Design, simulation and application of multichannel microfluidic chip for cell migration / 生物医学工程学杂志

Cell migration is defined as the directional movement of cells toward a specific chemical concentration gradient, which plays a crucial role in embryo development, wound healing and tumor metastasis. However, current research methods showed low flux and are only suitable for single-factor assessment, and it was difficult to comprehensively consider the effects of other parameters such as different concentration gradients on cell migration behavior. In this paper, a four-channel microfluidic chip was designed. Its characteristics were as follows: it relied on laminar flow and diffusion mechanisms to establish and maintain a concentration gradient; it was suitable for observation of cell migration in different concentration gradient environment under a single microscope field; four cell isolation zones (20 μm width) were integrated into the microfluidic device to calibrate the initial cell position, which ensured the accuracy of the experimental results. In particular, we used COMSOL Multiphysics software to simulate the structure of the chip, which demonstrated the necessity of designing S-shaped microchannel and horizontal pressure balance channel to maintain concentration gradient. Finally, neutrophils were incubated with advanced glycation end products (AGEs, 0, 0.2, 0.5, 1.0 μmol·L -1), which were closely related to diabetes mellitus and its complications. The migration behavior of incubated neutrophils was studied in the 100 nmol·L -1 of chemokine (N-formylmethionyl-leucyl-phenyl-alanine) concentration gradient. The results prove the reliability and practicability of the microfluidic chip.

Identification of microchips through diagnostic imaging helps to determine the longevity of the golden lancehead (Bothrops insularis) in captivity / Identificação de microchips por meio de diagnóstico por imagem auxilia a determinar a longevidade da jararaca-ilhoa (Bothrops insularis) em cativeiro

As serpentes vindas da natureza e encaminhadas para centros de reabilitação ou de pesquisa têm uma idade incerta. Na natureza, esses animais, muitas vezes atingem um tamanho corpóreo menor do que os indivíduos cativos devido a uma menor frequência de alimentação. Assim, a idade de uma cobra recém-chegada da natureza é geralmente estimada com base em seu tamanho corpóreo, o qual é comparado com dados em cativeiro. A utilização dos meios de diagnóstico por imagem tem auxiliado a medicina na análise das serpentes em cativeiro, mediante avaliação da estrutura óssea, dos órgãos e de alterações. Este trabalho relata o uso de diagnóstico por imagem (radiografia e ultrassonografia) para identificar a presença de microchips e, consequentemente, estimar a idade de uma Bothrops insularis em cativeiro.(AU)

Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model / Terapia de magneto-hipertermia no modelo de tumor de glioblastoma on-a-Chip

ABSTRACT Objective: To evaluate the magnetic hyperthermia therapy in glioblastoma tumor-on-a-Chip model using a microfluidics device. Methods: The magnetic nanoparticles coated with aminosilane were used for the therapy of magnetic hyperthermia, being evaluated the specific absorption rate of the magnetic nanoparticles at 300 Gauss and 305kHz. A preculture of C6 cells was performed before the 3D cells culture on the chip. The process of magnetic hyperthermia on the Chip was performed after administration of 20μL of magnetic nanoparticles (10mgFe/mL) using the parameters that generated the specific absorption rate value. The efficacy of magnetic hyperthermia therapy was evaluated by using the cell viability test through the following fluorescence staining: calcein acetoxymethyl ester (492/513nm), for live cells, and ethidium homodimer-1 (526/619nm) for dead cells dyes. Results: Magnetic nanoparticles when submitted to the alternating magnetic field (300 Gauss and 305kHz) produced a mean value of the specific absorption rate of 115.4±6.0W/g. The 3D culture of C6 cells evaluated by light field microscopy imaging showed the proliferation and morphology of the cells prior to the application of magnetic hyperthermia therapy. Fluorescence images showed decreased viability of cultured cells in organ-on-a-Chip by 20% and 100% after 10 and 30 minutes of the magnetic hyperthermia therapy application respectively. Conclusion: The study showed that the therapeutic process of magnetic hyperthermia in the glioblastoma on-a-chip model was effective to produce the total cell lise after 30 minutes of therapy.

