An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitride membrane

The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.     

Automatic transwell assay by an EIS cell chip to monitor cell migration

Here an EIS (electrochemical impedance spectroscopy) biochip to detect cell migration is demonstrated. This biochip has been inspired by a traditional transwell assay/modified Boyden chamber and consists of two compartments separated by a porous membrane. This structure (PDMS-based) is aligned to EIS sensors. Cells are seeded in the upper chamber through microfluidic channels. During migration cells go through the pores of the membrane and get in touch with the electrodes that detect migrated cells. The performance of our cell-chip was tested by investigating the migratory ability of hepatocellular carcinoma (HCC) cells as a function of microenvironment. For this purpose we challenged HCC cells to migrate on different extra-cellular matrix (ECM) components including laminin 1, collagen IV and laminin 5. The results reveal that our cell chip provides reliable results that consistently overlap with those obtained with traditional standardized Boyden chambers. Thus, we demonstrate a new, easy tool to study cell migration and to perform automatic assays. This approach is easier and faster than traditional transwell assays and can be suitable for high-throughput studies in drug discovery applications.     

Co-culture Based Blood-brain Barrier In Vitro Model,a Tissue Engineering Approach using Immortalized Cell Lines for Drug Transport Study

This study evaluated the feasibility of using commercially available immortalized cell lines in building an in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Astrocytes-derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel biomimetic basement membrane for the endothelial cells to form tight junctions. Trans-Endothelial Electrical Resistance (TEER) and solute mass transport studies quantitatively evaluated the tight junction formation. Immuno-fluorescence microscopy and Western blot analysis qualitatively verified the expression of occludin, one of the tight junction proteins on the samples. Experimental data from a total of 13 experiments conclusively showed that the novel BBB in vitro co-culture model with aECM (CO + aECM) is promising in terms of establishing tight junction formation represented by TEER values, transport profiles, and tight junction protein expression when compared with traditional co-culture (CO) model setup or the endothelial cells cultured alone (EC). In vitro colorimetric sulforhodamine B (SRB) assay also revealed that the “CO + aECM” samples resulted in less cell loss on the basal sides of the insert membranes than traditional co-culture models. Our novel approach using immortalized cell lines with the addition of aECM was proven to be a feasible and repeatable alternative to the traditional BBB in vitro modeling.     

High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel

In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.     

Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function

This study describes a novel assay to visualize the macromolecular permeability of epithelial and endothelial cell layers with subcellular lateral resolution. Defects within the cell layer and details about the permeation route of the migrating solute are revealed. The assay is based on silicon chips with densely packed, highly ordered, dead-ended pores of μm-diameters on one side. The cells under study are grown on the porous side of the chip such that the pores in the growth surface serve as an array of femtolitre-sized cuvettes in which the permeating probe accumulates at the site of permeation. The pattern of pore filling reveals the permeability characteristics of the cell layer with a lateral resolution in the μm range. Coating of the chip surface with a thin layer of gold allows for impedance analysis of the adherent cells in order to measure their tightness for inorganic ions at the same time. The new assay provides an unprecedented look on epithelial and endothelial barrier function.     

A parametric study of human fibroblasts culture in a microchannel bioreactor

The culture of cells in a microbioreactor can be highly beneficial for cell biology studies and tissue engineering applications. The present work provides new insights into the relationship between cell growth, cell morphology, perfusion rate, and design parameters in microchannel bioreactors. We demonstrate the long-term culture of mammalian (human foreskin fibroblasts, HFF) cells in a microbioreactor under constant perfusion in a straightforward simple manner. A perfusion system was used to culture human cells for more than two weeks in a plain microchannel (130 microm x 1 mm x 2 cm). At static conditions and at high flow rates (>0.3 ml h(-1)), the cells did not grow in the microchannel for more than a few days. For low flow rates (<0.2 ml h(-1)), the cells grew well and a confluent layer was obtained. We show that the culture of cells in microchannels under perfusion, even at low rates, affects cell growth kinetics as well as cell morphology. The oxygen level in the microchannel was evaluated using a mass transport model and the maximum cell density measured in the microchannel at steady state. The maximum shear stress, which corresponds to the maximum flow rate used for long term culture, was 20 mPa, which is significantly lower than the shear stress cells may endure under physiological conditions. The effect of channel size and cell type on long term cell culture were also examined and were found to be significant. The presented results demonstrate the importance of understanding the relationship between design parameters and cell behavior in microscale culture system, which vary from physiological and traditional culture conditions.     

Construction of Tumor Tissue Array on An Open-Access Microfluidic Chip

An open-access microfluidic chip which enabled automatic cell distribution and complex multi-step operations was developed. The microfluidic chip featured a key structure in which a nanoporous membrane was sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach took advantage of the characteristics of nanoporous membrane. On one side, this membrane permitted the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution. On the other side, it allowed diffusion-based media exchange and thus, mimicked the endothelial layer. In synergy with a liquid transferring platform, the open-access microfluidic system enabled complex multi-step operations involving medium exchange, drug treatment, and cell viability testing. By using this microfluidic protocol, a 10 × 10 tissue arrays was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays results indicated that the resultant tumor tissue was faithful to that in vivo. Drug testing assays showed that the microfluidic tissue array promised multi-step cell assays under biomimetic microenvironment, thus providing an advantageous tool for cell research.     

