RESUMO
Smart microfluidic pipette tip: A microfluidic method for removing cells and recovering liquid medium is presented and applied for blood cell rejection and cytotoxicity assay. This method enables continuous cell rejection by manual operation, potentially providing the means for rapid, inexpensive sample preparation for personalized diagnostics and mobile laboratory.
Assuntos
Microfluídica/instrumentação , Eritrócitos , HumanosRESUMO
Cell focusing into a narrow stream is an essential step prior to counting and sorting cells in microfluidic devices for flow cytometry and cell sorting applications. Hydrodynamic focusing techniques, however, rely on the need for large volumes of sheath liquid and complex mechanical setup for flow control, preventing miniaturization of the systems. Although microfluidic methods based on active or passive particle control offer sheathless and efficient focusing, they often accompany fabrication complexities or bulky external setups, and operate in a certain range of flow rates. We present here a microfluidic device to focus cells into a narrow stream. The device employs hydrophoresis to guide cells by locally patterned slanted grooves, and channel expansion to improve focusing efficiency and produce a narrow stream of cells. This device principle allows easy improvement of focusing efficiency by adding more expansion steps. Adjusting channel expansion also ensures successful cell focusing without defocusing by inertial effects even at high Reynolds numbers. Using this device, we successfully produced a narrow stream of cells having size variation of >11% in a coefficient of variation (CV), achieving a narrow cell stream with a focusing variation below CV of 3.0%.
Assuntos
Rastreamento de Células/instrumentação , Citometria de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Separação Celular/métodos , Rastreamento de Células/métodos , Citometria de Fluxo/métodos , Humanos , Técnicas Analíticas Microfluídicas/métodosRESUMO
Cell cycle studies for examining regulatory mechanisms and progression invariably require synchronization of cell cultures at a specific phase of the cell cycle. Current implementations to produce synchronous cell populations, however, tend to perturb normal cellular progression and metabolism and typically require complex, time-consuming preparations. Thus, it is challenging for the development of a simple, noninvasive, and effective means for cell cycle synchronization. We demonstrate the use of hydrophoretic size separation to sort cells in target phases of the cell cycle entirely based on a hydrodynamic principle. With this method, we found that there is a linear relationship between a cell's size and its position distribution in the hydrophoretic device. We also demonstrate the robustness of the hydrophoretic method for practical applications by sorting cells in the G(0)/G(1) and G(2)/M phases out of the original, asynchronous cells with a high level of synchrony of 95.5% and 85.2%, respectively. These results show that the hydrophoretic size separation can be used in order to collect cells at the same phase of the cell cycle in a gentle, noninvasive way.
Assuntos
Ciclo Celular/fisiologia , Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Animais , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Células HeLa , Humanos , Células VeroRESUMO
We describe a hydrophoretic device that uses rotational flows induced by regularly patterned obstacles only on the top wall for preparing samples of biological particles, including micrometer and submicrometer particles, and DNA molecules. Many of the current continuous separation devices based on physical fields are limited to the separation of cells and micrometer-sized particles due to their dependence on a particle volume, and the purely hydrodynamic separation of macromolecules such as DNA or protein complexes remains a challenge. Hydrophoresis is entirely based on hydrodynamics using rotational flows induced by anisotropic obstacles. Different sizes of micrometer and submicrometer beads, as well as DNA molecules, were separated into distinct trajectories using two kinds of hindrance mechanisms. Continuous separation of these particles was achieved using the obstacles, demonstrating the potential of hydrophoresis for biological sample preparation on the micro- and nanoscales, with the advantages of continuous flow and sheathless passive operation.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Anisotropia , Bacteriófago lambda/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Dimetilpolisiloxanos/química , Micrococcus/genética , Técnicas Analíticas Microfluídicas/instrumentação , Microtecnologia/métodos , Tamanho da Partícula , Poliestirenos/isolamento & purificaçãoRESUMO
This paper presents a microfluidic device for sheathless focusing of microbeads and blood cells based on a hydrophoretic platform comprising a V-shaped obstacle array (VOA). The VOA generates lateral pressure gradients that induce helical recirculations. Following the focusing flow particles passing through the VOA are focused in the center of the channel. In the device, the focusing pattern can be modulated by varying the gap height of the VOA. To achieve complete focusing within 4.4% coefficient of variation, the relative size differences between the gap and the particle were 3 and 4 microm for 10 and 15 microm beads, respectively. Red blood cells were used to study the hydrophoretic focusing pattern of biconcave, disk-shaped particles.
