Gelatin-based cell culture device for construction and X-ray irradiation of a three-dimensional oral cancer model.

Bioassays using three-dimensional (3D) tissue models offer several advantages over 2D culture assays because they can reproduce the structure and function of native tissues. In this study, we used our newly designed gelatin device to generate a miniature 3D model of human oral squamous cell carcinoma with stroma and blood vessels. To enable air-liquid interface culture, we conceived a new device structure in which three wells were lined up and separated by a dividing thread; the wells could be connected by removing the dividing thread. Cells were seeded in the center well with the dividing thread to form a multilayer, followed by the supply of media from the side wells after thread removal. Human oral squamous cell carcinoma (HSC-4) cells, human umbilical vein endothelial cells (HUVECs), and normal human dermal fibroblasts (NHDFs) were successfully cocultured, resulting in structures that mimicked 3D-cancer tissues. This 3D-cancer model was subjected to an X-ray sensitivity assay, followed by the evaluation of DNA damage using confocal microscopy and section-scanning electron microscopy.

Fabrication of a Gelatin-Based Microdevice for Vascular Cell Culture.

This study presents a novel technique for fabricating microfluidic devices with microbial transglutaminase-gelatin gels instead of polydimethylsiloxane (PDMS), in which flow culture simulates blood flow and a capillary network is incorporated for assays of vascular permeability or angiogenesis. We developed a gelatin-based device with a coverslip as the bottom, which allows the use of high-magnification lenses with short working distances, and we observed the differences in cell dynamics on gelatin, glass, and PDMS surfaces. The tubes of the gelatin microfluidic channel are designed to be difficult to pull out of the inlet hole, making sample introduction easy, and the gelatin channel can be manipulated from the cell introduction to the flow culture steps in a manner comparable to that of a typical PDMS channel. Human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) were successfully co-cultured, resulting in structures that mimicked blood vessels with inner diameters ranging from 10 µm to 500 µm. Immunostaining and scanning electron microscopy results showed that the affinity of fibronectin for gelatin was stronger than that for glass or PDMS, making gelatin a suitable substrate for cell adhesion. The ability for microscopic observation at high magnification and the ease of sample introduction make this device easier to use than conventional gelatin microfluidics, and the above-mentioned small modifications in the device structure are important points that improve its convenience as a cell assay device.

Bead-based Padlock Rolling Circle Amplification under Molecular Crowding Conditions: The Effects of Crowder Charge and Size.

Bead-based padlock rolling circle amplification under molecular crowding conditions, which we have developed for ultrasensitive detection of DNA, is examined to improve the detection efficiency and sensitivity of the method as well as to gain insight into the mechanism of the method. Both non-magnetic and magnetic sepharose microbeads were employed. Biotinylated DNA had to be pre-immobilized onto the microbeads in order to obtain products on the magnetic beads. The optimal concentration of biotinylated DNA was found to be about 5 µM, above which the number of products decreased. The effect of the crowder charge was examined, and neutral polymers were found to be effective on ligation and the hybridization step, while charged polymers were only effective on the hybridization step and inhibited the ligation and primer extension. The effect of the molecular weight of neutral dextran on the number of products was investigated, and the number of products was found to be increased with an increase in the molecular weight of dextran.

Nitric Oxide and a Conditioned Medium Affect the Hematopoietic Development in a Microfluidic Mouse Embryonic Stem Cell/OP9 Co-Cultivation System.

A microfluidic co-culture system, consisting of mouse embryonic stem cells (mESCs)/OP9 cells, was evaluated as a platform for studying hematopoietic differentiation mechanisms in vitro. mESC differentiation into blood cells was achieved in a microchannel that had the minimum size necessary to culture cells. The number of generated blood cells increased or decreased based on the nitric oxide (NO) donor or inhibitor used. Conditioned medium from OP9 cell cultures also promoted an increase in the number of blood cells. The number of generated blood cells under normal medium flow conditions was lower than that observed under the static condition. However, when using a conditioned medium, the number of generated blood cells under flow conditions was the same as that observed under the static condition. We conclude that secreted molecules from OP9 cells have a large influence on the differentiation of mESCs into blood cells. This is the first report of a microfluidic mESC/OP9 co-culture system that can contribute to highly detailed hematopoietic research studies by mimicking the cellular environment.

Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions.

We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.

Characterizing the non-crosslinked aggregation of DNA-modified gold nanoparticles: effects of DNA length and terminal base pair.

