Academic literature on the topic 'Asymmetric Cell Focusing'

Inertial Microfluidics offer a high throughput, label-free, easy to design, and cost-effective solutions, and are a promising technique based on hydrodynamic forces (passive techniques) instead of external ones, which can be employed in the lab-on-a-chip and micro-total-analysis-systems for the focusing, manipulation, and separation of microparticles in chemical and biomedical applications. The current study focuses on the focusing behavior of the microparticles in an asymmetric curvilinear microchannel with curvature angle of 280°. For this purpose, the focusing behavior of the microparticles with three different diameters, representing cells with different sizes in the microchannel, was experimentally studied at flow rates from 400 to 2700 µL/min. In this regard, the width and position of the focusing band are carefully recorded for all of the particles in all of the flow rates. Moreover, the distance between the binary combinations of the microparticles is reported for each flow rate, along with the Reynolds number corresponding to the largest distances. Furthermore, the results of this study are compared with those of the microchannel with the same curvature angle but having a symmetric geometry. The microchannel proposed in this study can be used or further modified for cell separation applications.

Metasurfaces exhibiting spatially asymmetric inner structures have been shown to host unidirectional scattering effects, benefiting areas where directional control of waves is desired. In this work, we propose a non-Hermitian planar elastic metasurface to achieve unidirectional focusing of flexural waves. The unit cells are constructed by piezoelectric disks and metallic blocks that are asymmetrically loaded. A tunable material loss is then introduced by negative capacitance shunting. By suitably engineering the induced loss profile, a series of unit cells are designed, which can individually access the exceptional points manifested by unidirectional zero reflection. We then construct a planar metasurface by tuning the reflected phase to ensure constructive interference at one side of the metasurface. Unidirectional focusing of the incident waves is demonstrated, where the reflected wave energy is focused from one direction, and zero reflection is observed in the other direction. The proposed metasurface enriches the flexibility in asymmetric elastic wave manipulation as the loss and the reflected phase can be tailored independently in each unit cell.

In the microcirculation, a plasma layer forms near the vessel walls that is free of red blood cells (RBCs). This region, often termed as the cell-free layer (CFL), plays important haemorheological and biophysical roles, and has been the subject of extensive research. Many previous studies have considered the CFL development in single, isolated vessels that are straight tubes or channels, as well as in isolated bifurcations and mergers. In the body, blood vessels are typically winding and sequentially bifurcate into smaller vessels or merge to form larger vessels. Because of this geometric complexity, the CFL in vivo is three-dimensional (3D) and asymmetric, unlike in fully developed flow in straight tubes. The three-dimensionality of the CFL as it develops in a vascular network, and the underlying hydrodynamic mechanisms, are not well understood. Using a high-fidelity model of cellular-scale blood flow in microvascular networks with in vivo-like topologies, we present a detailed analysis of the fully 3D and asymmetric nature of the CFL in such networks. We show that the CFL significantly varies over different aspects of the networks. Along the vessel lengths, such variations are predominantly non-monotonic, which indicates that the CFL profiles do not simply become more symmetric over the length as they would in straight vessels. We show that vessel tortuosity causes the CFL to become more asymmetric along the length. We specifically identify a curvature-induced migration of the RBCs as the underlying mechanism of increased asymmetry in curved vessels. The vascular bifurcations and mergers are also seen to change the CFL profiles, and in the majority of them the CFL becomes more asymmetric. For most bifurcations, this is generally observed to occur such that the CFL downstream narrows on the side of the vessel nearest the upstream bifurcation, and widens on the other side. The 3D aspects of such behaviour are elucidated. For many bifurcations, a discrepancy exists between the CFL in the daughter vessels, which arises from a disproportionate partitioning between the flow rate and RBC flux. For most mergers, the downstream CFL narrows in the plane of the merger, but widens away from this plane. The dominant mechanism by which such changes occur is identified as the geometric focusing of the two merging streams. To our knowledge, this work provides the first simulation-based analysis of the 3D CFL structure in complex in vivo-like microvascular networks, including the hydrodynamic origins of the observed behaviour.