Assuntos
Separação Celular/métodos , Eritrócitos/citologia , Eritrócitos/fisiologia , Citometria de Fluxo/métodos , Microfluídica/métodos , Nanotecnologia/métodos , Células Cultivadas , Humanos , Microesferas , PressãoRESUMO
In this study, we investigated the anti-inflammatory effect of various peroxisome proliferator activated receptor gamma (PPARgamma) agonists (15-deoxy-Delta12,14-prostaglandin J(2), troglitazone, rosiglitazone, ciglitazone) on human aortic endothelial cells. Pretreatment with PPARgamma agonists abrogated tumor necrosis factor alpha (TNFalpha)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and subsequent monocytic adhesion by endothelial cells. Because reactive oxygen species (ROS) have been reported to play important roles in pro-inflammatory signal transduction, the involvement of ROS was investigated as a potential mechanism of anti-inflammatory effect of PPARgamma ligands. Consistent with previous reports in other cell types, blockade of TNFalpha-induced ROS by treatment with N-acetylcysteine, diphenylene iodonium or NADPH oxidase 4 (NOX4) siRNA suppressed TNFalpha-induced ICAM-1 expression and subsequent monocytic adhesion, indicating that TNFalpha mediates pro-inflammatory signals via NOX4-dependent ROS generation in human endothelial cells. Finally, pretreatment with PPARgamma agonists significantly suppressed TNFalpha-induced increases of intracellular ROS. Our results collectively suggest that PPARgamma agonists might exert an anti-inflammatory effect on endothelial cells in a ROS-dependent manner.
Assuntos
Anti-Inflamatórios/farmacologia , Endotélio Vascular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , PPAR gama/agonistas , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Células U937RESUMO
Among tumor necrosis factor (TNF) superfamily, TNF-related apoptosis inducing ligand (TRAIL) along with TNF-alpha and FasL is known as death ligand due to its selective cytotoxicity against transformed tumor cells. TRAIL can also induce alternative angiogenic and/or proinflammatory signals other than apoptosis, however, the molecular mechanisms responsible for the alternative signals have not been detailed yet. Intercellular adhesion molecule-1 (ICAM-1) is thought to be involved in the processes of metastasis and angiogenesis in various tumors. We investigated the molecular mechanisms responsible for ICAM-1 expression by death ligands in human astroglial cells to delineate the alternative signals of these ligands. Here, we demonstrate that (1) death ligands induced expression of ICAM-1 at the mRNA and protein levels in human astroglial cells; (2) pre-treatment of z-VAD-fmk and/or SB202190 suppressed death ligand-induced ICAM-1 expression and subsequent adhesion of activated monocytic cells; and (3) inhibition of caspase suppressed death ligand-induced phosphorylation of p38 MAPK and IKK. These findings suggest biological function of death receptors other than apoptosis in human astroglial cells, and the involvement of caspase and/or p38 MAPK in alternative signaling through death receptors.
