A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.     

A digital microfluidic platform for primary cell culture and analysis

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.     

Methods to array photonic crystal microcavities for high throughput high sensitivity biosensing on a silicon-chip based platform

We experimentally demonstrate a method to create large-scale chip-integrated photonic crystal sensor microarrays by combining the optical power splitting characteristics of multi-mode interference (MMI) power splitters and transmission drop resonance characteristics of multiple photonic crystal microcavities arrayed along the length of the same photonic crystal waveguide. L13 photonic crystal microcavities are employed which show high Q values (~9300) in the bio-ambient phosphate buffered saline (PBS) as well as high sensitivity, experimentally demonstrated to ~98 atto-grams. Two different probe antibodies were specifically detected simultaneously with a control sample, in the same experiment.     

A novel electric ion resonance cell design with high signal-to-noise ratio and low distortion for Fourier transform mass spectrometry

Performing wideband ion image current detection mass spectrometry experiments with an electric ion trap—e. g., the Paul trap—is a difficult task, as there is a strong crosstalk current induced by the high voltages of the radio frequency (rf) storage field. In a classic Paul trap the metallic hyperbolic electrodes (a ring electrode and two end cap electrodes) are shaped following the isopotential lines of the quadrupole potential distribution. In our new design the ring electrode is replaced by a cylindrical series of ring electrodes with a parabolic potential distribution, whereas the end cap electrodes are used without modification. Thus the quadrupole field within the trap remains unchanged but the capacitances between the electrodes and therefore the crosstalk currents are significantly reduced. The remaining crosstalk is balanced out by an electronic compensation technique. As a consequence the weak signals of the ion-induced charge can be detected with a wideband low-noise amplifier to perform Fourier transform mass spectrometry experiments with improved signal-to-noise ratio.     

Determination of monoclonal antibody production in cell culture using novel microfluidic and traditional assays

This study compares microfluidic technology (Protein 200 LabChip Assay kit, Agilent 2100 Bioanalyzer, referred to here as Protein 200) to the traditional approach for protein analysis, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), for the sizing and quantification of immunoglobulin G (IgG) in hybridoma cell cultures. Internal references differ between each method: purified IgG was used alone in SDS-PAGE while myosin (the upper marker) was added to each sample in Protein 200. The IgG used here were produced in cultures propagated in either a serum-free or a serum-containing medium. With serum-containing samples, there was a significant difference in the IgG concentrations (p < 0.05) between SDS-PAGE and Protein 200. The concentration determined by SDS-PAGE was significantly higher (> 30%) than by Protein 200 or by high-pressure liquid chromatography (HPLC) because the large amounts of serum albumin in the samples affect the accuracy of SDS-PAGE. Protein 200 can determine size similarly to SDS-PAGE in serum-free samples (standard error of the mean, SEM, < 1%, 95% confidence < +/-1%), unlike in serum-containing samples. The Protein 200 assay was more effective than the traditional one-dimensional SDS-PAGE in determining concentration and size of IgG in cell culture samples and it provided a miniaturized and convenient platform for rapid analysis.     

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L^?1. The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.     

Micro- and nanometer-scale patterned surface in a microchannel for cell culture in microfluidic devices

A novel microdevice which had a micro- and nanometer-scale patterned surface for cell adhesion in a microchip was developed. The surface had a metal pattern fabricated by electron-beam lithography and metal sputtering and a chemical pattern consisting of a self-assembled monolayer of alkanethiol. The metal patterned surface had a gold stripe pattern which was as small as 300 nm wide and 150 nm high and both topography and chemical properties could be controlled. Mouse fibroblast NIH/3T3 cells were cultured on the patterned surface and elongated along the gold stripes. These cells recognized the size of the pattern and the chemical properties on the pattern though it was much smaller than they were. There was satisfactory cell growth under fresh medium flow in the microchip. The combination of the patterned surface and the microchip provides cells with a novel environment for their growth and will facilitate many cellular experiments. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

How to embed three-dimensional flexible electrodes in microfluidic devices for cell culture applications

This communication describes a simple, rapid and cost effective method of embedding a conductive and flexible material within microfluidic devices as a means to realize uniform electric fields within cellular microenvironments. Fluidic channels and electrodes are fabricated by traditional soft-lithography in conjunction with chemical etching of PDMS. Devices can be deformable (thus allowing for a combination of electro-mechanical stimulation), they are made from inexpensive materials and easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets.     

