Visualization of cellular and microvascular relationships

Understanding the structure of microvasculature structures and their relationship to cells in biological tissue is an important and complex problem. Brain microvasculature in particular is known to play an important role in chronic diseases. However, these networks are only visible at the microscopic level and can span large volumes of tissue. Due to recent advances in microscopy, large volumes of data can be imaged at the resolution necessary to reconstruct these structures. Due to the dense and complex nature of microscopy data sets, it is important to limit the amount of information displayed. In this paper, we describe methods for encoding the unique structure of microvascular data, allowing researchers to selectively explore microvascular anatomy. We also identify the queries most useful to researchers studying microvascular and cellular relationships. By associating cellular structures with our microvascular framework, we allow researchers to explore interesting anatomical relationships in dense and complex data sets.     

3D-printed membrane microvalves and microdecoder

A microfluidic system for multichannel switching and multiphase flow control has potential uses in pneumatic soft robotics and biological sampling systems. At present, the membrane microvalves used in microfluidic systems are mostly constructed using a multilayer bonding process so that the device cannot withstand high pressures. In this paper, we demonstrate a design method and the properties of a bondless membrane microvalve fabricated using a commercial 3D printer. We used a multijet (MJP) 3D printer to print a 100-μm-thick and 6-mm-diameter membrane from a relatively hard material (1700 MPa). The membrane’s high toughness ensures that it does not need negative pressure to reopen. The measured operation frequency was less than 2.5 Hz under a pneumatic pressure of 14.5 kPa. We also 3D-printed an integrated Quake-style microfluidic decoder network by combining 8 valves in series to demonstrate the integrability of the microvalve. The decoder chip was demonstrated to control the ON/OFF state of the four coded fluidic channels, with the droplets being generated from selected channels according to the valve action. Therefore, such 3D-printed microvalves are highly integrable, have a high manufacturing efficiency, and can be applied in pneumatic controllers, sample switchers and integrated print heads.

     

On-chip fabrication of magnetic alginate hydrogel microfibers by multilayered pneumatic microvalves

Alginate hydrogel has widespread applications in tissue engineering, cancer therapy, wound management and drug/cell/growth factor delivery due to its biocompatibility, hydrated environment and desirable viscoelastic properties. However, the lack of controllability is still an obstacle for utilizing it in the fabrication of 3D tissue constructs and accurate targeting in mass delivery. Here, we proposed a new method for achieving magnetic alginate hydrogel microfibers by dispersing magnetic nanoparticles in alginate solution and solidifying the magnetic alginate into hydrogel fiber inside microfluidic devices. The microfluidic devices have multilayered pneumatic microvalves with hemicylindrical channels to fully stop the fluids. In the experiments, the magnetic nanoparticles and the alginate solution were mixed and formed a uniform suspension. No aggregation of magnetic nanoparticles was found, which is crucial for flow control inside microfluidic devices. By regulating the flow rates of different solutions with the microvalves inside the microfluidic device, magnetic hydrogel fibers and nonmagnetic hydrogel fibers were fabricated with controlled sizes. The proposed method for fabricating magnetic hydrogel fiber holds great potential for engineering 3D tissue constructs with complex architectures and active drug release.     

A microfluidic system for the study of the response of endothelial cells under pressure

Hydrostatic pressure can affect the structure and function of endothelial cells (ECs). A microfluidic system was built to study how ECs respond to applied pressure. The system included a syringe pump, a PDMS-glass microfluidic chip, and a digital manometer for pressure monitoring. The manometer was connected with the chip in two ways (one was before the inlet and the other after the outlet of the microchannel). The static control and flowing control systems were also set up. Human umbilical vein endothelial cells (HUVECs) were cultured in the 4 cm × 2 mm × 100 μm channel. Pressure of 12 ± 0.5 or 18 ± 0.5 kPa was applied on the cells for 8 h. The F-actin cytoskeleton and the nuclei of the cells were stained for examination and endothelin-1 (ET-1) released from the cells in the channel was assayed by ELISA. The results showed that the cell area and ET-1 concentration increased with the pressure and a higher pressure caused more damages to the cells. This microfluidic system provides a convenient and cost-effective platform for the studies of cell response to pressure.     

