The combination of optical tweezers and microwell array for cells physical manipulation and localization in microfluidic device

A microfluidic device combined with the microwell array and optical tweezers was set up for cell manipulation, localization and cultivation. Yeast cells were manipulated by a 1,064 nm laser and transferred to microwell array as a demonstration. The flow velocities at which the yeast cell can be confined in microwells of different sizes are charactered. The simulation of the cell’s flow trace in the microwell at different flow velocities is consisting with our experiment result. And we also proved a trapping laser power of 0.30 W is harmless for yeast cell cultivation. As a simple approach, this method can push forward the cell cultivation, cell interaction and other cell biology or biomedical studies in microfluidic system.     

Preparation of monodispersed chitosan microspheres and in situ encapsulation of BSA in a co-axial microfluidic device

This work describes a novel microfluidic method to prepare monodispersed chitosan microspheres by using the solvent extraction method. Our strategy is that a chitosan/acetic acid aqueous solution is emulsified in an organic phase containing the extractant by using the co-flowing shear method in a co-axial microfluidic device. The formed droplets are in situ solidified within a synthesizing channel by the extraction of acetic acid from the chitosan aqueous droplets to the organic solution. Based on this approach, the size of chitosan microspheres can be successfully controlled from 100 μm to 700 μm in diameter with a variation of less than 4%. Furthermore, high loading efficiency (>95%) of Bovine serum albumin (BSA) can be in situ encapsulated. The present method has the advantages of actively controlling the droplet diameter, narrow size distribution, good sphericity, and having a simple and low cost process, with a high throughput. This approach for the preparation of chitosan microspheres will provide many potential applications for pharmaceutical area.     

Simultaneous detection of five indices of hepatitis B based on an integrated automatic microfluidic device

Immunophenotyping evaluation is of particular importance for the clinical diagnosis, therapy, and prognosis of viral hepatitis. In this study, an integrated micro flow device has been developed to detect the differentiated antigens/antibodies for immunophenotyping of viral hepatitis. The sensors were fabricated with plasma-polymerized ethylenediamine film (PPF) and nanometer-sized gold particles (nanogold) on which the different hepatitis B antigens/antibodies (markers) were subsequently immobilized. Monitoring the changes in the potential signals before and after the antigen-antibody interaction provides the basis for an immunoassay that is simple, rapid, and cost-effective. It permits the detection of hepatitis B in the dynamic concentration range of 2 orders of magnitude (10(-6) g x L(-1) - 10(-4) g x L(-1)). Up to 7 successive assay cycles with retentive sensitivity were achieved for the sensors regenerated by 8 M urea. Moreover, the microfluidic device was applied to evaluate a number of practical specimens with analytical results in acceptable agreement with those clinically classified. The newly proposed multiparameter analysis technique provides a feasible alternative tool for the diagnosis and monitoring of hepatitis B.     

Multicompartmented microfluidic device for characterization of dose-dependent cadmium cytotoxicity in BALB/3T3 fibroblast cells

This paper describes the development of a miniaturized multicompartmented microfluidic device for high-throughput cell cytotoxicity assays and its applicability to the investigation of cadmium-induced cytotoxicity. A steady gradient of cadmium was generated inside the compartments to study the effects of cadmium ion on BALB/3T3 fibroblast cells in a dose-dependent fashion. The device allowed the performance of multiplexed assays to probe the dosage effect of cadmium, morphological alterations of live cells, regulation of proliferation and viability of cells, determination of reactive oxygen species, mechanisms of cell death, i.e. apoptosis and/or necrosis, and immunocytochemical staining of cells in parallel and/or serially, or on a single population simultaneously. The outcomes of all the microfluidic assays were compared to conventional plates-based cytotoxicity assays. The results indicated that the cells cultured in this device were morphologically healthy with greater than 90% viability. They further suggested that the basic mode of cell death behind cadmium-induced cytotoxicity was apoptosis, which was regulated by intracellular oxidative stress via cytoskeleton disorganization and nuclear condensation. Such microenvironments resemble the in vivo physiological conditions very closely and thus offer a unique platform for more accurate observations of cytotoxicity assays and more precise estimation of the IC₅₀ value in comparison to conventional analytical assays. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