Integration of intra- and extravasation in one cell-based microfluidic chip for the study of cancer metastasis

Most studies of cancer metastasis focus on cancer cell invasion utilizing adhesion assays that are performed independently, and are thus limited in their ability to mimic complex cancer metastasis on a chip. Here we report the development of an integrated cell-based microfluidic chip for intra- and extravasation that combines two assays on one chip for the study of the complex cascade of cancer metastasis. This device consists of two parts; one is an intravasation chamber for the three-dimensional (3-D) culture of cancer cells using a Matrigel matrix, and the other is an extravasation chamber for the detection of metastasized cancer cells by adhesion molecules expressed by epithelial cells. In this novel system, the intravasation and extravasation processes of cancer metastasis can be studied simultaneously using four screw valves. Metastatic LOVO and non-metastatic SW480 cells were used in this study, and the invasion of LOVOs was found to be higher compared to SW480. In contrast, invasion of cells treated with metalloproteinase (MMP) inhibitors decreased within the intravasation chamber. Degraded cancer cells from the intravasation chamber were detected within the extravasation chamber under physiological conditions of shear stress, and differences in binding efficiency were also detected when CA19-9 antibody, an inhibitor of cancer cell adhesion, was used to treat degraded cancer cells. Our results support the potential usefulness of this new 3D cell-based microfluidic system as a drug screening tool to select targets for the development of new drugs and to verify their effectiveness.     

Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 μl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.     

Characterization of a microfluidic in vitro model of the blood-brain barrier (μBBB)

The blood-brain barrier (BBB), a unique selective barrier for the central nervous system (CNS), hinders the passage of most compounds to the CNS, complicating drug development. Innovative in vitro models of the BBB can provide useful insights into its role in CNS disease progression and drug delivery. Static transwell models lack fluidic shear stress, while the conventional dynamic in vitro BBB lacks a thin dual cell layer interface. To address both limitations, we developed a microfluidic blood-brain barrier (μBBB) which closely mimics the in vivo BBB with a dynamic environment and a comparatively thin culture membrane (10 μm). To test validity of the fabricated BBB model, μBBBs were cultured with b.End3 endothelial cells, both with and without co-cultured C8-D1A astrocytes, and their key properties were tested with optical imaging, trans-endothelial electrical resistance (TEER), and permeability assays. The resultant imaging of ZO-1 revealed clearly expressed tight junctions in b.End3 cells, Live/Dead assays indicated high cell viability, and astrocytic morphology of C8-D1A cells were confirmed by ESEM and GFAP immunostains. By day 3 of endothelial culture, TEER levels typically exceeded 250 Ω cm(2) in μBBB co-cultures, and 25 Ω cm(2) for transwell co-cultures. Instantaneous transient drop in TEER in response to histamine exposure was observed in real-time, followed by recovery, implying stability of the fabricated μBBB model. Resultant permeability coefficients were comparable to previous BBB models, and were significantly increased at higher pH (>10). These results demonstrate that the developed μBBB system is a valid model for some studies of BBB function and drug delivery.     

Electrosynthesis and Microanalysis in Thin Layer: An Electrochemical Pipette for Rapid Electrolysis and Mechanistic Study of Electrochemical Reactions

Electrochemistry represents unique approaches for the promotion and mechanistic study of chemical reactions and has garnered increasing attention in different areas of chemistry. This expansion necessitates the enhancement of the traditional electrochemical cells that are intrinsically constrained by mass transport limitations. Herein, we present an approach for designing an electrochemical cell by limiting the reaction chamber to a thin layer of solution, comparable to the thickness of the diffusion layer. This thin layer electrode (TLE) provides a modular platform to bypass the constraints of traditional electrolysis cells and perform electrolysis reactions in the timescale of electroanalytical techniques. The utility of the TLE for electrosynthetic applications benchmarked using NHPI-mediated electrochemical C−H functionalization. The application of microscale electrolysis for the study of drug metabolites was showcased by elucidating the oxidation pathways of the paracetamol drug. Moreover, hosting a microelectrode in the TLE, was shown to enable real-time probing of the profiles of redox-active components of these rapid electrosynthesis reactions.     