RESUMO Objetivo: Avaliar a terapia de magneto-hipertermia em modelo de tumor de glioblastoma on-a-Chip. Métodos: As nanopartículas magnéticas recobertas com aminosilana foram utilizadas para a terapia da magneto-hipertermia, sendo avaliada a taxa de absorção específica das nanopartículas magnéticas em 300 Gauss e 305kHz. Uma pré-cultura de células C6 foi realizada e, seguidamente, foi feito o cultivo das células 3D no chip. O processo de magneto-hipertermia no chip foi realizado após administração de 20μL de nanopartículas magnéticas (10mgFe/mL), utilizando os parâmetros que geraram o valor da taxa de absorção específica. A eficácia da terapia de magneto-hipertermia foi avaliada pela viabilidade celular por meio dos corantes fluorescentes acetoximetiléster de calceína (492/513nm), para células vivas, e etídio homodímero-1 (526/619nm), para células mortas. Resultados: As nanopartículas magnéticas, quando submetidas ao campo magnético alternado (300 Gauss e 305kHz), produziram um valor médio da taxa de absorção específica de 115,4±6,0W/g. A cultura 3D das células C6 avaliada por imagem de microscopia de campo claro mostrou a proliferação e a morfologia das células antes da aplicação da terapia de magneto-hipertermia. As imagens de fluorescência mostraram diminuição da viabilidade das células cultivadas no organ-on-a-Chip em 20% e 100% após 10 e 30 minutos, respectivamente, da aplicação da terapia de magneto-hipertermia. Conclusão: O processo terapêutico da magneto-hipertermia no modelo de tumor glioblastoma on-a-chip foi eficaz para produzir lise total das células após 30 minutos de terapia.

Automated microfluidic chip system for radiosynthesis of PET imaging probes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Positron emission tomography (PET) is a powerful non-invasive molecular imaging technique for the early detection, characterization, and "real-time" monitoring of disease, and for investigating the efficacy of drugs (Phelps, 2000; Ametamey et al., 2008). The development of molecular probes bearing short-lived positron-emitting radionuclides, such as 18F (half-life 110 min) or 11C (half-life 20 min), is crucial for PET imaging to collect in vivo metabolic information in a time-efficient manner (Deng et al., 2019). In this regard, one of the main challenges is rapid synthesis of radiolabeled probes by introducing the radionuclides into pharmaceuticals as soon as possible before injection for a PET scan. Although many potential PET probes have been discovered, only a handful can satisfy the demand for a highly efficient synthesis procedure that achieves radiolabeling and delivery for imaging within 1-2 radioisotope half-lives. Only a few probes, such as 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) and [18F]fluorodopa, are routinely produced on a commercial scale for daily clinical diagnosis (Grayson et al., 2018; Carollo et al., 2019).

Inhibition of the Hedgehog Signaling Pathway Depresses the Cigarette Smoke-Induced Malignant Transformation of 16HBE Cells on a Microfluidic Chip / 中华医学杂志(英文版)

<p><b>Background</b>The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells.</p><p><b>Methods</b>In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells. Chip A contained a concentration gradient generator, while chip B had four cell chambers with a central channel. The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation. The 16HBE cells in chip B were cultured with 12.25% CSE (Group A), 12.25% CSE + 5 μmol/L cyclopamine (Group B), or normal complete medium as control for 8 months (Group C), to establish the in vitro lung inflammatory-cancer transformation model. The transformed cells were inoculated into 20 nude mice as cells alone (Group 1) or cells with cyclopamine (Group 2) for tumorigenesis testing. Expression of HHS proteins was detected by Western blot. Data were expressed as mean ± standard deviation. The t-test was used for paired samples, and the difference among groups was analyzed using a one-way analysis of variance.</p><p><b>Results</b>The optimal concentration of CSE was 12.25%. Expression of HHS proteins increased during the process of malignant transformation (Group B vs. Group A, F = 7.65, P < 0.05). After CSE exposure for 8 months, there were significant changes in cellular morphology, which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice. Cyclopamine could effectively depress the expression of HHS proteins (Group C vs. Group B, F = 6.47, P < 0.05) and prevent tumor growth in nude mice (Group 2 vs. Group 1, t = 31.59, P < 0.01).</p><p><b>Conclusions</b>The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells. Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.</p>

Neural Stem Cell Differentiation Using Microfluidic Device-Generated Growth Factor Gradient