Focusing particles into a tight stream is critical for many microfluidic particle-handling devices such as flow cytometers and particle sorters. This work presents a fundamental study of the passive focusing of polystyrene particles in ratchet microchannels via direct current dielectrophoresis (DC DEP). We demonstrate using both experiments and simulation that particles achieve better focusing in a symmetric ratchet microchannel than in an asymmetric one, regardless of the particle movement direction in the latter. The particle focusing ratio, which is defined as the microchannel width over the particle stream width, is found to increase with an increase in particle size or electric field in the symmetric ratchet microchannel. Moreover, it exhibits an almost linear correlation with the number of ratchets, which can be explained by a theoretical formula that is obtained from a scaling analysis. In addition, we have demonstrated a DC dielectrophoretic focusing of yeast cells in the symmetric ratchet microchannel with minimal impact on the cell viability.

Many experimental findings on heterogeneity, flexibility, and plasticity of tissue stem cells are currently challenging stem cell concepts that assume a cell intrinsically predefined, unidirectional differentiation program. In contrast to these classical concepts, nonhierarchical self-organizing systems provide an elegant and comprehensive alternative to explain the experimental data. Here we present the application of such a self-organizing concept to quantitatively describe the hematopoietic stem cell system. Focusing on the analysis of individual-stem-cell fates and clonal dynamics, we particularly discuss implications of the theoretical results on the interpretation of experimental findings. We demonstrate that it is possible to understand hematopoietic stem cell organization without assumptions on unidirectional developmental hierarchies, preprogrammed asymmetric division events or other assumptions implying the existence of a predetermined stem cell entity. The proposed perspective, therefore, changes the general paradigm of thinking about stem cells.

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis have been gleaned by assessing the rate of phenotypic assortment. Though powerful, this experimental approach relies on the availability of phenotypic markers. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dualism and a highly polyploid somatic nucleus, we probe the limits of single-cell whole-genome sequencing to study the consequences of amitosis. To this end, we first evaluate the suitability of single-cell sequencing to study the AT-rich genome of P. tetraurelia, focusing on common sources of genome representation bias. We then asked: can alternative rearrangements of a given locus eventually assort after a number of amitotic divisions? To address this question, we track somatic assortment of developmentally acquired Internal Eliminated Sequences (IESs) up to 50 amitotic divisions post self-fertilization. To further strengthen our observations, we contrast empirical estimates of IES retention levels with in silico predictions obtained through mathematical modeling. In agreement with theoretical expectations, our empirical findings are consistent with a mild increase in variation of IES retention levels across successive amitotic divisions of the macronucleus. The modest levels of somatic assortment in P. tetraurelia suggest that IESs retention levels are largely sculpted at the time of macronuclear development, and remain fairly stable during vegetative growth. In forgoing the requirement for phenotypic assortment, our approach can be applied to a wide variety of amitotic species and could facilitate the identification of environmental and genetic factors affecting amitosis.

Some representative models of radiation-induced cell death, which is a crucial endpoint in radiobiology, were reviewed. The basic assumptions were identified, their consequences on predicted cell survival were analyzed, and the advantages and drawbacks of each approach were outlined. In addition to “historical” approaches such as the Target Theory, the Linear-Quadratic model, the Theory of Dual Radiation Action and Katz' model, the more recent Local Effect Model was discussed, focusing on its application in Carbon-ion hadrontherapy. Furthermore, a mechanistic model developed at the University of Pavia and based on the relationship between cell inactivation and chromosome aberrations was presented, together with recent results; the good agreement between model predictions and literature experimental data on different radiation types (photons, protons, alpha particles, and Carbon ions) supported the idea that asymmetric chromosome aberrations like dicentrics and rings play a fundamental role for cell death. Basing on these results, a reinterpretation of the TDRA was also proposed, identifying the TDRA “sublesions” and “lesions” as clustered DNA double-strand breaks and (lethal) chromosome aberrations, respectively.