Microfluidic chips for capillary electrophoresis with integrated electrodes for capacitively coupled conductivity detection based on printed circuit board technology

A simple and low budget microfabrication method compatible with mass production was developed for the integration of electrodes for capacitively coupled contactless conductivity detection (C4D) in Lab on a Chip devices. Electrodes were patterned on a printed circuit board using standard processing. This was followed by lamination-photolithography-lamination to cover the electrodes in dry film photoresist (DFR) using an office laminator. This resulted in a flush, smooth surface on top of the detection electrodes, enabling subsequent integration of a microfluidic network at a distance dictated by the thickness of the DFR (17 μm, 30 μm and 60 μm were used in this work). This process was applied to create two types of detectors, re-usable detectors to be used in combination with a separate microfluidic network and integrated detectors where the microfluidic network is irreversibly sealed to the detector. A poly(dimethylsiloxane) (PDMS) slab containing the microfluidic network was positioned on top of the re-usable detectors to create the PDMS hybrid devices. The integrated DFR devices were created by patterning and sealing the microchannel in DFR using subsequent lamination and lithographic steps. The sensitivity of the C4D made using this new technology for small inorganic cations was between 6 and 20 μM, which is comparable with devices made using significantly more advanced technologies. Where the 17 μm film slightly improved the sensitivity, the use of 30 μm thick insulating films was preferred as these did not impose significant restrictions on the applicable field strengths.     

Microvalve and liquid membrane double-controlled integrated microfluidics for observing the interaction of breast cancer cells

The methodical development of cell biology has resulted in significant advancements in the study of breast tumors. However, the dynamic investigation of the self-seeding process remains largely out of reach. In the present study, we describe a microvalve and liquid membrane double-controlled integrated microfluidic device that provides for the versatile assessment of breast tumor cell invasion dynamics. The liquid membrane formation was first optimized to obtain a high level of control, and was then applied to different types of homotypic and heterotypic cell seeding with precise selective positioning for monoculture and coculture. Using this device, the interaction between breast cancer cells MDA231-LM2 and MDA-MB231 was successfully observed to investigate self-seeding dynamics, including migration, infiltration, and coexistence. The results quantitatively demonstrate the mutual signal-induced attraction between MDA-MB231 and MDA231-LM2 cells, as well as the progressive infiltration of MDA231-LM2 cells into the MDA-MB231 cell population. These results are valuable in the development of spatiotemporal-controlled microfluidic systems and to many microscale-based biological and diagnostic studies involving cell growth, cell differentiation, cell interaction, and cell signal.     

Optimal fuzzy sliding-mode control for bio-microfluidic manipulation

In biometric and biomedical applications, a special transporting mechanism must be designed for the micro total analysis system (μTAS) to move samples and reagents through the microchannels that connect the unit procedure components in the system. An important issue for this miniaturization and integration is the microfluid management technique, i.e., microfluid transportation, metering, and mixing. In view of this, an optimal fuzzy sliding-mode control (OFSMC) based on the 8051 microprocessor is designed and a complete microfluidic manipulated biochip system is implemented in this study, with a pneumatic pumping actuator, two feedback-signal photodiodes and flowmeters for better microfluidic management. This new technique successfully improved the efficiency of biochemical reaction by increasing the effective collision into the probe molecules as the target molecules flow back and forth. The new technique was used in DNA extraction. When the number of Escherichia coli cells was 2×10²–10⁴ in 25 μl of whole blood, the extraction efficiency of immobilized beads with solution flowing back and forth was 600-fold larger than that of free beads.     

基于逐像素点深度卷积网络分割模型的上皮和间质组织分割

上皮和间质组织是乳腺组织病理图像中最基本的两种组织,约80%的乳腺肿瘤起源于乳腺上皮组织.为了构建基于乳腺组织病理图像分析的计算机辅助诊断系统和分析肿瘤微环境,上皮和间质组织的自动分割是重要的前提条件.本文构建一种基于逐像素点深度卷积网络（CN-PI）模型的上皮和间质组织的自动分割方法.1）以病理医生标注的两类区域边界附近具有类信息为标签的像素点为中心,构建包含该像素点上下文信息的正方形图像块的训练集.2）以每个正方形图像块包含的像素的彩色灰度值作为特征,以这些图像块中心像素类信息为标签训练CN模型.在测试阶段,在待分割的组织病理图像上逐像素点地取包含每个中心像素点上下文信息的正方形图像块,并输入到预先训练好的CN网络模型,以预测该图像块中心像素点的类信息.3）以每个图像块中心像素为基础,逐像素地遍历图像中的每一个像素,将预测结果作为该图像块中心像素点类信息的预测标签,实现对整幅图像的逐像素分割.实验表明,本文提出的CN-PI模型的性能比基于图像块分割的CN网络（CN-PA）模型表现出了更优越的性能.     