We previously reported that fully complementary DNA duplexes formed on gold nanoparticle (GNP) surfaces aggregate at high salt concentrations. We previously reported that DNA-functionalized gold nanoparticles (GNPs) aggregate by hybridization with fully complementary DNA at high salt concentrations. Although this behavior has been applied to some precise naked-eye colorimetric analyses of DNA-related molecules, the aggregation mechanism is still unclear and comprehensive studies are needed. In this paper, we reveal the key factors that influence GNP aggregation. The effects of temperature, electrolyte concentration, probe length, and particle size, which control the stabilities of double-stranded DNAs and GNPs, were investigated. Larger GNPs aggregated more easily, and GNP aggregates were easily formed with ∼15-mer-long probes, while longer probes prevented aggregation, perhaps by preventing the formation of rigid double-stranded DNA layers, compared to shorter probes. Furthermore, GNPs with purine bases at their 5' ends aggregated more easily than those with these bases at their 3' ends. This phenomenon is different from that based on the melting-temperature trend calculated using the nearest-neighbor method.

Pancreatic stellate cells derived from human pancreatic cancer demonstrate aberrant SPARC-dependent ECM remodeling in 3D engineered fibrotic tissue of clinically relevant thickness.

Desmoplasia is a hallmark of pancreatic cancer and consists of fibrotic cells and secreted extracellular matrix (ECM) components. Various in vitro three-dimensional (3D) models of desmoplasia have been reported, but little is known about the relevant thickness of the engineered fibrotic tissue. We thus measured the thickness of fibrotic tissue in human pancreatic cancer, as defined by the distance from the blood vessel wall to tumor cells. We then generated a 3D fibrosis model with a thickness reaching the clinically observed range using pancreatic stellate cells (PSCs), the main cellular constituent of pancreatic cancer desmoplasia. Using this model, we found that Collagen fiber deposition was increased and Fibronectin fibril orientation drastically remodeled by PSCs, but not normal fibroblasts, in a manner dependent on Transforming Growth Factor (TGF)-ß/Rho-Associated Kinase (ROCK) signaling and Matrix Metalloproteinase (MMP) activity. Finally, by targeting Secreted Protein, Acidic and Rich in Cysteine (SPARC) by siRNA, we found that SPARC expression in PSCs was necessary for ECM remodeling. Taken together, we developed a 3D fibrosis model of pancreatic cancer with a clinically relevant thickness and observed aberrant SPARC-dependent ECM remodeling in cancer-derived PSCs.

Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel.

Microfluidic devices have emerged as a new cell culture tool, which can mimic the structure and physiology of living human organs. However, no standardized culture method for a microfluidic device has yet been established. Here, we describe the effects of various conditions on cell proliferation in a microchannel with a depth smaller than 100 µm. Primary endothelial cell proliferation was suppressed with a decrease in the culture medium volume per cell culture area. Moreover, cell growth was compared with or without medium flow, and the optimum culture condition was determined to be 1 µL/h flow in a 65-µm-deep microchannel. In addition, glucose consumption was greater under fluidic conditions than under static conditions, and the ability of tumor (HeLa) cells to convert glucose into lactate appeared to be higher in a static culture than that in a fluidic culture. Overall, our results will serve as a useful guide for designing a microfluidic cell culture platform in a channel smaller than 100 µm.

Effects of Microchannel Shape and Ultrasonic Mixing on Microfluidic Padlock Probe Rolling Circle Amplification (RCA) Reactions.

The fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we aimed to improve the efficiency of padlock/RCA by determining the effects of microchannel shape and ultrasonic solution mixing. Using a circular-shaped microchamber and ultrasonic mixing, the efficiency of microfluidic padlock/RCA was improved, and the consumption of the expensive probe solution was reduced from 10 µL to approximately 3.5 µL. Moreover, the fluorescent probe hybridization time was reduced to 5 min, which is four times faster than that of the standard protocol. We used this method to successfully detect mitochondrial DNA and transcripts of ß-actin and K-ras proto-oncogene codon 12 in cells. Our method offers improvements over current padlock/RCA methods and will be helpful in optimizing other microfluidics-based FISH-related analyses.

Recent Progress in the Development of Microfluidic Vascular Models.

The blood vessel is part of the circulatory system, and systemic circulation provides the blood supply to all tissues. Arteries are pathways through which the blood is carried, and the capillaries have a key role in material exchange to maintain the tissue environment. Blood vessels have structures appropriate for their functions, and their sizes and cell types are different. In this review, we introduced recent studies of the microfluidic vascular models. The model structures are classified mainly as poly(dimethylsiloxane) and hydrogel microchannels and self-assembled networks. Basic phenomena and functions were realized in vascular models, including fluid shear stress, cell strain, interstitial flow, endothelial permeation, angiogenesis, and thrombosis. In some models, endothelial cells were co-cultured with smooth muscle cells, pericytes, and fibroblasts in an extracellular matrix. Examples of vascular models involving the brain, lung, liver, kidney, placenta, and cancer were also introduced.

Development of a Simple Permeability Assay Method for Snake Venom-induced Vascular Damage.

We have developed a novel bioassay method for the detection of snake venom based on the permeability of endothelial cell monolayers cultured in Transwell cell culture inserts. This assay relies on the proteolytic degradation of capillary basement membrane proteins, a pathophysiological event that occurs due to snakebites in vivo. Transwell permeability assays with fluorescence measurements are advantageous with regard to ethical considerations for the use of animals. The assay time was reduced from 24 h for animal tests to 2 h, and many samples could be assayed easily.