Abstract Background. During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. Enucleation has been thought to be a process similar to cytokinesis. However, more concrete evidence has been difficult to obtain because of a lack of an ex vivo experimental system capable of confirming cytokinesis. Focusing on the mechanism of cell division, we investigated the redistribution of cytoplasmic proteins and integral membrane proteins during enucleation, using ex vivo generation system of mature human blood cells from hematopoietic stem cells. Materials and Methods. The highly purified human CD34+ cells were grown in the presence of interleukin-3, stem cell factor and erythropoietin (EPO) in a liquid phase. After 7 days of culture, the generated cells (day 7 cells) were replaced in a medium with EPO alone. The cells matured and terminally differentiated into reticulocytes during a 13–15-day culture period. We mainly used non-gravity and non-pipetting system to avoid physical stress that may disrupt the connection between the nucleus and reticulocyte. Day 9 cells, predominantly consisted of polychromatophilic erythroblasts and expressed glycophorin A (GPA) at a purity of 97%, were labeled with DNA-staining dye SYTO21 for the direct monitoring of the enucleation process, using differential interference contrast microscopy. We also cultured day 9 cells until day 14, on 4-well culture slides or on the membrane of a cell culture insert system, and removed culture medium by aspiration without centrifugation and pipetting. The day 14 cells on the slide were analyzed using immunohistochemical staining, whereas the cells on the membrane were embedded in O.C.T. compound for confocal microscopy. Results. Approximately a half of erythroblasts enucleated until day 14. The monitoring of the enucleation process at day 13 showed autonomous extrusion of SYTO21 positive nucleus from single erythroblast. Some of the expelled nuclei were still connected with reticulocyte through strings that were positive for antibody against tubulin, actin, GPA, band 3 and glycophorin C (GPC). The expelled nuclei were covered by lamin, a protein specific for nuclear membrane, which were surrounded by a substance positive for GPA, band 3, GPC, p55, 4.1R80, actin, tubulin, b-spectrin, calnexin and cytochrome C, although the distribution of each proteins were asymmetric between nuclei and reticulocytes. An intense area of GPA, GPC, band 3, 4.1R80, actin, tubulin, myosin and b-spectrin was found in the region of the constriction between the extruding nucleus and incipient reticulocyte in enucleating cells. In cells just before enucleation, tubulin and actin formed a radial array around the nucleus. The center of the radial array was positive for centrin and NuMA, indicating that the cenriole formation occurred during an enucleation process. Conclusion. Our investigations show that a part of human erythroblasts enucleate independent of an interaction with accessory cells. The appearance of cenriole and the asymmetric redistribution of cytoplasmic and integral membrane proteins during enucleation strongly suggest that enucleation of human erythroblasts is a process of asymmetric cytokinesis.

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta-mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39-kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39-kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.

The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7–17 keV (1.8–0.7 Å) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. High-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline.

Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency.
Master of Science

The development of Lab-on-a-Chip devices with integrated bio-analysis functions requires a complex network of microfluidic transport and processes. Many of these functions call for the isolation or separation of specific bio-particles or cells. The design of a miniaturized cell-sorting device for handheld operation must follow the strict parameters associated with Lab-on-a-Chip technology. The limitations include applied voltage, high efficiency of cell-separation, repeatability, size, flow control, and cost, among others. Currently used designs have achieved successful levels of cell-isolation. However, further improvements in the microfluidic chip design are important for incorporation into larger systems. This study evaluates specific design modifications that contribute to the reduction of required applied potential aiming for developing portable devices, improved operation reliability by minimizing induced pressure disturbance when electrokinetic pumping is employed and incorporating online filters to reduce channel blockage, and improved flow control by incorporating directing streams achieving dynamic sorting and counting. The chip designs fabricated in glass and polymeric materials include asymmetric channel widths for sample focusing, nonuniform channel depth for minimizing induced pressure disturbance, directing streams to assist particle flow control, and online filters for reducing channel blockage. Fluorescence-based visualization of electrokinetic focusing, flow field phenomena, and dynamic cell-sorting demonstrate the advantages of the chip design. Numerical simulations in COMSOL are validated by the experimental data and used to investigate the effects of channel geometry and fluid properties on the flow field.