External force-assisted cell positioning inside microfluidic devices

This paper describes straightforward approaches to positioning cells within microfluidic devices that can be implemented without special equipment or fabrication steps. External forces can effectively transport and position cells in preferred locations inside microfluidic channels. Except for centrifugal force-based positioning that can be used with any microfluidic channels, hydrodynamic and gravitational force-based positioning yield reproducible and biocompatible results when implemented with a microfluidic “module” that contains a barrier with embedded microgrooves. Primary rat cortical neurons, metastatic human breast cancer cells MDA-MB-231, NIH 3T3 mouse fibroblasts, and human umbilical vein endothelial cells (HUVECs) were compatible with the positioning processes. After positioning, cells attached, proliferated and migrated like control cells that were cultured on tissue culture dishes or glass coverslips. No apparent morphological differences were observed in positioned cells compared with control cells. Finally, to demonstrate a practical application of the methods, cells were placed in a single row along a wall inside a microfluidic chemotaxis chamber (MCC), and were exposed to stable concentration gradient of chemoattractant. Cell positioning allows that all cells get exposed to the same level of chemoattractant at the start of the experiment helping standardize cellular response.     

Generation of monodisperse alginate microbeads and <Emphasis Type="Italic">in situ</Emphasis> encapsulation of cell in microfluidic device

A microfluidic method for the in situ production of monodispersed alginate hydrogels using chaotic mixing is described. Aqueous droplets comprising of alginate and calcium as a cross-linking agent were formed as an immiscible continuous phase, and then the alginate and calcium in the droplet came into contact and were rapidly mixed. Gelation of the hydrogel was achieved in situ by the chaotic mixing of the droplets in the microfluidic device. Important operating parameters included: the capillary number (Ca) and the flow rate of the continuous phase, which mainly influenced the formation of three distinctive flow regimes, such as fluctuation, stable droplets, and laminar flow. Under the stable formation of droplets regime, monodispersed alginate microbeads having a narrow size distribution (below 3% of CV) were produced in the microfluidic device and the size of the microbeads, ranging from 60 to 95 μm, could be easily modulated by varying the flow rate, viscosity, and interfacial tension. In addition, this approach can be applied to the encapsulation of yeast cells in alginate hydrogels with a high monodispersity. This simple microfluidic technique for the production of monodispersed hydrogels and encapsulation of biomolecules shows strong potential for use in biosensors, cell sensors, drug delivery systems, and cell transplantation applications.     

Sperm motion in a microfluidic fertilization device

Microfluidics has shown promise as a new platform for assisted reproduction. To assess the potential of microfluidics for fertilization, we studied sperm and fluid motion in microchannels to better understand the flow characteristics in a microfluidic device, how sperm interacted with this flow, and how sperm-oocyte attachment occurs in the device. There is a threshold fluid velocity where sperm transition from traveling with the fluid to a regime in which the sperm can move independently of the flow. A significant population of sperm remained in the inlet well area. Based on the lack of progressive forward movement, it was presumed that these sperm may have defects. Also of extreme interest was the tendency of sperm to travel along surface contours. These observations provide an improved understanding of sperm motion in microchannels and provide a basis for improved device designs that take advantage of the sperm/flow and sperm/geometry interactions.     

A model for predicting magnetic particle capture in a microfluidic bioseparator

A model is presented for predicting the capture of magnetic micro/nano-particles in a bioseparation microsystem. This bioseparator consists of an array of conductive elements embedded beneath a rectangular microfluidic channel. The magnetic particles are introduced into the microchannel in solution, and are attracted and held by the magnetic force produced by the energized elements. Analytical expressions are obtained for the dominant magnetic and fluidic forces on the particles as they move through the microchannel. These expressions are included in the equations of motion, which are solved numerically to predict particle trajectories and capture time. This model is well-suited for parametric analysis of particle capture taking into account variations in particle size, material properties, applied current, microchannel dimensions, fluid properties, and flow velocity.     

Laboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25 × 10² and 2.40 × 10⁴ plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4–5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.     

Dye Dilution Proliferation Assay: Application of the DDPA to Identify Tumor-Specific T Cell Precursor Frequencies in Clinical Trials

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8⁺ cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8⁺ lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8⁺ as well as CD4⁺ lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8⁺ and CD4⁺ precursor frequency (PF) data in patients being treated on novel immunotherapy trials.     

Dye Dilution Proliferation Assay: Application of the DDPA to Identify Tumor-Specific T Cell Precursor Frequencies in Clinical Trials

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8⁺ cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8⁺ lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8⁺ as well as CD4⁺ lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8⁺ and CD4⁺ precursor frequency (PF) data in patients being treated on novel immunotherapy trials.