Assuntos
Astrócitos/metabolismo , Caspases/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Astrócitos/citologia , Astrócitos/imunologia , Inibidores de Caspase , Caspases/imunologia , Adesão Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Piridinas/farmacologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transfecção , Células U937 , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Vascular smooth muscle cell (VSMC) proliferation and migration in response to platelet-derived growth factor (PDGF) play an important role in the development of atherosclerosis and restenosis. Recent evidence indicates that PDGF increases intracellular levels of reactive oxygen species in VSMCs and that both PDGF-induced VSMC proliferation and migration are reactive oxygen species-dependent. Danshen is a representative oriental medicine used for the treatment of vascular disease. Previously, we reported that magnesium lithospermate B, an active component of Danshen, is a potent antioxidant. Thus we investigated the therapeutic potential of magnesium lithospermate B in neointimal formation after carotid artery injury in rats along with its effects on the PDGF signaling pathway for stimulating VSMC proliferation and migration in vitro. PDGF is dimeric glycoprotein composed of two A or two B chains. In this study, we used PDGF-BB, which is one of the isoforms of PDGF (i.e., PDGF-AA, PDGF-BB, and PDGF-AB). Our results demonstrated that magnesium lithospermate B directly scavenged reactive oxygen species in a xanthine/xanthine oxidase system and reduced PDGF-BB-induced intracellular reactive oxygen species generation in VSMCs. In a rat carotid artery balloon injury model, magnesium lithospermate B treatment (10 mg/kg/day, i.p) showed a significant effect on the prevention of neointimal formation compared with vehicle treatment. In cultured VSMCs, magnesium lithospermate B significantly attenuated PDGF-BB-induced cell proliferation and migration as measured by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and transwell migration assays, respectively. Further, magnesium lithospermate B inhibited PDGF-BB-induced phosphorylation of phospatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways by scavenging reactive oxygen species. Together, these data indicated that magnesium lithospermate B, a potent reactive oxygen species scavenger, prevented both injury-induced neointimal formation in vivo and PDGF-BB-induced VSMC proliferation and migration in vitro, suggesting that magnesium lithospermate B may be a promising agent to prevent atherosclerosis and restenosis following angioplasty.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antioxidantes/uso terapêutico , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/patologia , Medicamentos de Ervas Chinesas/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Western Blotting , Cateterismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Medicamentos de Ervas Chinesas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Raízes de Plantas/química , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacosRESUMO
We propose a new hydrophoretic method for continuous blood cell separation using a microfluidic device composed of slanted obstacles and filtration obstacles. The slanted obstacles have a larger height and gap than the particles in order to focus them to a sidewall by hydrophoresis. In the successive structure, the height and gap of the filtration obstacles with a filtration pore are set between the diameters of small and large particles, which defines the critical separation diameter. Accordingly, the particles smaller than the criterion freely pass through the gap and keep their focused position. In contrast, the particles larger than the criterion collide against the filtration obstacle and move into the filtration pore. The microfluidic device was characterized with polystyrene beads with a minimum diameter difference of 7.3%. We completely separated polystyrene microbeads of 9 and 12 microm diameter with a separation resolution of approximately 6.2. This resolution is increased by 6.4-fold compared with our previous separation method based on hydrophoresis (S. Choi and J.-K. Park, Lab Chip, 2007, 7, 890, ref. 1). In the isolation of white blood cells (WBCs) from red blood cells (RBCs), the microfluidic device isolated WBCs with 210-fold enrichment within a short filtration time of approximately 0.3 s. These results show that the device can be useful for the binary separation of a wide range of biological particles by size. The hydrophoretic filtration as a sample preparation unit offers potential for a power-free cell sorter to be integrated into disposable lab-on-a-chip devices.
Assuntos
Separação Celular/métodos , Filtração/métodos , Leucócitos/citologiaRESUMO
This communication presents a microfluidic method for size-based cell sorting, which provides a simple and robust approach for cell cycle synchronization by manual and stand-alone operation.