A microfluidic platform with permeable walls for the analysis of vascular and extravascular mass transport

The interface between the blood pool and the extravascular matrix is fundamental in regulating the transport of molecules, nanoparticles and cells under physiological and pathological conditions. In this work, a microfluidic chip is presented comprising two parallel microchannels connected laterally via an array of high aspect ratio micropillars, constituting the permeable vascular membrane. A double-step lithographic process combined with a replica molding approach is employed to realize 80 different arrays of micropillars exhibiting three cross-sectional geometries (rectangular, elliptical and curved); two orientations (normal and parallel) with respect to the flow; and a variety of width and gap sizes, respectively, ranging from 10 to 20 μm and 2 to 5 μm. As compared to conventional rectangular structures, the curved pillars provide higher bending stiffness, lower adhesive interactions, and smaller intra-channel separation distances. Specifically, 10-μm-wide curved pillars, laying parallel to the flow, offered the highest mechanical stability. To assess vascular permeability, the extravascular channel was filled with a hyaluronic acid hydrogel, while fluorescent Dextran molecules and calibrated polystyrene beads were injected in the vascular channel. Membrane permeability was observed to reduce with the molecular weight of Dextran and diameter of the beads, ranging from about 6 × 10^?5 to 2 × 10^?5 cm/s for 40 and 250 kDa Dextran and up to zero for 1.5 μm beads. The presented data demonstrate the potential of the proposed microfluidic chip for analyzing the vascular and extravascular mass transport, over multiple spatial and temporal scales, in a variety of diseases involving differential permeation across vascular walls.     

Controllable fission of droplets and bubbles by pneumatic valve

We report a feasible method that can precisely control the fission of droplets by modulating the flow resistance using pneumatic valves. Multilayer soft lithography was used to fabricate the valves. They can be used as variable microfluidic resistor (VMR) to dramatically change the flow resistance. A simulation has been done to forecast the behavior of droplets. We used this technique to control break-up of generated droplets and direct their motion. Droplets with different volume ratios were obtained in one chip. To investigate the mechanism, an equivalent electrical circuit was introduced to compare with the fluid network. This method could potentially be applied to different geometries, especially for the microfluidic network consisting of a set of two parallel channels with a common inlet and different outlets in bifurcating channels. Besides, manipulation of bubbles was also demonstrated.     

On-chip cell migration assay for quantifying the effect of ethanol on MCF-7 human breast cancer cells

Ethanol consumption is associated with the risk of breast cancer progression; however, the mechanism of relationship has not yet been fully explained. Research on breast cancer cell migration after ethanol stimulation may give hope for a better understanding of the disease and oncotherapy. Conventional cell migration assays such as Boyden chamber and wound-healing assays are easy to conduct for this purpose; however, these assays have inherent limitations. In this study, we quantified the effect of ethanol on MCF-7 hunam breast cancer cells using a microfluidics-based wound-healing assay. Wounds were prepared by partially digesting a confluent cell sheet using parallel laminar flows in the presence of protease trypsin. The cells at the leading edge of the wound remained intact. Cell image analysis indicates that all the cells cultured in the microdevice took on a good morphology and monolayer growth status. Cell viability assay demonstrates that cell viability decreased with the increase in ethanol concentration and treatment time. For 0, 22, 43, and 65 mmol/l of ethanol, cell viability after being cultured for 24 h was 100%, 99.6%, 99.4%, and 98.4%, respectively. Studying MCF-7 human breast cancer cell migration when treated with different ethanol concentrations revealed that the cell migration distance is directly proportional with ethanol concentration. After being cultured for 24 h at 37°C and 5% CO₂, the maximal cell migration distance was 231, 283, and 332 μm for 22, 43, and 65 mmol/l ethanol, respectively; all results were higher than the blank test (i.e., ethanol-free test, 218 μm). These findings will be beneficial in developing microfluidic device applications for future research on breast tumor therapy in a biomimetic microenvironment and for developing new methods for breast cancer therapy.     

An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

Micromachining of biocompatible polymer substrate for cancer cell separation applications

Isolation of circulating tumor cells from human blood plays a significant role in diagnosis and treatment of cancer. The most effective way of isolating cells is the use of lab on-chip microfluidic devices. This paper presents the development of cyclic olefin polymer membrane with an array of micropores for separating circulating tumour cells using ultrashort laser ablation process. Initially, the substrate is tested for its cell viability. The laser Fluence is varied from 1–12 J/cm² and its observed that at a fluence of 2.5 J/cm² the desired diameter of 12 μm with ablation depth of 1 μm is obtained which is necessary for CTC separation. The treated and untreated structure is characterized using Fourier transform infrared spectrometer and it shows that the treated region shows no significant shift in the spectrum after femtosecond laser ablation.

