Microfluidic chip of concentration gradient and fluid shear stress on a single cell level

Concentration gradient and fluid shear stress(FSS) for cell microenvironment were investigated through microfluidic technology. The Darcy–Weisbach equation combined with computational fluid dynamics modeling was exploited to design the microfluidic chip, and the FSS distribution on the cell model with varying micro-channels(triangular, conical, and elliptical). The diffusion with the incompressible laminar flow model by solving the time-dependent diffusion–convection equation was applied to simu...     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

基于微流控芯片的乳腺癌细胞微环境酸化检测

建立了基于微流控芯片的乳腺癌微环境酸化模型和动态检测微环境酸化情况的分析方法。设计了一种多层复合式微流控芯片,将乳腺癌细胞悬液引入含有水凝胶前体的芯片培养室后,在硝酸纤维素薄膜上固化形成3D培养支架。芯片通道连续灌流模拟血流供应,并将非电化学的pH检测器引入芯片,通过图像分析得到实时的pH变化。通过观察癌细胞的存活率、增殖率、乳酸水平及pH值,分析微环境的酸化情况,同时与正常细胞进行比较。结果表明,连续灌流培养7 d,乳腺癌细胞的存活率保持在90%以上;随着培养天数的增加,芯片上癌细胞微环境的pH值逐渐降低,且灌流速度越低,pH值下降越明显,而正常细胞微环境的pH值无明显变化。基于微流控芯片的微环境酸化检测平台可实时动态检测微环境的pH值,有望成为相关肿瘤研究的有力工具。     

Mechanical stimulation of epithelial cells using polypyrrole microactuators

The importance of mechanotransduction for physiological systems is becoming increasingly recognized. The effect of mechanical stimulation is well studied in organs and tissues, for instance by using flexible tissue culture substrates that can be stretched by external means. However, on the cellular and subcellular level, dedicated technology to apply appropriate mechanical stimuli is limited. Here we report an organic electronic microactuator chip for mechanical stimulation of single cells. These chips are manufactured on silicon wafers using traditional microfabrication and photolithography techniques. The active unit of the chip consists of the electroactive polymer polypyrrole that expands upon the application of a low potential. The fact that polypyrrole can be activated in physiological electrolytes makes it well suited as the active material in a microactuator chip for biomedical applications. Renal epithelial cells, which are responsive to mechanical stimuli and relevant from a physiological perspective, are cultured on top of the microactuator chip. The cells exhibit good adhesion and spread along the surface of the chip. After culturing, individual cells are mechanically stimulated by electrical addressing of the microactuator chip and the response to this stimulation is monitored as an increase in intracellular Ca(2+). This Ca(2+) response is caused by an autocrine ATP signalling pathway associated with mechanical stimulation of the cells. In conclusion, the present work demonstrates a microactuator chip based on an organic conjugated polymer, for mechanical stimulation of biological systems at the cellular and sub-cellular level.     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells’ autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.     

A three-dimensional biomimetic microfluidic chip to study the behavior of hepatic stellate cell under the tumor microenvironment

In this paper, we designed a three-dimensional cell co-cultured microfluidic chip, which generated interstitial flow and oxygen gradient to simulate the complex tumor microenvironment. It consisted of five parallel cell culture channels and one hypoxic channel. These channels were constructed for the culture of mouse liver tumor cells (Hepa1-6), mouse liver stellate cells (JS-1), the simulation of extracellular matrix, complex biochemical factors (hypoxia and interstitial flow), and the supply of cellular nutrients. The 3D-interstitial flow-hypoxia model was used to study the behavior of JS-1 cells under the effect of tumor microenvironment (TME). The results showed that by co-cultured with Hepa1-6 cells, hypoxia of Hepa1-6 cells, and adding TGF-β1 by interstitial flow, the migration of JS-1 cells could be promoted. Similarly, activated JS-1 cells could led to the epithelial-mesenchymal transformation in co-cultured Hepa1-6 cells, which secreted more TGF-β1.     

Electrochemical enantiorecognition of tryptophan enantiomers based on graphene quantum dots–chitosan composite film

A graphene quantum dots (GQDs)–chitosan (CS) composite film was prepared via successive electrodeposition of GQDs and CS on the surface of a glassy carbon electrode (GCE). The strong interactions between GQDs and CS resulted in the formation of a regular and uniform film, which can be applied in the electrochemical chiral recognition of tryptophan (Trp) enantiomers. CS in the composite film provides a chiral microenvironment, meanwhile, GQDs can amplify the electrochemical signals and improve the recognition efficiency. Due to the synergetic effect of GQDs and CS, chiral recognition of Trp enantiomers is achieved successfully. Compared with previous reports utilizing GQDs in photoluminescent research, this work opens a new avenue for broadening the applications of GQDs in the electrochemically chiral sensors.     

