Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well ...characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell–matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.
Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is ...still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization ...and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.
A major technical hurdle in microfluidics is the difficulty in achieving high fidelity lithographic patterning on polydimethylsiloxane (PDMS). Here, we report a simple yet highly precise and ...repeatable PDMS surface micromachining method using direct photolithography followed by reactive ion etching (RIE). Our method to achieve surface patterning of PDMS applied an O(2) plasma treatment to PDMS to activate its surface to overcome the challenge of poor photoresist adhesion on PDMS for photolithography. Our photolithographic PDMS surface micromachining technique is compatible with conventional soft lithography techniques and other silicon-based surface and bulk micromachining methods. To illustrate the general application of our method, we demonstrated fabrication of large microfiltration membranes and free-standing beam structures in PDMS.
Microcontact printing (μCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and tissue engineering. However, different types of μCPs may create ...micropatterns with varied protein–substrate adhesion, which may change cell behaviors and pose uncertainty in result interpretation. Here, we characterize two μCP methods for coating extracellular matrix (ECM) proteins (stamp-off and covalent bond) and demonstrate for the first time the important role of protein–substrate adhesion in determining cell behavior. We found that, as compared to cells with weaker traction force (e.g., endothelial cells), cells with strong traction force (e.g., vascular smooth muscle cells) may delaminate the ECM patterns, which reduced cell viability as a result. Importantly, such ECM delamination was observed on patterns by stamp-off but not on the patterns by covalent bonds. Further comparisons of the displacement of the ECM patterns between the normal VSMCs and the force-reduced VSMCs suggested that the cell traction force plays an essential role in this ECM delamination. Together, our results indicated that μCPs with insufficient adhesion may lead to ECM delamination and cause cell death, providing new insight for micropatterning in cell–biomaterial interaction on biointerfaces.
The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor ...(4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3 days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15 μM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15 μM) and in vitro in mammalian Neuro-2A cells (0.1 μM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects.
There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important ...contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma.
Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus.
Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings.
Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.
In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential ...oxygenator. A gas-filled microchannel network functioning as an oxygen−nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated).
Prefocusing of the cell mixture is necessary for achieving a high-efficiency and continuous dielectrophoretic (DEP) cell separation. However, prefocusing through sheath flow requires a complex and ...tedious peripheral system for multi-channel fluid control, hindering the integration of DEP separation systems with other microfluidic functionalities for comprehensive clinical and biological tasks. This paper presented a simplified sheathless cell separation approach that combines gravitational-sedimentation-based sheathless prefocusing and DEP separation methods. Through gravitational sedimentation in a tubing, which was inserted into the inlet of a microfluidic chip with an adjustable steering angle, the cells were focused into a stream at the upstream region of a microchannel prior to separation. Then, a DEP force was applied at the downstream region of the microchannel for the active separation of the cells. Through this combined strategy, the peripheral system for the sheath flow was no longer required, and thus the integration of cell separation system with additional microfluidic functionalities was facilitated. The proposed sheathless scheme focused the mixture of cells with different sizes and dielectric properties into a stream in a wide range of flow rates without changing the design of the microfluidic chip. The DEP method is a label-free approach that can continuously separate cells on the basis of the sizes or dielectric properties of the cells and thus capable of greatly flexible cell separation. The efficiency of the proposed approach was experimentally assessed according to its performance in the separation of human acute monocytic leukemia THP-1 cells from yeast cells with respect to different sizes and THP-1 cells from human acute myelomonocytic leukemia OCI-AML3 cells with respect to different dielectric properties. The experimental results revealed that the separation efficiency of the method can surpass 90% and thus effective in separating cells on the basis of either size or dielectric property.
Interfacial bonding integrity between different materials is critical to maintain the functionality of the entire physical system in any scale, ranging from building structures down to semiconductor ...transistors. For example, micro-patterned polymers embedded with conductive nanoparticles e.g., carbon nanotubes (CNTs) bonded with integrated circuits have been applied as many emerging chemical/biological microelectronic sensors. Nonetheless, it is challenging to measure and ensure the interfacial bonding integrity between materials for consistent and sustainable operations. Herein, we apply multiple interface characterization methods based on micro-engineering and microscopy as an integrative approach to reveal the mechanism of interfacial reinforcement by adding CNTs in a matrix material. An epoxy/CNT micro-beam is fabricated onto a silicon substrate, sandwiching a gold layer as an interfacial precrack. Superlayers of chromium are then repeatedly deposited onto the microstructure, inducing stepwise increasing stress over the materials and the corresponding micro-beam bending after detachment from the bonded interface. Accordingly, we can quantify key interfacial fracture parameters such as crack length, steady-state energy release rate, and fracture toughness. By further examining the formation and distribution of the micro-/nanostructures along the debonded interface using bright-field microscopy, 3D fluorescence imaging, and scanning electron microscopy, we can identify the underlying dominant interfacial strengthening and fracture toughening mechanisms. We further compare experimental results and theoretical predictions to quantify the interfacial bonding properties between epoxy/CNT and silicon and unveil the underlying reinforcement mechanisms. The results provide insights to develop polymer/nanoparticle composites with reinforced interfacial bonding integrity for more sustainable and reliable applications including microelectronics, surface coatings, and adhesive materials.
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