Disrupting the wall accumulation of human sperm cells by artificial corrugation

Many self-propelled microorganisms are attracted to surfaces. This makes their dynamics in restricted geometries very different from that observed in the bulk. Swimming along walls is beneficial for directing and sorting cells, but may be detrimental if homogeneous populations are desired, such as in counting microchambers. In this work, we characterize the motion of human sperm cells ∼60 μm long, strongly confined to ∼25 μm shallow chambers. We investigate the nature of the cell trajectories between the confining surfaces and their accumulation near the borders. Observed cell trajectories are composed of a succession of quasi-circular and quasi-linear segments. This suggests that the cells follow a path of intermittent trappings near the top and bottom surfaces separated by stretches of quasi-free motion in between the two surfaces, as confirmed by depth resolved confocal microscopy studies. We show that the introduction of artificial petal-shaped corrugation in the lateral boundaries removes the tendency of cells to accumulate near the borders, an effect which we hypothesize may be valuable for microfluidic applications in biomedicine.     

Hybrid capillary-inserted microfluidic device for sheathless particle focusing and separation in viscoelastic flow

A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.     

Measurement of cell compressibility changes during epithelial–mesenchymal transition based on acoustofluidic microdevice

Epithelial–mesenchymal transition (EMT) confers migratory and invasiveness abilities on cancer cells, as well as leading to changes in biomechanical properties and cytoskeletal structure. Cell mechanical properties are considered to be promising label-free markers for diagnosis of cancer metastasis. In this work, cell compressibility, a novel and important parameter of cell mechanical properties, was measured directly and quickly using a specially designed acoustofluidic microdevice. The compressibilities of cells with different metastatic potentials were investigated. Based on a comparison of the measurement results, non-metastatic cells exhibited lower compressibility than metastatic cells. The correlation between cell compressibility and EMT status was further studied; the results showed that the acquisition of mesenchymal status was accompanied by an increase in cell compressibility. These findings imply strong correlations among cell compressibility, EMT status, and invasiveness. Therefore, cell compressibility represents a novel biomechanical marker for evaluating malignant transformation and metastasis of cancer.     

An insulator-based dielectrophoretic microdevice for the simultaneous filtration and focusing of biological cells

Manipulating and discriminating biological cells of interest using microfluidic and micro total analysis system (μTAS) devices have potential applications in clinical diagnosis and medicine. Cellular focusing in microfluidic devices is a prerequisite for medical applications, such as cell sorting, cell counting, or flow cytometry. In the present study, an insulator-based dielectrophoretic microdevice is designed for the simultaneous filtration and focusing of biological cells. The cells are introduced into the microchannel and hydrodynamically pre-confined by funnel-shaped insulating structures close to the inlet. There are ten sets of X-patterned insulating structures in the microfluidic channel. The main function of the first five sets of insulating structures is to guide the cells by negative dielectrophoretic responses (viable HeLa cells) into the center region of the microchannel. The positive dielectrophoretic cells (dead HeLa cells) are attracted to regions with a high electric-field gradient generated at the edges of the insulating structures. The remaining five sets of insulating structures are mainly used to focus negative dielectrophoretic cells that have escaped from the upstream region. Experiments employing a mixture of dead and viable HeLa cells are conducted to demonstrate the effectiveness of the proposed design. The results indicate that the performance of both filtration and focusing improves with the increasing strength of the applied electric field and a decreasing inlet sample flow rate, which agrees with the trend predicted by the numerical simulations. The filtration efficiency, which is quantitatively investigated, is up to 88% at an applied voltage of 50 V peak-to-peak (1 kHz) and a sample flow rate of 0.5 μl/min. The proposed device can focus viable cells into a single file using a voltage of 35 V peak-to-peak (1 kHz) at a sample flow rate of 1.0 μl/min.     

Embryo formation from low sperm concentration by using dielectrophoretic force

A biochip system imitates the oviduct of mammals with a microfluidic channel to achieve fertilization in vitro of imprinting-control-region (ICR) mice. We apply a method to manipulate and to position the oocyte and the sperm of ICR mice at the same time in our microfluidic channel with a positive dielectrophoretic (DEP) force. The positive dielectrophoretic response of the oocyte and sperm was exhibited under applied bias conditions AC 10 V_pp waveform, 1 MHz, 10 min. With this method, the concentration of sperm in the vicinity of the oocyte was increased and enhanced the probability of natural fertilization. We used commercial numerical software (CFDRC-ACE+) to simulate the square of the electric field and analyzed the location at which the oocyte and sperm are trapped. The microfluidic devices were designed and fabricated with poly(dimethylsiloxane). The results of our experiments indicate that a positive DEP served to drive the position of the oocyte and the sperm to natural fertilization (average rate of fertilization 51.58%) in our microchannel structures at insemination concentration 1.5 × 10⁶ sperm ml⁻¹. Embryos were cultured to two cells after 24 h and four cells after 48 h.     

