Tim C. Chang

3D-printed microfluidic automation

Microfluidic automation - the automated routing, dispensing, mixing, and/or separation of fluids through microchannels - generally remains a slowly-spreading technology because device fabrication requires sophisticated facilities and the technologys use demands expert operators. Integrating microfluidic automation in devices has involved specialized multi-layering and bonding approaches. Stereolithography is an assembly-free, 3D-printing technique that is emerging as an efficient alternative for rapid prototyping of biomedical devices. Here we describe fluidic valves and pumps that can be stereolithographically printed in optically-clear, biocompatible plastic and integrated within microfluidic devices at low cost. User-friendly fluid automation devices can be printed and used by non-engineers as replacement for costly robotic pipettors or tedious manual pipetting. Engineers can manipulate the designs as digital modules into new devices of expanded functionality. Printing these devices only requires the digital file and electronic access to a printer.

Parallel microfluidic chemosensitivity testing on individual slice cultures

There is a critical unmet need to tailor chemotherapies to individual patients. Personalized approaches could lower treatment toxicity, improve the patients quality of life, and ultimately reduce mortality. However, existing models of drug activity (based on tumor cells in culture or animal models) cannot accurately predict how drugs act in patients in time to inform the best possible treatment. Here we demonstrate a microfluidic device that integrates live slice cultures with an intuitive multiwell platform that allows for exposing the slices to multiple compounds at once or in sequence. We demonstrate the response of live mouse brain slices to a range of drug doses in parallel. Drug response is measured by imaging of markers for cell apoptosis and for cell death. The platform has the potential to allow for identifying the subset of therapies of greatest potential value to individual patients, on a timescale rapid enough to guide therapeutic decision-making.

Indium-tin oxide coated microfabricated device for the injection of a single cell into a fused silica capillary for chemical cytometry

A microfabricated device is described for the capture and injection of a single mammalian cell into a fused silica capillary for subsequent analysis by chemical cytometry. The device consists of a 500 μm diameter well made from polydimethylsiloxane on an indium-tin oxide coated microscope slide. The bottom of the well contains a 2 μm high aperture, which was designed to block passage of cells. A cellular suspension was allowed to settle on the device, and aspiration through the aperture was used to trap a single NG-108 cell. Untrapped cells were washed from the device, and a 150 μm outer diameter and 50 μm inner diameter capillary was placed in the well. To inject a cell, voltage was applied to the indium-tin oxide while simultaneously applying vacuum at the distal end of the capillary.

Microwell arrays reveal cellular heterogeneity during the clonal expansion of transformed human cells.

We developed micromolded microwell arrays to study the proliferation and senescence of single cells. Microwell arrays were designed to be compatible with conventional cell culture protocols to simplify cell loading, cell culture, and imaging. We demonstrated the utility of these arrays by measuring the proliferation and senescence of isogenic cells which expressed or had been depleted of the human Werner syndrome protein. Our results allowed us to reveal cell-to-cell heterogeneity in proliferation in WRN+ and WRN-depleted fibroblasts during clonal growth.

Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRads ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.

An open-chamber flow-focusing device for focal stimulation of micropatterned cells

Microfluidic devices can deliver soluble factors to cell and tissue culture microenvironments with precise spatiotemporal control. However, enclosed microfluidic environments often have drawbacks such as the need for continuous culture medium perfusion which limits the duration of experiments, incongruity between microculture and macroculture, difficulty in introducing cells and tissues, and high shear stress on cells. Here, we present an open-chamber microfluidic device that delivers hydrodynamically focused streams of soluble reagents to cells over long time periods (i.e., several hours). We demonstrate the advantage of the open chamber by using conventional cell culture techniques to induce the differentiation of myoblasts into myotubes, a process that occurs in 7-10 days and is difficult to achieve in closed chamber microfluidic devices. By controlling the flow rates and altering the device geometry, we produced sharp focal streams with widths ranging from 36 μm to 187 μm. The focal streams were reproducible (∼12% variation between units) and stable (∼20% increase in stream width over 10 h of operation). Furthermore, we integrated trenches for micropatterning myoblasts and microtraps for confining single primary myofibers into the device. We demonstrate with finite element method (FEM) simulations that shear stresses within the cell trench are well below values known to be deleterious to cells, while local concentrations are maintained at ∼22% of the input concentration. Finally, we demonstrated focused delivery of cytoplasmic and nuclear dyes to micropatterned myoblasts and myofibers. The open-chamber microfluidic flow-focusing concept combined with micropatterning may be generalized to other microfluidic applications that require stringent long-term cell culture conditions.