     

Two-layer multiplexed peristaltic pumps for high-density integrated microfluidics

The integration and operation of a large number of components is needed to enable ever more complex and integrated chemical and biological processes on a single microfluidic chip. The capabilities of these chips are often limited by the maximum number of pumps and valves that can be controlled on a single chip, a limitation typically set by the number of pneumatic interconnects available from ancillary hardware. Here, we report a multiplexing approach that greatly reduces the number of external pneumatic connections needed for the operation of a large number of peristaltic pumps. The utility of the approach is demonstrated with a complex microfluidic network capable of generating and routing liquid droplets in a two-phase flow. We also report a set of design rules for the design and operation of multiplexed peristaltic pumps, based on a study of the effect of the number of valves per pump and the valve-to-valve distance on the performance of peristaltic pumps. The multiplexing approach reported here may find application in a wide range of microfluidic chips for chemical and biological applications, especially those that require the integration of many different operations on a single chip and those that need to perform similar operations massively in parallel, in sub-nanoliter volumes.     

Highly integrated polymeric microliquid flow controller for droplet microfluidics

Microfluidic applications demand accurate control and measurement of small fluid flows and volumes, and the majority of approaches found in the literature involve materials and fabrication methods not suitable for a monolithic integration of different microcomponents needed to make a complex Lab-on-a-Chip (LoC) system. The present work leads to a design and manufacturing approach for problem-free monolithic integration of components on thermoplastics, allowing the production of excellent quality devices either as stand-alone components or combined in a complex structures. In particular, a polymeric liquid flow controlling system (LFCS) at microscale is presented, which is composed of a pneumatic microvalve and an on-chip microflow sensor. It enables flow regulation between 30 and 230 μl/min with excellent reproducibility and accuracy (error lower than 5%). The device is made of a single Cyclic Olefin Polymer (COP) piece, where the channels and cavities are hot-embossed, sealed with a single COP membrane by solvent bonding and metalized, after sealing, to render a fully functional microfluidic control system that features on-chip flow sensing. In contrast with commercially available flow control systems, the device can be used for high-quality flow modulation in disposable LoC devices, since the microfluidic chip is low cost and replaceable from the external electronic and pneumatic actuators box. Functionality of the LFCS is tested by connecting it to a microfluidic droplet generator, rendering highly stable flow rates and allowing generation of monodisperse droplets over a wide range of flow rates. The results indicate the successful performance of the LFCS with significant improvements over existing LFCS devices, facing the possibility of using the system for biological applications such as generating distinct perfusion modes in cell culture, novel digital microfluidics. Moreover, the integration capabilities and the reproducible fabrication method enable straightforward transition from prototype to product in a way that is lean, cost-effective and with reduced risk.     

Mathematical analysis of oxygen transfer through polydimethylsiloxane membrane between double layers of cell culture channel and gas chamber in microfluidic oxygenator

For successful cell culture in microfluidic devices, precise control of the microenvironment, including gas transfer between the cells and the surrounding medium, is exceptionally important. The work is motivated by a polydimethylsiloxane (PDMS) microfluidic oxygenator chip for mammalian cell culture suggesting that the speed of the oxygen transfer may vary depending on the thickness of a PDMS membrane or the height of a fluid channel. In this paper, a model is presented to describe the oxygen transfer dynamics in the PDMS microfluidic oxygenator chip for mammalian cell culture. Theoretical studies were carried out to evaluate the oxygen profile within the multilayer device, consisting of a gas reservoir, a PDMS membrane, a fluid channel containing growth media, and a cell culture layer. The corresponding semi-analytical solution was derived to evaluate dissolved oxygen concentration within the heterogeneous materials, and was found to be in good agreement with the numerical solution. In addition, a separate analytical solution was obtained to investigate the oxygen pressure drop (OPD) along the cell layer due to oxygen uptake of cells, with experimental validation of the OPD model carried out using human umbilical vein endothelial cells cultured in a PDMS microfluidic oxygenator. Within the theoretical framework, the effects of several microfluidic oxygenator design parameters were studied, including cell type and critical device dimensions.     