Neural stem cells (NSCs) have the ability to self-renew and differentiate into multiple nervous system cell types. During embryonic development, the concentrations of soluble biological molecules have a critical role in controlling cell proliferation, migration, differentiation and apoptosis. In an effort to find optimal culture conditions for the generation of desired cell types in vitro, we used a microfluidic chip-generated growth factor gradient system. In the current study, NSCs in the microfluidic device remained healthy during the entire period of cell culture, and proliferated and differentiated in response to the concentration gradient of growth factors (epithermal growth factor and basic fibroblast growth factor). We also showed that overexpression of ASCL1 in NSCs increased neuronal differentiation depending on the concentration gradient of growth factors generated in the microfluidic gradient chip. The microfluidic system allowed us to study concentration-dependent effects of growth factors within a single device, while a traditional system requires multiple independent cultures using fixed growth factor concentrations. Our study suggests that the microfluidic gradient-generating chip is a powerful tool for determining the optimal culture conditions.

Relationship between Pericytes and Endothelial Cells in Retinal Neovascularization: A Histological and Immunofluorescent Study of Retinal Angiogenesis

PURPOSE: To evaluate the relationship between pericytes and endothelial cells in retinal neovascularization through histological and immunofluorescent studies. METHODS: C57BL/6J mice were exposed to hyperoxia from postnatal day (P) 7 to P12 and were returned to room air at P12 to induce a model of oxygen-induced retinopathy (OIR). The cross sections of enucleated eyes were processed with hematoxylin and eosin. Immunofluorescent staining of pericytes, endothelial cells, and N-cadherin was performed. Microfluidic devices were fabricated out of polydimethylsiloxane using soft lithography and replica molding. Human retinal microvascular endothelial cells, human brain microvascular endothelial cells, human umbilical vein endothelial cells and human placenta pericyte were mixed and co-cultured. RESULTS: Unlike the three-layered vascular plexus found in retinal angiogenesis of a normal mouse, angiogenesis in the OIR model is identified by the neovascular tuft extending into the vitreous. Neovascular tufts and the three-layered vascular plexus were both covered with pericytes in the OIR model. In this pathologic vascularization, N-cadherin, known to be crucial intercellular adhesion molecule, was also present. Further evaluation using the microfluidic in vitro model, successfully developed a microvascular network of endothelial cells covered with pericytes, mimicking normal retinal angiogenesis within 6 days. CONCLUSIONS: Pericytes covering endothelial cells were observed not only in vasculature of normal retina but also pathologic neovascularization of OIR mouse at P17. Factors involved in the endothelial cell-pericyte interaction can be evaluated as an attractive novel treatment target. These future studies can be performed using microfluidic systems, which can shorten the study time and provide three-dimensional structural evaluation.

Flow Shear Stress Enhances the Proliferative Potential of Cultured Radial Glial Cells Possibly Via an Activation of Mechanosensitive Calcium Channel

Radial glial cells (RGCs) which function as neural stem cells are known to be non-excitable and their proliferation depends on the intracellular calcium (Ca²⁺) level. It has been well established that Inositol 1,4,5-trisphosphate (IP3)-mediated Ca²⁺ release and Ca²⁺ entry through various Ca²⁺ channels are involved in the proliferation of RGCs. Furthermore, RGCs line the ventricular wall and are exposed to a shear stress due to a physical contact with the cerebrospinal fluid (CSF). However, little is known about how the Ca²⁺ entry through mechanosensitive ion channels affects the proliferation of RGCs. Hence, we hypothesized that shear stress due to a flow of CSF boosts the proliferative potential of RGCs possibly via an activation of mechanosensitive Ca²⁺ channel during the embryonic brain development. Here, we developed a new microfluidic two-dimensional culture system to establish a link between the flow shear stress and the proliferative activity of cultured RGCs. Using this microfluidic device, we successfully visualized the artificial CSF and RGCs in direct contact and found a significant enhancement of proliferative capacity of RGCs in response to increased shear stress. To determine if there are any mechanosensitive ion channels involved, a mechanical stimulation by poking was given to individual RGCs. We found that a poking on radial glial cell induced an increase in intracellular Ca²⁺ level, which disappeared under the extracellular Ca²⁺-free condition. Our results suggest that the shear stress by CSF flow possibly activates mechanosensitive Ca²⁺ channels, which gives rise to a Ca²⁺ entry which enhances the proliferative capacity of RGCs.

