samples were simultaneously analysed on a single device. The sample-to-result capability of the proposed microdevice provides great usefulness in the fields of clinical diagnostics, food safety and environment monitoring.
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DNA Bacteriano/isolamento & purificação , Dispositivos Lab-On-A-Chip , Leite/microbiologia , Salmonella typhimurium/isolamento & purificação , Extração em Fase Sólida/instrumentação , Vibrio parahaemolyticus/isolamento & purificação , Microbiologia da Água , Animais , Técnicas Biossensoriais/instrumentação , Colorimetria/instrumentação , DNA Bacteriano/análise , DNA Bacteriano/genética , Desenho de Equipamento , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Fitas Reagentes/análise , Infecções por Salmonella/diagnóstico , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Vibrioses/diagnóstico , Vibrioses/microbiologia , Vibrio parahaemolyticus/genéticaRESUMO
We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.
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Técnicas Biossensoriais , DNA Bacteriano/isolamento & purificação , Doenças Transmitidas por Alimentos/microbiologia , Dispositivos Lab-On-A-Chip , Colorimetria , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Doenças Transmitidas por Alimentos/diagnóstico , Humanos , Limite de Detecção , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/patogenicidade , Vibrio parahaemolyticus/isolamento & purificação , Vibrio parahaemolyticus/patogenicidadeRESUMO
This work describes fully automated and colorimetric foodborne pathogen detection on an integrated centrifugal microfluidic device, which is called a lab-on-a-disc. All the processes for molecular diagnostics including DNA extraction and purification, DNA amplification and amplicon detection were integrated on a single disc. Silica microbeads incorporated in the disc enabled extraction and purification of bacterial genomic DNA from bacteria-contaminated milk samples. We targeted four kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus and Listeria monocytogenes) and performed loop-mediated isothermal amplification (LAMP) to amplify the specific genes of the targets. Colorimetric detection mediated by a metal indicator confirmed the results of the LAMP reactions with the colour change of the LAMP mixtures from purple to sky blue. The whole process was conducted in an automated manner using the lab-on-a-disc and a miniaturized rotary instrument equipped with three heating blocks. We demonstrated that a milk sample contaminated with foodborne pathogens can be automatically analysed on the centrifugal disc even at the 10 bacterial cell level in 65 min. The simplicity and portability of the proposed microdevice would provide an advanced platform for point-of-care diagnostics of foodborne pathogens, where prompt confirmation of food quality is needed.
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Colorimetria/instrumentação , Colorimetria/métodos , Doenças Transmitidas por Alimentos/microbiologia , Leite/microbiologia , Animais , Automação , Centrifugação , DNA Bacteriano/genética , Escherichia coli O157/genética , Microbiologia de Alimentos/instrumentação , Microbiologia de Alimentos/métodos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Listeria monocytogenes/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmonella typhimurium/genética , Vibrio parahaemolyticus/genéticaRESUMO
In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 µL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.
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Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/microbiologia , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Animais , Centrifugação , Desenho de Equipamento/métodos , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Genes Bacterianos , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Recombinases/genética , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidadeRESUMO
An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice.