RESUMEN
Liver cancer represents a significant global burden in terms of cancer-related mortality, with resistance to anti-angiogenic drugs such as Sorafenib and Lenvatinib presenting a formidable challenge. Tumor angiogenesis, characterized by the formation of new blood vessels within tumors, plays a pivotal role in cancer progression and metastasis. Tumor endothelial cells, specialized endothelial cells lining tumor blood vessels, exhibit unique phenotypic and functional traits that drive aberrant vessel formation and contribute to therapy resistance. CD105, a cell-surface glycoprotein that is highly expressed on endothelial cells during angiogenesis, including tumor endothelial cells, regulates endothelial cell proliferation, migration, and vessel formation by modulating transforming growth factor-beta (TGF-ß) signaling pathways. Elevated CD105 expression on tumor endothelial cells correlates with increased angiogenic activity and poor prognosis in cancer patients. Targeting CD105 with antibodies presents a promising strategy to inhibit tumor angiogenesis and disrupt tumor vasculature, offering potential therapeutic benefits by interfering with the tumor microenvironment and inhibiting its progression. This study investigates tumor angiogenesis through a three-dimensional (3D) microfluidic co-culture system incorporating endothelial cells and hepatocellular carcinoma (HCC) cells. The primary focus is on the role of CD105 expression within the liver tumor microenvironment and its contribution to increased chemoresistance. Additionally, this research examines the influence of CD105 expression on the efficacy of tyrosine kinase inhibitors (TKIs) and its pivotal function in facilitating angiogenesis in liver tumors. The proposed microfluidic chip model investigates liver cancer cell interactions within a microfluidic chip model designed to simulate aspects of liver tumor angiogenesis.
Asunto(s)
Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Dispositivos Laboratorio en un Chip , Neoplasias Hepáticas , Inhibidores de Proteínas Quinasas , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Inhibidores de Proteínas Quinasas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endoglina/metabolismo , Endoglina/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Sorafenib/farmacología , Línea Celular Tumoral , Compuestos de Fenilurea , QuinolinasRESUMEN
Microalgae that have recently captivated interest worldwide are a great source of renewable, sustainable and economical biofuels. The extensive potential application in the renewable energy, biopharmaceutical and nutraceutical industries have made them necessary resources for green energy. Microalgae can substitute liquid fossil fuels based on cost, renewability and environmental concern. Microfluidic-based systems outperform their competitors by executing many functions, such as sorting and analysing small volumes of samples (nanolitre to picolitre) with better sensitivities. In this review, we consider the developing uses of microfluidic technology on microalgal processes such as cell sorting, cultivation, harvesting and applications in biofuels and biosensing.
Asunto(s)
Microalgas , Biocombustibles , Biomasa , Combustibles Fósiles , MicrofluídicaRESUMEN
Cancer cell-immune cell hybrids and cancer immunotherapy have attracted much attention in recent years. The design of efficient cell pairing and fusion chips for hybridoma generation has been, subsequently, a subject of great interest. Here, we report a three-layered integrated Microfluidic Flip-Chip (MFC) consisting of a thin through-hole membrane sandwiched between a mirrored array of microfluidic channels and saw-tooth shaped titanium electrodes on the glass. We discuss the design and operation of MFC and show its applicability for cell fusion. The proposed device combines passive hydrodynamic phenomenon and gravitational sedimentation, which allows the transportation and trapping of homotypic and heterotypic cells in large numbers with pairing efficiencies of 75~78% and fusion efficiencies of 73%. Additionally, we also report properties of fused cells from cell biology perspectives, including combined fluorescence-labeled intracellular materials from THP1 and A549, mixed cell morphology, and cell viability. The MFC can be tuned for pairing and fusion of cells with a similar protocol for different cell types. The MFC can be easily disconnected from the test setup for further analysis.
Asunto(s)
Fusión Celular , Hidrodinámica , Microfluídica , Células A549 , Fusión Celular/instrumentación , Supervivencia Celular , Electricidad , Humanos , Imagenología Tridimensional , Microfluídica/instrumentación , Células THP-1RESUMEN
Globally, non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths. Despite advancements in chemotherapy and targeted therapies, the 5-year survival rate has remained at 16% for the past forty years. Minimal residual disease (MRD) is described as the existence of either isolated tumour cells or circulating tumour cells in biological liquid of patients after removal of the primary tumour without any clinical signs of cancer. Recently, liquid biopsy has been promising as a non-invasive method of disease monitoring and treatment guidelines as an MRD marker. Liquid biopsy could be used to detect and assess earlier stages of NSCLC, post-treatment MRD, resistance to targeted therapies, immune checkpoint inhibitors (ICIs) and tumour mutational burden. MRD surveillance has been proposed as a potential marker for lung cancer relapse. Principally, biosensors provide the quantitative analysis of various materials by converting biological functions into quantifiable signals. Biosensors are usually operated to detect antibodies, enzymes, DNA, RNA, extracellular vesicles (EVs) and whole cells. Here, we present a category of biosensors based on the signal transduction method for identifying biosensor-based biomarkers in liquid biopsy specimens to monitor lung cancer treatment.
Asunto(s)
Técnicas Biosensibles , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/terapia , Humanos , Biopsia Líquida , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Neoplasia ResidualRESUMEN
Agglutination is an antigen-antibody reaction with visible expression of aggregation of the antigens and their corresponding antibodies. Applications extend to the identification of acute bacterial infection, hemagglutination, such as blood grouping, and diagnostic immunology. Our finger-powered agglutination lab chip with external CMOS image sensing was developed to support a platform for inexpensive, rapid point-of-care (POC) testing applications related to agglutination effects. In this paper, blood grouping (ABO and Rh grouping) was utilized to demonstrate the function of our finger-powered agglutination lab chip with CMOS image sensing. Blood antibodies were preloaded into the antibody reaction chamber in the lab chip. The blood sample was pushed through the antibody reaction chamber using finger-powered pressure actuation to initiate a hemagglutination reaction to identify the blood type at the on-chip detection area using our homemade CMOS image sensing mini-system. Finger-powered actuation without the need for external electrical pumping is excellent for low-cost POC applications, but the pumping liquid volume per finger push is hard to control. In our finger-powered agglutination lab chip with CMOS image sensing, we minimized the effects of different finger push depths and achieved robust performance for the test results with different push depths. The driving sample volume per finger push is about 0.79 mm3. For different chips and different pushes, the driven sample volume per finger push was observed to vary in the range of 0.64 to 1.18 mm3. The red blood cells were separated from the plasma on-chip after the whole blood sample was finger pumped and before the red blood cells reached the antibody chamber via an embedded plasma-separation membrane. Our homemade CMOS image mini-system robustly read and identified the agglutination results on our agglutination lab chip.
Asunto(s)
Anticuerpos/inmunología , Antígenos/inmunología , Eritrocitos/inmunología , Dispositivos Laboratorio en un Chip , Imagen Óptica , Pruebas en el Punto de Atención , Aglutinación , Reacciones Antígeno-Anticuerpo , Eritrocitos/citología , HumanosRESUMEN
The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1ß and human TNF-α, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520â¯nL; the total duration of the on-chip process is less than 40â¯min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-α change little from day 3 to day 5-6, but the concentrations of IL-1ß for some embryos might double from day 3 to day 5-6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1ß, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.
Asunto(s)
Técnicas Biosensibles , Interleucina-1beta/aislamiento & purificación , Microfluídica , Factor de Necrosis Tumoral alfa/aislamiento & purificación , Medios de Cultivo/química , Femenino , Humanos , Interleucina-1beta/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A cancer immunotherapy µ-environment LabChip, equipped with titanium oxide phthalocyanine (TiOPc)-based optoelectronic tweezers (OET) to achieve direct cell-cell contact, can be used to study the interaction between immune cells and other cells for real-time analysis of NK cells' behavior. In microfluidic devices, it is difficult to solve dead zone problems and observe dynamic cell-cell interactions. We have created a stable and static culture µ-environment which can enhance NK cell activities. In addition, OET is used to solve dead zone problems by manipulating a single cell into four-leaf-clover-shaped (FLCS) microwells made of poly(ethylene glycol) diacrylate (PEG-DA) through optofluidic maskless lithography, causing direct cell-cell contact. Our design reconstructed an in vitro human immune system for the study of dynamic immunological response. When the NK cells came into contact with the target cells in the µ-environment LabChip, we observed that the target cells showed apoptotic characteristics (i.e. cell shrinkage and blebbing within 2 h and then die within 3 h). In addition, our µ-environment LabChip demonstrated higher NK cell activity compared with conventional analysis. We have created an innovative cancer immunotherapy µ-environment LabChip to provide a stable and static µ-environment for cell-cell interaction study. Furthermore, our µ-environment LabChip showed the potential to enhance NK cell activity and to study immunological interactions between immune cells and cancer cells dynamically.