A passive pump-assisted microfluidic assay for quantifying endothelial wound healing in response to fluid shear stress

There as an urgent need to quantify the endothelial wound-healing process in response to fluid shear stress to improve the biological and clinical understanding of healing mechanisms, which is of great importance for preventing healing impairment, chronic wounds, and postoperative in-stent restenosis. However, current experimental platforms not only require expensive, cumbersome, and powered pumping devices (to, e.g., generate cell scratches and load shear stress stimulation) but also lack quantitative controls for quantitative analysis. In this paper, a passive pump-assisted microfluidic assay is developed to quantify endothelial wound healing in response to fluid shear stress. Our assay consists of passive constant-flow pumps based on the siphon principle and a three-inlet microfluidic chip for cell wound-healing experiments. We also propose a method for quantitatively adjusting cell scratch size by controlling trypsin flow. Both numerical simulations and fluorescein experiments validate the effectiveness of this method. Moreover, we use the designed microfluidic assay to successfully generate cell scratches, load a 12-h shear stress of 5 dyn/cm² to the cells, and observe wound healing. The results indicate that the healing of a cell scratch is significantly accelerated under the stimulation of shear stress. In conclusion, our passive pump-assisted microfluidic assay shows versatility, applicability, and the potential for quantifying endothelial wound healing in response to fluid shear stress.     

One‐Step Electrochemically Deposited Nanocomposite Film of CS‐Fc/MWNTs/GOD for Glucose Biosensor Application

A simple and controllable electrodeposition approach was proposed for one‐step construction of glucose biosensors by in situ co‐deposition of ferrocene‐branched chitosan derivatives (CS‐Fc), multiwalled carbon nanotubes (MWNTs), and glucose oxidase (GOD) onto electrode surface. The formation of CS‐Fc could not only effectively prevent the leakage of Fc and retain its electrochemical activity, but also provide a biocompatible microenvironment for retaining the native activity of the immobilized biomolecules. Further entrapment of MWNTs into the CS matrix improved electronic conductivity of the biocomposite significantly. The facile procedure of immobilizing GOD and the promising feature of biocomposite will offer a versatile platform to fabricate biosensors and bioelectronic devices.     

新型介孔羟基磷灰石/壳聚糖-万古霉素药物释放系统复合材料的制备及体外抗菌和成骨能力

采用冷冻干燥法合成了介孔羟基磷灰石(HA)/壳聚糖(CS)-万古霉素(VCM)药物释放系统复合材料, 利用SEM, XRD和FTIR等方法对材料进行了表征. 结果证实CS与HA混合复合材料具有良好的孔径和孔隙率, 万古霉素吸附于复合材料的表面和内部. 细胞毒性实验[噻唑蓝(MTT)比色法]结果表明, 材料可以促进成骨细胞增殖且具有良好的细胞相容性. 体外抑菌实验结果证实此材料可长时间抑制耐甲氧西林金葡菌(MRSA)的生长, 具有良好的抑菌和杀菌能力. 细胞黏附实验结果表明, 成骨细胞附着于材料表面增殖并通过孔道延伸. 实时聚合酶链式反应(RT-PCR)实验结果表明, 在成骨相关标志产物胶原蛋白-1(COL-1)及骨形态发生蛋白-2(BMP-2)基因上均有较高的表达, 表明材料在体外可以促进成骨细胞生长, 具有良好的成骨能力.     

A simple elastic membrane-based microfluidic chip for the proliferation and differentiation of mesenchymal stem cells under tensile stress

This work presents a simple membrane-based microfluidic chip for the investigation of proliferation and differentiation of mesenchymal stem cells (MSCs) under mechanical stimuli. The cyclic tensile stress was generated by the deformation of elastic PDMS membrane sandwiched between the two layer microfluidic chip via actuated negative pressure, and the cultured MSCs on membrane were subjected to different orders of tensile stress. The results suggest that mechanical stimuli are attributed to the different phenomena of MSCs in cell proliferation and differentiation. The higher tensile stress (>3.5) promoted obvious proliferation, osteogenesis and reduced adipogenesis in MSCs, indicating the possible regulative role of tensile stress in modifying the osteogenesis/adipogenesis balance in the development of tissue organ.     

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.     

Asymmetric cancer-cell filopodium growth induced by electric-fields in a microfluidic culture chip

We combine a micro-fluidic electric-field cell-culture (MEC) chip with structured-illumination nano-profilometry (SINAP) to quantitatively study the variations of cancer cell filopodia under external direct-current electric field (dcEF) stimulations. Because the lateral resolution of SINAP is better than 150 nm in bright-field image modality, filopodia with diameters smaller than 200 nm can be observed clearly without fluorescent labeling. In the MEC chip, a homogeneous EF is generated inside the culture area that simulates the endogenous EF environment. With this MEC chip-SINAP system, we directly observe and quantify the biased growth of filopodia of lung cancer cells toward the cathode. The epidermal growth factor receptors around the cell edges are also redistributed to the cathodal side. These results suggest that cancer-cell filopodia respond to the changes in EFs in the microenvironment.     