Assuntos
Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Separação Celular/instrumentação , Fase G1 , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas/instrumentação , SeringasRESUMO
Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing differentiation of VSMCs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micropatterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micropatterned groove substrate to modulate the morphology and function of VSMCs. Later than 7(th) passage of VSMCs showed typical synthetic phenotype characterized by epithelial morphology, increased proliferation rates and corresponding gene expression profiles; while short-term culture of these cells on a micropatterned groove induced a change to an intermediate phenotype characterized by low proliferation rates, increased migration, a spindle-like morphology associated with cytoskeletal rearrangement and expression of muscle-specific genes. Microarray analysis showed preferential expression of contractile and smooth muscle cell-specific genes in cells cultured on the micropatterned groove. Culture on a patterned groove may provide a valuable model for the study the role of VSMCs in normal vascular physiology and a variety of proliferative vascular diseases.
Assuntos
Células Cultivadas/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Masculino , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Transcriptoma/genéticaRESUMO
We describe the effects of geometric and operational parameters on the performances of hydrophoresis devices for optimal size-based cell sorting and sheathless cell focusing. Hydrophoresis has been recently demonstrated to precisely control cells in a continuous flow with advantages of sheathless, high resolution, and easy parallelization. To date, key parameters for optimal design and operation of hydrophoresis systems have yet to be fully studied. In this study we have investigated geometric parameters such as channel width and oblique angle of slanted grooves, and an operational parameter, flow rate that can potentially influence the device performances. The channel width is found to be the most significant geometric factor that affects the device performances, while the oblique angle of slanted grooves has no significant influence. Size-based separation of cells having size diversity (≈11% in a coefficient of variation (CV)), as well as sheathless cell focusing, was performed with optimal designs, demonstrating the potential use of hydrophoresis as a microfluidic component to precisely control cells for integrated cell sorting and analysis systems.
Assuntos
Separação Celular/métodos , Linhagem Celular , Humanos , Microfluídica/métodosRESUMO
A gentle, but fast means for low-stress, high-throughput platelet purification is of significant clinical and biotechnological utility. Current implementations to sort platelets, however, require an external physical field, specialized buffer, or the harsh separation condition of high shear stress that tends to cause platelet stimulation. Here we report the use of hydrophoretic size separation in a wider channel and its parallelization to augment its throughput capability, maintaining physiological shear-stress range. We demonstrate a parallelized device comprising 10 stacks of the wide-channel hydrophoresis device, yielding a throughput of 2.9 million cells s(-1) and a platelet purity of 76.8%. The use of the wide channel for hydrophoresis also facilitates clogging-free separation by sorting blood clots and plaques. The wide-channel hydrophoresis offers the potential for gentle, fast, clogging-free sorting of rare blood cells with extreme throughput capabilities.
Assuntos
Plaquetas/química , Ensaios de Triagem em Larga Escala/métodos , Ativação Plaquetária , Animais , Biotecnologia , Separação Celular , Micropartículas Derivadas de Células , Citometria de Fluxo , Ratos , Ratos Sprague-Dawley , Resistência ao Cisalhamento , Estresse MecânicoRESUMO
AIMS: A reduction in the level of thioredoxin 1 (Trx1) has been proposed as a possible mechanism for the tumor-specific growth arrest caused by inhibition of histone deacetylases (HDACs). In this study, we investigated the effect of trichostatin A (TSA), a potent HDAC inhibitor, on the proliferation and migration of vascular smooth muscle cells (VSMCs), and we examined the role of reduced Trx1 levels in this effect. METHODS AND RESULTS: TSA treatment time-dependently decreased Trx1 expression in rat VSMCs at both the mRNA and protein levels. It also enhanced platelet-derived growth factor (PDGF)-induced proliferation and migration of the VSMCs. By potentiating Akt phosphorylation, the siRNA-induced downregulation of Trx1 also enhanced VSMC proliferation and migration in response to PDGF or serum treatment. Consistent with these results, TSA administration increased neointimal thickening in a murine model of post-angioplastic restenosis. CONCLUSION: These data demonstrate that TSA enhances vascular proliferative activity by downregulating Trx1, thus activating an Akt-dependent pathway. Our results indicate that, in addition to its apoptotic effects in tumour cells, the downregulation of Trx1 has a proliferative role in primary VSMCs.