Asunto(s)
Inmunoterapia , Dispositivos Laboratorio en un Chip , Modelos Biológicos , Pinzas Ópticas , Microambiente Tumoral , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Línea Celular , Diseño de Equipo , Humanos , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Here, we describe a technique to manipulate a low number of beads to achieve high washing efficiency with zero bead loss in the washing process of a digital microfluidic (DMF) immunoassay. Previously, two magnetic bead extraction methods were reported in the DMF platform: (1) single-side electrowetting method and (2) double-side electrowetting method. The first approach could provide high washing efficiency, but it required a large number of beads. The second approach could reduce the required number of beads, but it was inefficient where multiple washes were required. More importantly, bead loss during the washing process was unavoidable in both methods. Here, an improved double-side electrowetting method is proposed for bead extraction by utilizing a series of unequal electrodes. It is shown that, with proper electrode size ratio, only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. It allows using only about 25 magnetic beads in DMF immunoassay to increase the number of captured analytes on each bead effectively. In our human soluble tumor necrosis factor receptor I (sTNF-RI) model immunoassay, the experimental results show that, comparing to our previous results without using the proposed bead extraction technique, the immunoassay with low bead number significantly enhances the fluorescence signal to provide a better limit of detection (3.14 pg/ml) with smaller reagent volumes (200 nl) and shorter analysis time (<1 h). This improved bead extraction technique not only can be used in the DMF immunoassay but also has great potential to be used in any other bead-based DMF systems for different applications.
RESUMEN
Dendritic cells/tumor fusions have shown to elicit anti-cancer immunity in different cancer types. However, the application of these vaccines for human cancer immunotherapy are limited by the instable quality and insufficient quanity of fusion cells. We present a cell electrofusion chip fabricated using soft lithography technique, which combines the rapid and precise cell pairing microstructures and the high yield electrofusion micro-electrodes to improve the cell fusion. The design uses hydrodynamic trapping in combination with positive dielectrophoretic force (pDEP) to achieve cell fusion. The chip consists of total 960 pairs of trapping channels, which are capable of pairing and fusing both homogeneous and heterogeneous types of cells. The fused cells can be easily taken out of the chip that makes this device a distinguishable from other designs. We observe pairing efficiency of 68% with fusion efficiency of 64%.
Asunto(s)
Fusión Celular/métodos , Hibridomas/citología , Inmunoterapia/métodos , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Fusión Celular/instrumentación , Línea Celular Tumoral , Humanos , Hibridomas/inmunología , Leucemia Monocítica Aguda/patología , Neoplasias Pulmonares/patología , Microelectrodos , Microfluídica/instrumentaciónRESUMEN
Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application.
Asunto(s)
Electrohumectación/instrumentación , Electrohumectación/métodos , Técnicas de Cultivo de Embriones/instrumentación , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/fisiología , Microfluídica/instrumentación , Microfluídica/métodos , Animales , Blastocisto/citología , Supervivencia Celular , Transferencia de Embrión , Femenino , Ratones , SeudoembarazoRESUMEN
How to release growth factors (GFs) scientifically to promote stem cell proliferation and differentiation is one of the most significant research focuses in the field of regenerative medicine. In a controlled release system, growth factors, extracellular matrices or biomaterial carriers, and sometimes stem cells together form a geometric entirety. Biomaterial carriers provide GFs with a support structure to be adhered, immobilized, encapsulated or/and protected. As a unity, the release rate and rhythm of GFs on cells are normally very delicate and precise. Up to now, the best strategy for clinical applications is the combination systems that encapsulate GFs in microspheres, particularly the nano- or micro-encapsulation techniques integrated GFs with biomaterial carriers. In this mini review, we summarize the current progress in GF delivery systems for regenerative medicine and provide an outlook on two main aspects: one is the classes of stem cells and GFs that have been used frequently in regenerative medicine, including their respective application conditions and functions; the other is the controlled GF release systems, in which various GFs are released orderly and continuously without diffusing simply and rapidly, including their respective opportunities and challenges.