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

We developed a new method for releasing viable cells from affinity-based microfluidic devices. The lumen of a microchannel with a U-shape and user-designed microstructures was coated with supported lipid bilayers functionalized by epithelial cell adhesion molecule antibodies to capture circulating epithelial cells of influx solution. After the capturing process, air foam was introduced into channels for releasing target cells and then carrying them to a small area of membrane. The results show that when the air foam is driven at linear velocity of 4.2 mm/s for more than 20 min or at linear velocity of 8.4 mm/s for more than 10 min, the cell releasing efficiency approaches 100%. This flow-induced shear stress is much less than the physiological level (15 dyn/cm²), which is necessary to maintain the intactness of released cells. Combining the design of microstructures of the microfluidic system, the cell recovery on the membrane exceeds 90%. Importantly, we demonstrate that the cells released by air foam are viable and could be cultured in vitro. This novel method for releasing cells could power the microfluidic platform for isolating and identifying circulating tumor cells.     

Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.     

大学生性侵害防治理论基础及其实施路径

现代大学开放式管理模式的兴起,给了女大学生更多的交往空间。但受社会层面经验尚浅、有效防范性侵害理论基础知识薄弱等限制,往往首当其冲成为犯罪分子的危害对象。鉴于大学生性侵害包含暴力型侵害、胁迫型侵害、网恋型侵害、社交型侵害等类型,同时受社会、法律法规等层面问题的影响,事后保护和关爱不足,直接导致了二次伤害。因此,探寻大学生性侵害防治理论基础知识,并通过对有效实施手段加以研究,成为规避大学生性侵害事件发生的关键点,对有效防治大学生性侵害具有重要的推广价值。     

Characterization of a microflow cytometer with an integrated three-dimensional optofluidic lens system

Flow cytometry is a standard analytical method in cell biology and clinical diagnostics and is widely distributed for the experimental investigation of microparticle characteristics. In this work, the design, realization, and measurement results of a novel planar optofluidic flow cytometric device with an integrated three-dimensional (3D) adjustable optofluidic lens system for forward-scattering∕extinction-based biochemical analysis fabricated by silicon micromachining are presented. To our knowledge, this is the first planar cytometric system with the ability to focus light three-dimensionally on cells∕particles by the application of fluidic lenses. The single layer microfluidic platform enables versatile 3D hydrodynamic sample focusing to an arbitrary position in the channel and incorporates integrated fiber grooves for the insertion of glass fibers. To confirm the fluid dynamics and raytracing simulations and to characterize the sensor, different cell lines and sets of microparticles were investigated by detecting the extinction (axial light loss) signal, demonstrating the high sensitivity and sample discrimination capability of this analysis system. The unique features of this planar microdevice enable new biotechnological analysis techniques due to the highly increased sensitivity.     

旋花科植物雄性细胞的细胞质DNA存在状况及其系统学意义

用DAPI荧光染色方法检测了旋花科4属4种植物——空心菜Ipomoea aquatica Forsk. 、茑萝 Qua- moclit pennata (Desr．)Boj．、月光花 Calonyction aculeatum(L．)House和日本菟丝子Cuscuta japonica Choisy 中生殖细胞和精细胞中细胞质DNA存在的状况。证明除日本菟丝子外，其余3种植物的雄性细胞中都 存在细胞质DNA。此外，在我们曾研究过的5种旋花科种植物的生殖细胞和精细胞中亦显示含有细胞质 DNA，因而可认为这是旋花科较普遍的特性，日本菟丝子精细胞中缺少细胞质DNA，可作为—个新的胚胎学证据，支持此属从旋花科分出独立为菟丝子科。     

A microdevice for the creation of patent, three-dimensional endothelial cell-based microcirculatory networks

Microvascular network formation is a significant and challenging goal in the engineering of large three-dimensional artificial tissue structures. We show here the development of a fully patent, 3D endothelial cell (microvascular) microfluidic network that has a single inlet and outlet, created in only 28 h in a microdevice involving fluid flow equivalent to natural vasculature. Our microdevice features a tailored "multi-rung ladder" network, a stylized mimic of an arterial-to-venous pedicle, designed to also allow for systematic and reproducible cell seeding. Immunofluorescence staining revealed a highly contiguous endothelial monolayer (human umbilical vein endothelial cells) throughout the whole network after 24 h of continuous perfusion. This network persisted for up to 72 h of culture, providing a useful template from which the effects of surface chemistry, fluid flow, and environmental conditions on the development of artificial vascular networks ex vivo may be rapidly and robustly evaluated.     