Applications of Microfluidic Devices for Urology / 대한배뇨장애요실금학회지

Microfluidics is considered an important technology that is suitable for numerous biomedical applications, including cancer diagnosis, metastasis, drug delivery, and tissue engineering. Although microfluidics is still considered to be a new approach in urological research, several pioneering studies have been reported in recent years. In this paper, we reviewed urological research works using microfluidic devices. Microfluidic devices were used for the detection of prostate and bladder cancer and the characterization of cancer microenvironments. The potential applications of microfluidics in urinary analysis and sperm sorting were demonstrated. The use of microfluidic devices in urology research can provide high-throughput, high-precision, and low-cost analyzing platforms.

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named "MitoQuant". This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.

Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses / 대한배뇨장애요실금학회지

PURPOSE: We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. METHODS: A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. RESULTS: A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. CONCLUSIONS: In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately.

Indirect Co-Culture of Stem Cells from Human Exfoliated Deciduous Teeth and Oral Cells in a Microfluidic Platform

Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

Organ-on-a-chip and the kidney

Traditional approaches to pathophysiology are advancing but still have many limitations that arise from real biologic systems and their associated physiological phenomena being too complicated. Microfluidics is a novel technology in the field of engineering, which provides new options that may overcome these hurdles. Microfluidics handles small volumes of fluids and may apply to various applications such as DNA analysis chips, other lab-on-a-chip analyses, micropropulsion, and microthermal technologies. Among them, organ-on-a-chip applications allow the fabrication of minimal functional units of a single organ or multiple organs. Relevant to the field of nephrology, renal tubular cells have been integrated with microfluidic devices for making kidneys-on-a-chip. Although still early in development, kidneys-on-a-chip are showing potential to provide a better understanding of the kidney to replace some traditional animal and human studies, particularly as more cell types are incorporated toward the development of a complete glomerulion-a-chip.

Microfluidic System Based High Throughput Drug Screening System for Curcumin/TRAIL Combinational Chemotherapy in Human Prostate Cancer PC3 Cells

We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells.

Flexible print circuit technology application in biomedical engineering / 生物医学工程学杂志

Flexible print circuit (FPC) technology has been widely applied in variety of electric circuits with high precision due to its advantages, such as low-cost, high specific fabrication ability, and good flexibility, etc. Recently, this technology has also been used in biomedical engineering, especially in the development of microfluidic chip and microelectrode array. The high specific fabrication can help making microelectrode and other micro-structure equipment. And good flexibility allows the micro devices based on FPC technique to be easily packaged with other parts. In addition, it also reduces the damage of microelectrodes to the tissue. In this paper, the application of FPC technology in biomedical engineering is introduced. Moreover, the important parameters of FPC technique and the development trend of prosperous applications is also discussed.

The development of new standard plane light source for calibration of biochip readers / 中国医疗器械杂志

Biochip reader is an important instrument for taking pictures of biochip. The accuracy of the instrument influences diagnosis of doctors. So it is important to reduce the nonuniformity of images and the error between the biochip readers. This paper describes the development of new standard plane light source, which can reduce the work load of calibration, improve the accuracy and speed of calibration, made the biochip readers more reliably.

Recent Progress in Lab-on-a-Chip Technology and Its Potential Application to Clinical Diagnoses / 대한배뇨장애요실금학회지

We present the construction of the lab-on-a-chip (LOC) system, a state-of-the-art technology that uses polymer materials (i.e., poly[dimethylsiloxane]) for the miniaturization of conventional laboratory apparatuses, and show the potential use of these microfluidic devices in clinical applications. In particular, we introduce the independent unit components of the LOC system and demonstrate how each component can be functionally integrated into one monolithic system for the realization of a LOC system. In specific, we demonstrate microscale polymerase chain reaction with the use of a single heater, a microscale sample injection device with a disposable plastic syringe and a strategy for device assembly under environmentally mild conditions assisted by surface modification techniques. In this way, we endeavor to construct a totally integrated, disposable microfluidic system operated by a single mode, the pressure, which can be applied on-site with enhanced device portability and disposability and with simple and rapid operation for medical and clinical diagnoses, potentially extending its application to urodynamic studies in molecular level.

Biochip micromachining technology adopting laser technology / 生物医学工程学杂志

This paper deals with the manufacturing state of the art of biochip, and introduces a new method--laser microtechnology, including its developing procedure, characteristics and function in biochip production.