An instant cell recognition system using a microfabricated coordinate standard chip useful for combinable cell observation with multiple microscopic apparatuses

Disposable coordinate standard (CS) chips were fabricated by the ejection of melted polystyrene into a metal mold. The CS chip surface was divided into four parts different in height and width. The edge lines of these parts could be recognized as straight lines 2 mum in width in the microscope view and used as the X and Y axes for the culture dish. The CS chip was attached on the bottom of a culture dish outside. Then the dish was set on the microscope stage and moved by means of a motorized automatic stage. The X-Y coordinates of many single-cells in a culture dish were registered, respectively. Once registered, any single-cell could instantly be brought to the center of the microscope view even after displacing the dish from the stage for a while and setting it again on the stage. Therefore, experimenters can easily search any single-cell in any culture dish on any microscope at any time. Such a system is remarkably useful for various modes of single-cell experiment and named "Suguwaculture," which means "instantly" ("sugu" in Japanese) + "recognizable" ("wakaru" in Japanese) + "culture" (during culture).     

Meniscus induced self organization of multiple deep concave wells in a microchannel for embryoid bodies generation

Embryonic stem cells (ESCs) have attracted great interest in the fields of tissue engineering, regenerative medicine, and organogenesis for their pluripotency and ability to self-renew. ESC aggregation, which produces an embryoid body (EB), has been widely utilized as a trigger of in vitro directed differentiation. In this paper, we propose a novel method for constructing large numbers of deep concave wells in PDMS microfluidic chips using the meniscus induced by the surface tension of a liquid PDMS prepolymer, and applied this chip for the mass production of uniform sized EBs. To investigate if the microenvironment in the deep concave well is suitable for ES cells, the oxygen diffusion to the deep concave well was analyzed by CFD simulation. Murine EBs were successfully formed in the deep concave wells without loss of cells and laborious careful intervention to refresh culture media. The size of the EBs was uniform, and retrieving of EBs was done just by flipping over the chip. All the processes including EB formation and harvest are easy and safe to cells, and their viability after completion of all processes was over 95%. The basic properties of the EBs were generated and their capacity to differentiate into 3 germ layers was investigated by analyzing the gene expression profile. The harvested EBs were found to differentiate into cardiac cells and neurons, and neurofilaments formed branches of elongated extensions more than 1.0 mm in length.     

An Endoplasmic Reticulum Targeting Type I Photosensitizer for Effective Photodynamic Therapy against Hypoxic Tumor Cells

Organelle-targeted type I photodynamic therapy (PDT) shows great potential to overcome the hypoxic microenvironment in solid tumors. The endoplasmic reticulum (ER) is an indispensable organelle in cells with important biological functions. When the ER is damaged due to the production of reactive oxygen species (ROS), the accumulation of misfolded proteins will interfere with ER homeostasis, resulting in ER stress. Here, an ER-targeted benzophenothiazine-based photosensitizer NBS-ER was presented. ER targeting modification significantly reduced the dark toxicity and improved phototoxicity index (PI). NBS-ER could effectively produce O₂⁻⋅ with near-infrared irradiation, making its phototoxicity under hypoxia close to that under normoxia. Meanwhile, the photoinduced ROS triggered ER stress and induced apoptosis. In addition, NBS-ER possessed excellent photodynamic therapeutic effect in 4T1-tumor-bearing mice.     

Collagen microsphere production on a chip

We have developed an integrated microfluidic material processing chip and demonstrated the rapid production of collagen microspheres encapsulating cells with high uniformity and cell viability. The chip integrated three material processing steps. Monodisperse microdroplets were generated at a microfluidic T junction between aqueous and mineral oil flows. The flow was heated immediately to 37 °C to initiate collagen fiber assembly within a gelation channel. Gelled microspheres were extracted from the mineral oil phase into cell culture media within an extraction chamber. Collagen gelation immediately after microdroplet generation significantly reduced coalescence among microdroplets that led to non-uniform microsphere production. The microfluidic extraction approach led to higher microsphere recovery and cell viability than when a conventional centrifugation extraction approach was employed. These results indicate that chip-based material processing is a promising approach for cell-ECM microenvironment generation for applications such as tissue engineering and stem cell delivery.     

A time-coded multi-concentration microfluidic chemical waveform generator for high-throughput probing suspension single-cell signaling

The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular matrix. Accurately reconstructing this process and analyzing the changes in receptor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical methodology. In this paper, a time-coded multiconcentration microfluidic chemical waveform generator was developed for the dynamic signaling ...