Selective Formation and Removal of Liquid Microlenses at Predetermined Locations Within Microfluidics Through Pneumatic Control

This paper reports on liquid (deionized water) microlenses that are intrinsically formed and integrated within microfluidics through pneumatic manipulation of fluids inside microchannels. Such microlenses are formed via liquid-air interfaces of liquid droplets, which are pinned at T-shaped junctions of channels. In addition to being tunable in focal lengths (a few hundreds of micrometers to infin) along the microchannels parallel to the substrate used, these microlenses can uniquely be repositioned, removed, and reformed at predetermined locations of the T-shaped junctions within microchannels on demand under pneumatic controls. The design and formation of a microfluidic channel network for the in situ formation are first described. Then, the pneumatic control of the fluids, including the formation, movement, and size control of a lens droplet, is discussed, and the in situ formation processes for single and multiple liquid microlenses are described. The in situ formation processes for a single microlens and a two-lens combination only take tens of seconds, eliminating various conventional microfabrication processes and multiple layers of materials. Finally, the detail of characterization of these microlenses is given.     

A novel three-dimensional microfluidic platform for on chip multicellular tumor spheroid formation and culture

The formation of three-dimensional (3D) multicellular cell spheroids such as microspheres and embryoid bodies has recently gained much attention as a useful cell culture technique, but few studies have investigated the suitability of glass for spheroids formation and culture. In this work, we present a novel three-dimensional microfluidic device made of poly(dimethylsiloxane) (PDMS) and glass for the easy and rapid synthesis and culture of tumor spheroid. The cell culture unit is composed of an array of microwells on the bottom of a glass plate, bigger microwells and elastomeric microchannels on the top of a PDMS plate. Cell suspension can be easily introduced into the cell culture unit and exchange with the external liquid environment by the microfluidic channels. A single tumor spheroid can be formed and cultured in each glass cell culture chamber, the surface of which was modified with poly(vinyl alcohol) to render it to be resistant to cell adhesion. As the cell culture medium could be replaced, spheroids of the human breast cancer (MCF-7) cells were cultured on the chip for 3 days, reaching the diameters of about 150 μm. Furthermore, the MCF-7 cells were successfully cultured on the chip in 2D and 3D culture modes. Results have shown that glass is well suitable for multicellular tumor spheroids culture. The established platform provides a convenient and rapid method for tumor spheroid culture, which is also adaptable for anticancer drug screening and fundamental biomedical research in cell biology.     

Hydrodynamic trap-and-release of single particles using dual-function elastomeric valves: design, fabrication, and characterization

This paper introduces a simple method for trapping and releasing single particles, such as microbeads and living cells, using dual-function elastomeric valves. Our key technique is the utilization of the elastomeric valve as a dual-function removable trap instead of a fixed trap and a separate component for releasing trapped particles, thereby enabling a simple yet effective trap-and-release of particles. We designed, fabricated, and characterized a microfluidic-based device for trapping and releasing single beads by controlling elastomeric valves driven by pneumatic pressure and a fluid flow action. The fluid flow is controlled to ensure that beads flowing in a main stream enter into a branch channel. A bead is trapped by deflected elastomeric valves positioned at the entrance of a branch channel. The trapped bead is easily released by removing the applied pressure. The trapping and releasing of single beads of 21?μm in diameter were successfully performed under an optimized pressure and flow rate ratio. Moreover, we confirmed that continuous trapping and releasing of single beads by repeatedly switching elastomeric valves enables the collection of a controllable number of beads. Our simple method can be integrated into microfluidic systems that require single or multiple particle arrays for quantitative and high-throughput assays in applications within the fields of biology and chemistry.     

Fabrication and testing of embedded microvalves within PMMA microfluidic devices

The integration of a PDMS membrane within orthogonally placed PMMA microfluidic channels enables the pneumatic actuation of valves within bonded PMMA–PDMS–PMMA multilayer devices. Here, surface functionalization of PMMA substrates via acid catalyzed hydrolysis and air plasma corona treatment were investigated as possible techniques to permanently bond PMMA microfluidic channels to PDMS surfaces. FTIR and water contact angle analysis of functionalized PMMA substrates showed that air plasma corona treatment was most effective in inducing PMMA hydrophilicity. Subsequent fluidic tests showed that air plasma modified and bonded PMMA multilayer devices could withstand fluid leakage at an operational flow rate of 9 μl/min. The pneumatic actuation of the embedded PDMS membrane was observed through optical microscopy and an electrical resistance based technique. PDMS membrane actuation occurred at pneumatic pressures of as low as 10 kPa and complete valving occurred at 14 kPa for ~100 μm by 100 μm channel cross-sections.