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face.

Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.

Molecular crowding improves bead-based padlock rolling circle amplification.

Bead-based padlock rolling circle amplification (RCA), an ultrasensitive and accurate DNA detection technique, was conducted in a molecular crowding environment created by poly(ethylene glycol) (PEG). The number of RCA products generated increased and exhibited a bell-shaped dependence on PEG concentration. Experiments using magnetic beads suggested that facilitation of DNA ligation and hybridization is the main reason for the observed increase. Selectivity of the technique was retained in the presence of PEG. This technique is simple and can be utilized to detect target DNA with high accuracy and sensitivity in a variety of areas such as medical diagnosis and food analysis.

A Membrane-integrated Microfluidic Device to Study Permeation of Nanoparticles through Straight Micropores toward Rational Design of Nanomedicines.

Nanoparticles have been widely utilized to deliver drugs from blood vessels to target tissues. A crucial issue concerning nanoparticle-based drug delivery is to discuss the relationship between experimentally-obtained permeability and physical parameters. Although nanoparticles can permeate vascular pores, because the size and shape of the pores are essentially non-uniform, conventional animal testing and recent cell-based microfluidic devices are unable to precisely evaluate the effects of physical parameters (e.g. pore size and nanoparticle size) on permeation. In this study, we present a membrane-integrated microfluidic device to study permeation of nanoparticles through straight micropores. Porous membranes possessing uniform straight pores were utilized. The effects of pore size and pressure difference across the pores on nanoparticle permeation were examined. The experimentally determined permeability coefficient of 1.0 µm-pore membrane against 100 nm-diameter nanoparticles agreed well with the theoretical value obtained for convectional permeation. Our method can be utilized to clarify the relationship between the experimentally-obtained permeability and physical parameters, and will help rational design of nanomedicines.

Fluidic Culture and Analysis of Pulmonary Artery Smooth Muscle Cells for the Study of Pulmonary Hypertension.

There is an urgent need to develop novel in-vitro models to mimic the disease conditions in pulmonary hypertension (PH). We developed a microfluidic cell culture device for PH studies that withstood high shear stress. Techniques were also developed for cell recovery from the microchannel and mRNA isolation from the collected cells. Using this device, we found that shear stress caused a 7.5-fold increase in the transcription levels of a PH-related molecule, Cyclin D1.

Patterned Co-culture of Live Cells on a Microchip by Photocrosslinking with Benzophenone.

The patterned coculture of different types of living cells in a microfluidic device is crucial for the analysis of cellular interactions and cell-cell communication. In the present study, cell patterning was achieved by photocrosslinking benzophenone derivatives in a microfluidic channel. Optimization of UV irradiation conditions enabled successful fixation of live cells. In addition, patterning and co-culture of non-adherent K562 cells and adherent RF-6A cells was achieved by successive rounds of patterning. The present approach is expected to be useful for the development of in vitro methods for studying cell signaling.

Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability.

We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate.

Microdevice in Cellular Pathology: Microfluidic Platforms for Fluorescence in situ Hybridization and Analysis of Circulating Tumor Cells.

Microfluidic devices enable the miniaturization, integration, automation, and parallelization of chemical and biochemical processes. This new technology also provides opportunity for expansion in the field of cellular pathology. Fluorescence in situ hybridization (FISH) is a well-known gene-based method to image genetic abnormalities. Development of a FISH microfluidic platform has offered the possibility of automation with significant time and cost reductions, which overcomes many drawbacks of the current protocols. Microfluidic devices are also powerful tools for single-cell analysis. Capturing the circulating tumor cells (CTCs) from blood samples is one of the most promising approaches to enable the early diagnosis of cancer. The microfluidic devices are also useful to isolate rare CTCs at high efficiency and purity. In this review, I outline recent FISH and CTC analyses using microfluidic devices, and describe their applications for the cellular diagnosis of cancers.

Alternating current cloud point extraction on a microchip: the effect of electrode geometry.

We report on the effect of electrode geometry on alternating current cloud point extraction (ACPE). ACPE is a technique utilized to extract membrane-associated biomolecules in an electrode-integrated microfluidic channel. In this study, we investigated the effect of gap size (4∼22 µm) between microband electrodes on ACPE. A decrease in gap size resulted in efficient and rapid concentration of fluorescent-labeled phospholipids, a model of membrane-associated biomolecules. We also investigated the effect of applied voltage amplitude on ACPE using devices with decreased electrode gap size. When the gap was small, ACPE was achieved with low applied voltages. ACPE of membrane proteins extracted from HeLa cells was also studied to demonstrate the applicability of the ACPE to real samples. The results provide a guideline to improve the performance of ACPE and facilitate application of the ACPE technique as part of an overall analytical process.

Microfluidics-based in situ padlock/rolling circle amplification system for counting single DNA molecules in a cell.

In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondrial DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 µL, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.