A 96-well microplate incorporating a replica molded microfluidic network integrated with photonic crystal biosensors for high throughput kinetic biomolecular interaction analysis

A nanoreplica molding process has been used to produce polymer microfluidic channels, with integrated label-free photonic crystal biosensors as the bottom surface of the channels. Multiple flow channels are gathered in parallel so that an imaging detection instrument may simultaneously monitor the binding kinetics of many biomolecular interactions. In this work, the flow channel pattern has been adapted to a 96-well microplate format in which, for each 12-element row of the microplate, a single well serves as a common access port for 11 flow channels that are connected to separate microplate wells. Application of pneumatic pressure or suction to the common well serves to drive forward or backward flow to the channels. The system is demonstrated by measuring the kinetic binding interaction of protein A with IgG molecules of high, medium, and low affinity. The approach offers a means for minimizing the volume of reagent required to functionalize the biosensor surface, while retaining compatibility with the microplate assay fluid-handling methods that are most commonly used in biological research.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.     

Pumping-induced perturbation of flow in microfluidic channels and its implications for on-chip cell culture

We study the rate of response to changes in the rate of flow and the perturbations in flow in polydimethylsiloxane (PDMS) microfluidic chips that are subjected to several common flow-control systems. We find that the flow rate of liquid delivered from a syringe pump equipped with a glass syringe responds faster to the changes in the conditions of flow than the same liquid delivered from a plastic syringe; and the rate of flow delivered from compressed air responds faster than that from a glass syringe. We discover that the rate of flow that is driven by a syringe pump and regulated by an integrated pneumatic valve responds even faster, but this flow-control method is characterized by large perturbations. We also examine the possible effects of these large perturbations on NIH 3T3 cells in microfluidic channels and find that they could cause the detachment of NIH 3T3 cells in the microchannels.     

Essential attributes identified in the design of a Laboratory Information Management System for a high throughput siRNA screening laboratory

In recent years high throughput screening operations have become a critical application in functional and translational research. Although a seemingly unmanageable amount of data is generated by these high-throughput, large-scale techniques, through careful planning, an effective Laboratory Information Management System (LIMS) can be developed and implemented in order to streamline all phases of a workflow. Just as important as data mining and analysis procedures at the end of complex processes is the tracking of individual steps of applications that generate such data. Ultimately, the use of a customized LIMS will enable users to extract meaningful results from large datasets while trusting the robustness of their assays. To illustrate the design of a custom LIMS, this practical example is provided to highlight the important aspects of the design of a LIMS to effectively modulate all aspects of an siRNA screening service. This system incorporates inventory management, control of workflow, data handling and interaction with investigators, statisticians and administrators. All these modules are regulated in a synchronous manner within the LIMS.     

Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets

A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM) in tablets. The formation of a colored charge-transfer (CT) complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA) as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml^-1. The limits of detection and quantitation were 0.3 and 1 μg ml^-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.     

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.     

A mass and energy balance to provide microbial growth yield efficiency in soil

The control on the CO₂ coming from soil handling, makes necessary the introduction of new methodologies that inform about the capacity of the soil as a carbon sink and about the carbon decay. It can be performed through the microbial growth yield efficiency concept by calorimetry and enthalpy balances. Here it is examined the sensitivity of these indicators to two metal layering phosphates, AZP [(NH)₄Zn₂(PO)₄(HPO)₄] and AIP [(NH)₄Fe(PO)₄H₂O] to assess about their soil impact. Both compounds caused metabolic changes on soil microbial biomass when compared to appropriated references indicating that the proposed methodology is sensitive to different inorganic sources of microbial growth.     

A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening

We developed a novel fluorescent bioprobe (SF44) that can specifically visualize the cellular lipid droplets in in vitro and in vivo systems and illustrated the mechanistic rationale of its fluorogenic property. Its application to image-based high throughput screening led us to the identification of a new small-molecule modulator of lipid droplet formation.     

A modular single-cell pipette microfluidic chip coupling to ETAAS and ICP-MS for single cell analysis

Accurate single-cell capture is a crucial step for single cell biological and chemical analysis. Conventional single-cell capturing often confront operational complexity, limited efficiency, cell damage, large scale but low accuracy, incompetence in the acquirement of nano-upgraded single-cell liquid. Flow cytometry has been widely used in large-scale single-cell detection, while precise single-cell isolation relies on both a precision operating platform and a microscope, which is not only extre...