Proliferation of endothelial cells on the plasma-treated segmented-polyurethane surface: attempt of construction of a small caliber hybrid vascular graft and antithrombogenicity

To prepare a porous segmented-polyurethane (SPU) tube, a solution of SPU containing different concentrations of NaCl was coated on a glass rod and the coated SPU was immediately immersed in water. When the surface of the porous SPU, where bovine aortic endothelial cells are not normally capable of adhering and proliferating, was modified by plasma treatment, the proliferation of endothelial cells could be drastically improved. The cells proliferated confluently on the porous SPU surface prepared at low concentrations of NaCl below 10 g per 100 ml, but poorly on the porous surface prepared at high concentrations of NaCl. The construction of a hybrid vascular graft consisting of a porous SPU tube (2 mm in inner diameter, 5 cm in length) and endothelial cells was attempted. The cells cultured on the inner surface of the tube proliferated to confluency everywhere. From an in vitro antithrombogenic evaluation test, which involved the use of human blood, the present hybrid graft can be considered to provide an inert surface against thrombus formation and blood coagulation. Negligible changes in shape of human leukocytes in contact with bovine aortic endothelial cell surface occurred, suggesting that the bovine aortic endothelial cells used are immunologically less active against human blood.     

Synthesis of Migrastatin Analogues as Inhibitors of Tumour Cell Migration: Exploring Structural Change in and on the Macrocyclic Ring

Migrastatin and isomigrastatin analogues have been synthesised in order to contribute to structure–activity studies on tumour cell migration inhibitors. These include macrocycles varying in ring size, functionality and alkene stereochemistry, as well as glucuronides. The synthesis work included application of the Saegusa–Ito reaction for regio‐ and stereoselective unsaturated macroketone formation, diastereoselective Brown allylation to generate 9‐methylmigrastatin analogues and chelation‐induced anomerisation to vary glucuronide configuration. Compounds were tested in vitro against both breast and pancreatic cancer cell lines and inhibition of tumour cell migration was observed in both wound‐healing (scratch) and Boyden chamber assays. One unsaturated macroketone showed low affinity for a range of secondary drug targets, indicating it is at low risk of displaying adverse side effects.     

A palmtop-sized microfluidic cell culture system driven by a miniaturized infusion pump

A palmtop-sized microfluidic cell culture system is presented. The system consists of a microfluidic device and a miniaturized infusion pump that possesses a reservoir of culture medium, an electrical control circuit, and an internal battery. The footprint of the system was downsized to 87 × 57 mm, which is, to the best of our knowledge, the smallest integrated cell culture system. Immortalized human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were cultured in the system. HMEC-1 in the system proliferated at the same speed as cells in a microchannel perfused by a syringe pump and cells in a culture flask. HUVEC in the system oriented along the direction of the fluid flow. Claudin-5, a tight junction protein, was localized along the peripheries of the HUVEC. We expect that the present system is applicable to various cell types as a stand-alone and easy-to-use system for microfluidic bioanalysis.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.     

Oxygen consumption of cell suspension in a poly(dimethylsiloxane) (PDMS) microchannel estimated by scanning electrochemical microscopy

A quantitative analysis of the oxygen concentration profile near a poly(dimethylsiloxane) (PDMS) microfluidic device was performed using scanning electrochemical microscopy (SECM). A microchannel filled with sodium sulfite (Na(2)SO(3)) aqueous solution was imaged by SECM, showing that the oxygen diffusion layer of the PDMS microchannel was observed to be hemicylindrical. Based on a theoretical analysis of the hemicylindrical diffusion layer of the microchannel, the total oxygen mass transfer rates of oxygen to the PDMS microchannel filled with the Na(2)SO(3) solution was calculated to be (4.01 +/- 0.30) x 10(-12) mol s(-1). This is the maximum value of the oxygen transfer rate for this PDMS microchannel device. The oxygen consumption rate increased almost linearly with the logarithm of the concentration of E. coli cells (10(6) approximately 10(8) cells). The respiratory activity for a single E. coli cell was estimated to be approximately 4.31 x 10(-20) mol s(-1) cell(-1).     

A high-throughput dielectrophoresis-based cell electrofusion microfluidic device

A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO? film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (～12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.     

A microfluidic-based device for study of transendothelial invasion of tumor aggregates in realtime

Circulating tumor aggregates exhibit a high metastatic potential and could potentially serve as an important target for cancer therapies. In this study, we developed a microfluidic model that reconstitutes and is representative of the principal components of biological blood vessels, including vessel cavity, endothelium, and perivascular matrix containing chemokines. Using this model, the transendothelial invasion of tumor aggregates can be observed and recorded in realtime. In this study we analyzed the extravasation process of salivary gland adenoid cystic carcinoma (ACC) cell aggregates. ACC aggregates transmigrated across the endothelium under the stimulation of chemokine CXCL12. The endothelial integrity was irreversibly damaged at the site of transendothelial invasion. The transendothelial invasion of ACC aggregates was inhibited by AMD3100, but the adhesion of ACC aggregates to the endothelium was not affected by the CXCR4 antagonist. This model allows for detailed study of the attachment and transendothelial invasion of tumor aggregates; thus, it would be a useful tool for analysis of the underlying mechanisms of metastasis and for testing novel anti-metastasis agents.     

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells(SACC),immunocytochemical staining was employed to detect proliferating cell nuclear antigen(PCNA),caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy(LSCM) was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased(P0.01) after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.