Asunto(s)
Sistemas de Liberación de Medicamentos , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Medicina Regenerativa/métodos , Animales , Materiales Biocompatibles/química , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Preparaciones de Acción Retardada , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre/citologíaRESUMEN
BACKGROUND: Fibrocytes express several chemokine receptors (CCR7 and CXCR4) that regulate their recruitment and trafficking into tissue-damage sites in response to specific chemokine gradients (CCL19 and CXCL12). OBJECTIVE: We investigated whether these chemoattractants and S100A9, through the receptor for advanced glycation end-products (RAGE; ie, its receptor), are involved in fibrocyte trafficking in patients with chronic obstructive asthma (COA) and during an acute exacerbation (AE) in patients without airflow obstruction (Asthma AE group). METHODS: We collected peripheral blood from 14 asthmatic patients with normal pulmonary function, 14 patients with COA, 11 patients in the Asthma AE group, and 14 healthy subjects. Isolated circulating fibrocytes were used for migration assay. Expression of CCR7, CXCR4, S100A9, and RAGE in fibrocytes was measured by using flow cytometry. CCL19 and CXCL12 expression in bronchial tissues was determined by using immunohistochemistry and RT-PCR. RESULTS: There were higher numbers of circulating fibrocytes in patients in the Asthma AE group and patients with COA. The expression of CXCL12 in bronchial tissues and CXCR4 in circulating fibrocytes was higher in the Asthma AE group and, to a lesser extent, in patients with COA. The expression of CCL19 in bronchial tissues and CCR7 in fibrocytes was higher in patients with COA. CXCL12/CXCR4 and CCL19/CCR7 enhanced fibrocyte transmigration in the Asthma AE group and in patients with COA, respectively. The upregulated expression of S100A9 and RAGE in fibrocytes of patients in the Asthma AE group and those with COA contributes to the enhanced basal migratory motility of fibrocytes. CONCLUSION: The CXCR4/CXCL12 axis contributes to chemotaxis of fibrocytes in patients in the Asthma AE group, whereas the CCR7/CCL19 axis plays an important role in patients with COA. S100A9 enhances the basal migratory motility of fibrocytes from patients in the Asthma AE group and patients with COA.
Asunto(s)
Asma/etiología , Asma/metabolismo , Movimiento Celular , Asma/patología , Asma/fisiopatología , Calgranulina B/metabolismo , Estudios de Casos y Controles , Movimiento Celular/genética , Quimiocina CCL19/metabolismo , Quimiocina CXCL12/metabolismo , Enfermedad Crónica , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Receptores CCR7/metabolismo , Receptores CXCR4/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
This study reports a biomimetic microsystem that reconstitutes the lung microenvironment for monitoring the role of eosinophil cationic protein (ECP) in lung inflammation. ECP induces the airway epithelial cell expression of CXCL-12, which in turn stimulates the migration of fibrocytes towards the epithelium. This two-layered microfluidic system provides a feasible platform for perfusion culture, and was used in this study to reveal that the CXCL12-CXCR4 axis mediates ECP induced fibrocyte extravasation in lung inflammation. This 'lung-on-a-chip' microdevice serves as a dynamic transwell system by introducing a flow that can reconstitute the blood vessel-tissue interface for in vitro assays, enhancing pre-clinical studies. We made an attempt to develop a new microfluidic model which could not only simulate the transwell for studying cell migration, but could also study the migration in the presence of a flow mimicking the physiological conditions in the body. As blood vessels are the integral part of our body, this model gives an opportunity to study more realistic in vitro models of organs where the blood vessel i.e. flow based migration is involved.
Asunto(s)
Dispositivos Laboratorio en un Chip , Pulmón/patología , Pulmón/fisiopatología , Neumonía/etiología , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Animales , Materiales Biomiméticos , Línea Celular , Movimiento Celular , Microambiente Celular/fisiología , Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiología , Técnicas de Cocultivo , Proteína Catiónica del Eosinófilo/fisiología , Diseño de Equipo , Humanos , Pulmón/irrigación sanguínea , Modelos Biológicos , Neumonía/patología , Neumonía/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/fisiologíaRESUMEN
Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Microfluídica/instrumentación , Esferoides Celulares/citología , Análisis de Matrices Tisulares/instrumentación , Animales , Adhesión Celular , Diseño de Equipo , Células Hep G2 , Humanos , Ratones , Células 3T3 NIH , Esferoides Celulares/metabolismoRESUMEN
A laser diffraction-induced dielectrophoresis (DEP) phenomenon for the patterning and manipulation of individual HepG2 cells and polystyrene beads via positive/negative DEP forces is reported in this paper. The optoelectronic substrate was fabricated using an organic photoconductive material, TiOPc, via a spin-coating process on an indium tin oxide glass surface. A piece of square aperture array grid grating was utilized to transform the collimating He-Ne laser beam into the multi-spot diffraction pattern which forms the virtual electrodes as the TiOPc-coating surface was illuminated by the multi-spot diffraction light pattern. HepG2 cells were trapped at the spot centers and polystyrene beads were trapped within the dim region of the illuminated image. The simulation results of light-induced electric field and a Fresnel diffraction image illustrated the distribution of trapped microparticles. The HepG2 morphology change, adhesion, and growth during a 5-day culture period demonstrated the cell viability through our manipulation. The power density inducing DEP phenomena, the characteristics of the thin TiOPc coating layer, the operating ac voltage/frequency, the sandwiched medium, the temperature rise due to the ac electric fields and the illuminating patterns are discussed in this paper. This concept of utilizing laser diffraction images to generate virtual electrodes on our TiOPc-based optoelectronic DEP chip extends the applications of optoelectronic dielectrophoretic manipulation.