Separation of tumor cells with dielectrophoresis-based microfluidic chip

The present work demonstrates the use of a dielectrophoretic lab-on-a-chip device in effectively separating different cancer cells of epithelial origin for application in circulating tumor cell (CTC) identification. This study uses dielectrophoresis (DEP) to distinguish and separate MCF-7 human breast cancer cells from HCT-116 colorectal cancer cells. The DEP responses for each cell type were measured against AC electrical frequency changes in solutions of varying conductivities. Increasing the conductivity of the suspension directly correlated with an increasing frequency value for the first cross-over (no DEP force) point in the DEP spectra. Differences in the cross-over frequency for each cell type were leveraged to determine a frequency at which the two types of cell could be separated through DEP forces. Under a particular medium conductivity, different types of cells could have different DEP behaviors in a very narrow AC frequency band, demonstrating a high specificity of DEP. Using a microfluidic DEP sorter with optically transparent electrodes, MCF-7 and HCT-116 cells were successfully separated from each other under a 3.2 MHz frequency in a 0.1X PBS solution. Further experiments were conducted to characterize the separation efficiency (enrichment factor) by changing experimental parameters (AC frequency, voltage, and flow rate). This work has shown the high specificity of the described DEP cell sorter for distinguishing cells with similar characteristics for potential diagnostic applications through CTC enrichment.     

The construction of an interfacial valve-based microfluidic chip for thermotaxis evaluation of human sperm

Thermotaxis has been demonstrated to be an important criterion for sperm evaluation, yet clinical assessment of thermotaxis capacity is currently lacking. In this article, the on-chip thermotaxis evaluation of human sperm is presented for the first time using an interfacial valve-facilitated microfluidic device. The temperature gradient was established and accurately controlled by an external temperature gradient control system. The temperature gradient responsive sperm population was enriched into one of the branch channels with higher temperature setting and the non-responsive ones were evenly distributed into the two branch channels. We employed air-liquid interfacial valves to ensure stable isolation of the two branches, facilitating convenient manipulation of the entrapped sperm. With this device, thermotactic responses were observed in 5.7%-10.6% of the motile sperm moving through four temperature ranges (34.0-35.3 °C, 35.0-36.3 °C, 36.0-37.3 °C, and 37.0-38.3 °C, respectively). In conclusion, we have developed a new method for high throughput clinical evaluation of sperm thermotaxis and this method may allow other researchers to derive better IVF procedure.     

Highly selective biomechanical separation of cancer cells from leukocytes using microfluidic ratchets and hydrodynamic concentrator

The separation of cells based on their biomechanical properties, such as size and deformability, is important in applications such as the identification of circulating tumor cells, where morphological differences can be used to distinguish target cancer cells from contaminant leukocytes. Existing filtration-based separation processes are limited in their selectivity and their ability to extract the separated cells because of clogging in the filter microstructures. We present a cell separation device consisting of a hydrodynamic concentrator and a microfluidic ratchet mechanism operating in tandem. The hydrodynamic concentrator removes the majority of the fluid and a fraction of leukocytes based on size, while the microfluidic ratchet mechanism separates cancer cells from leukocytes based on a combination of size and deformability. The irreversible ratcheting process enables highly selective separation and robust extraction of separated cells. Using cancer cells spiked into leukocyte suspensions, the complete system demonstrated a yield of 97%, while enriching the concentration of target cancer cells 3000 fold relative to the concentration of leukocytes.     

Membrane-integrated microfluidic device for high-resolution live cell imaging

The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane     

An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells

Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.     

On-chip actuation transmitter for enhancing the dynamic response of cell manipulation using a macro-scale pump

An on-chip actuation transmitter for achieving fast and accurate cell manipulation is proposed. Instead of manipulating cell position by a directly connected macro-scale pump, polydimethylsiloxane deformation is used as a medium to transmit the actuation generated from the pump to control the cell position. This actuation transmitter has three main advantages. First, the dynamic response of cell manipulation is faster than the conventional method with direct flow control based on both the theoretical modeling and experimental results. The cell can be manipulated in a simple harmonic motion up to 130 Hz by the proposed actuation transmitter as opposed to 90 Hz by direct flow control. Second, there is no need to fill the syringe pump with the sample solution because the actuation transmitter physically separates the fluids between the pump and the cell flow, and consequently, only a very small quantity of the sample is required (<1 μl). In addition, such fluid separation makes it easy to keep the experiment platform sterilized because there is no direct fluid exchange between the sample and fluid inside the pump. Third, the fabrication process is simple because of the single-layer design, making it convenient to implement the actuation transmitter in different microfluidic applications. The proposed actuation transmitter is implemented in a lab-on-a-chip system for red blood cell (RBC) evaluation, where the extensibility of red blood cells is evaluated by manipulating the cells through a constriction channel at a constant velocity. The application shows a successful example of implementing the proposed transmitter.     

A pneumatic valve controlled microdevice for bioanalysis

This paper describes a pneumatic valve controlled microdevice for performing mixing and reaction. This microdevice combined the degassed polydimethylsiloxane (PDMS) pumping method with a syringe-actuated valve system to control the dispensing and mixing of nanoliter solutions. The syringe was used to manually generate vacuum and to open the valves. Upon the opening of the valve, the microchamber was filled with the solution, which was driven by the external atmosphere through the degassed PDMS microchannel. With this microdevice, the enzymatic kinetics of alkaline phosphatase converting the fluorescein diphosphate was studied, and the Michaelis-Menten kinetics was analyzed. The microdevice has the advantages of simplicity and low cost in fabrication and operation.     

Phaseguide-assisted blood separation microfluidic device for point-of-care applications

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.