Chihchen Chen

Microfluidic isolation and transcriptome analysis of serum microvesicles

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

Gray-scale photolithography using microfluidic photomasks.

The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a “photomask” (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and/or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches.

Enhancing the performance of a point-of-care CD4+ T-cell counting microchip through monocyte depletion for HIV/AIDS diagnostics

CD4+ T cell counts are important tests used to stage HIV-positive patients, enabling clinicians to make informed antiretroviral treatment decisions and to monitor the therapeutic outcomes. However, state-of-the-art CD4 counting methods based on flow cytometry are not applicable in resource-limited settings, due to their high cost and technical requirements. In previous work, we reported the development of a cell isolation microchip that can be used at the point of care for CD4 counts. In that microfluidic chip, CD4+ T cells were separated from 10 microL of whole blood, and enumerated via either light microscopy or impedance sensing. The microchip counts matched flow cytometry results in the intermediate CD4 count range, between 200-800 cells/microL, but displayed a positive bias at absolute CD4 counts below 200 cells/microL, due largely to monocyte contamination. To enhance the performance in the low CD4 count range, we report here an improved design of a two-stage microfluidic device to deplete monocytes from whole blood, followed by CD4+ T cell capture. Using the double-stage device combined with a high viscosity rinsing solution, we obtained microchip CD4 counts comparable to flow cytometry results in the full clinically relevant range. In addition to CD4 counting, the strategy of contaminant depletion prior to target cell isolation can be easily adapted to immunoaffinity capture of other cell types that lack a unique surface marker from a complex biological fluid.

Microvortex for focusing, guiding and sorting of particles

We report a microvortex manipulator (MVM) that is a passive, scalable system with great potential for the manipulation and separation of particulate samples in microfluidic environments. The movement of particles is determined by a unique combination of helical flow, buoyant, and gravitational forces. Helical flows are induced by topographically patterned microchannel surfaces, which have previously been used for molecular mixing in microfluidic devices. We illustrate the mechanism of MVM and its applications in passive focusing of beads and cells into parallel streams and guiding of particles and cells. We also explore the application of the unique density-selectivity of microvortex focusing and successfully sort a mixture of two bead populations whose density difference is as small as 0.1 g cm(-3).

Paper-based devices for isolation and characterization of extracellular vesicles.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

Fast sorting of CD4+ T cells from whole blood using glass microbubbles

The isolation of CD4 positive T lymphocyte (CD4+) from peripheral blood is important for monitoring patients after HIV infection. Here, we demonstrate a fast isolation strategy for CD4+ cells that involves mixing blood and glass microbubbles. After the specific binding of target cells to the microbubbles carrying specific antibodies on their surface, target cells will spontaneously float to the top of the blood vial and can be quickly separated. Using this strategy, we demonstrate that the isolation of CD4+ cells in less than 5 minutes and with better than 90% efficiency. This strategy for cell isolation based on buoyancy and glass microbubbles is quick and inexpensive, minimizes blood handling, does not require magnetic fields, or centrifugation equipment, and could lead to new, efficient strategies for AIDS diagnosis in resource-limited areas.

A PDMS-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation.

The conventional hanging drop technique is the most widely used method for embryoid body (EB) formation. However, this method is labor intensive and limited by the difficulty in exchanging the medium. Here, we report a microfluidic chip-based approach for high-throughput formation of EBs. The device consists of microfluidic channels with 6 × 12 opening wells in PDMS supported by a glass substrate. The PDMS channels were fabricated by replicating polydimethyl-siloxane (PDMS) from SU-8 mold. The droplet formation in the chip was tested with different hydrostatic pressures to obtain optimal operation pressures for the wells with 1000 μm diameter openings. The droplets formed at the opening wells were used to culture mouse embryonic stem cells which could subsequently developed into EBs in the hanging droplets. This device also allows for medium exchange of the hanging droplets making it possible to perform immunochemistry staining and characterize EBs on chip.

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca2+ concentration. However, glucose evoked a rapid elevation of intracellular Ca2+ followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Paper-Based Microfluidic Platforms for Understanding the Role of Exosomes in the Pathogenesis of Major Blindness-Threatening Diseases

Emerging roles of exosomes in the pathogenesis of major blindness-threatening diseases, such as age-related macular degeneration, glaucoma, and corneal dystrophy, were discovered by aqueous humor analysis. A new diagnostic method using cellulose-based devices and microfluidic chip techniques for the isolation of exosomes from aqueous humor is less cumbersome and saves time. This method will enable more investigations for aqueous humor analysis in the future.