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.
Cell Movement ; Chemotaxis ; Equipment Design ; Lab-On-A-Chip Devices ; Microfluidic Analytical Techniques ; Microfluidics ; Neutrophils ; Reproducibility of Results

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).
Lab-On-A-Chip Devices ; Positron-Emission Tomography/methods* ; Radioisotopes/chemistry* ; Radiopharmaceuticals/chemical synthesis* ; Solid Phase Extraction

BackgroundThe 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.

MethodsIn 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.

ResultsThe 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).

ConclusionsThe 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.

Animals ; Cell Transformation, Neoplastic ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Hedgehog Proteins ; genetics ; metabolism ; Lab-On-A-Chip Devices ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microfluidics ; Signal Transduction ; genetics ; physiology ; Smoke ; Smoking ; adverse effects

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.
Animals ; Brain ; Cadherins ; Endothelial Cells ; Eosine Yellowish-(YS) ; Fungi ; Hematoxylin ; Human Umbilical Vein Endothelial Cells ; Humans ; Hyperoxia ; In Vitro Techniques ; Lab-On-A-Chip Devices ; Mice ; Microfluidics ; Microvessels ; Neovascularization, Pathologic ; Pericytes ; Placenta ; Retina ; Retinal Neovascularization ; Retinaldehyde

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.
Apoptosis ; Cell Culture Techniques ; Cell Proliferation ; Embryonic Development ; Female ; Fibroblasts ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Lab-On-A-Chip Devices ; Microfluidics* ; Nervous System ; Neural Stem Cells* ; Neurogenesis ; Neurons ; Pregnancy

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.
Diagnosis ; Lab-On-A-Chip Devices* ; Microfluidics* ; Neoplasm Metastasis ; Prostate ; Prostatic Neoplasms ; Spermatozoa ; Tissue Engineering ; Tumor Microenvironment ; Urinary Bladder Neoplasms ; Urology*

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.
Brain ; Calcium Channels* ; Calcium* ; Cerebrospinal Fluid ; Ependymoglial Cells* ; Inositol 1,4,5-Trisphosphate ; Ion Channels ; Lab-On-A-Chip Devices ; Microfluidics ; Neural Stem Cells

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.
Bacteria ; Cronobacter ; Diagnosis* ; Escherichia coli ; Lab-On-A-Chip Devices* ; Plastics* ; Polymerase Chain Reaction* ; Polymethyl Methacrylate

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.
Animals ; Axonal Transport ; physiology ; Cerebral Cortex ; cytology ; metabolism ; Drosophila melanogaster ; cytology ; metabolism ; Embryo, Mammalian ; Gene Expression ; Lab-On-A-Chip Devices ; Microscopy, Confocal ; Mitochondria ; metabolism ; ultrastructure ; Motor Neurons ; metabolism ; ultrastructure ; Movement ; Mutation ; Primary Cell Culture ; RNA-Binding Protein FUS ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Software

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.
Coculture Techniques* ; Dental Pulp ; Fibroblasts ; Gene Expression ; Humans* ; Indicators and Reagents ; Lab-On-A-Chip Devices ; Microfluidics* ; Miners ; Osteoblasts ; Periodontal Ligament ; Stem Cells* ; Tooth ; Tooth, Deciduous*