Asunto(s)
Separación Celular/instrumentación , Equipos y Suministros Eléctricos , Electroforesis/métodos , Rayos Láser , Fenómenos Ópticos , Compuestos Orgánicos/química , Análisis de Matrices Tisulares/instrumentación , Impedancia Eléctrica , Vidrio/química , Células Hep G2 , Humanos , Microesferas , Poliestirenos/química , Compuestos de Estaño/químicaRESUMEN
A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.
Asunto(s)
Materiales Biomiméticos/química , Hígado/citología , Técnicas Analíticas Microfluídicas/instrumentación , Supervivencia Celular , Citocromo P-450 CYP1A1/metabolismo , Electrodos , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hígado/metabolismo , Medicina Regenerativa , Ingeniería de TejidosRESUMEN
The acinus-mimicking microfluidic chip, which simulates the in vivo condition of the liver, was developed and reported in this paper. The gradient microenvironment of the liver acinus is replicated within this proposed microfluidic chip. The advantage of this acinus-mimicking chip is capable of adjusting the concentration gradient in a relatively short period of time at around 10 s. At the same instance the non-linear concentration gradient can be presented in the various zones within this microfluidic chip. The other advantage of this proposed design is in the convenience of allowing the direct injection of the cells into the chip. The environment within the chip is multi-welled and gel-free with high cell density. The multi-row pillar microstructure located at the entrance of the top and bottom flow channels is designed to be able to balance the pressure of the perfusion medium. Through this mechanism the shear stress experienced by the cultured cells can be minimized to reduce the potential damage flow from the perfusion process. The fluorescence staining and the observations of the cell morphology verify the life and death of the cells. The shear stress experienced by the cells in the various zones within the chip can be effectively mapped. The serum glutamic oxaloacetic transaminase (SGOT) collected from the supernatants was used to determine the effects of the degassing process and the shear stress of the medium flow on the cultured cells.
Asunto(s)
Hígado/citología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Aspartato Aminotransferasas/sangre , Línea Celular , Dimetilpolisiloxanos/química , Diseño de Equipo , Humanos , Perfusión , Presión , Resistencia al Corte , Estrés Mecánico , Propiedades de SuperficieRESUMEN
The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wild-type. This study has demonstrated that the C-terminal ß strands of MrkA are required for the assembly and structural stability of fimbriae.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Fimbrias/metabolismo , Klebsiella pneumoniae/metabolismo , Proteínas Bacterianas/química , Biopelículas , Proteínas Fimbrias/química , Estructura Terciaria de ProteínaRESUMEN
This study investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium. Transmission electron microscopy and optical tweezers were used to understand the ability of the bacterium to survive flushes. An individual K. pneumoniae type 3 fimbria exhibited a helix-like structure with a pitch of 4.1 nm and a three-phase force-extension curve. The fimbria was first nonlinearly stretched with increasing force. Then, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN (n = 22). Finally, the extension of the fimbria shifted to the third phase, with a characteristic force of 102 ± 9 pN (n = 14) at the inflection point. Compared with the P fimbriae and type 1 fimbriae of uropathogenic Escherichia coli, K. pneumoniae type 3 fimbriae have a larger pitch in the helix-like structure and stronger uncoiling and characteristic forces.
Asunto(s)
Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Mecánica , Microscopía Electrónica de Rastreo , Conformación Proteica , Escherichia coli Uropatógena/metabolismo , Factores de VirulenciaRESUMEN
Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.