Paul J. Hung

Microfluidic application-specific integrated device for monitoring direct cell-cell communication via gap junctions between individual cell pairs

Direct cell-cell communication between adjacent cells is vital for the development and regulation of functional tissues. However, current biological techniques are difficult to scale up for high-throughput screening of cell-cell communication in an array format. In order to provide an effective biophysical tool for the analysis of molecular mechanisms of gap junctions that underlie intercellular communication, we have developed a microfluidic device for selective trapping of cell-pairs and simultaneous optical characterizations. Two different cell populations can be brought into membrane contact using an array of trapping channels with a 2μm by 2μm cross section. Device operation was verified by observation of dye transfer between mouse fibroblasts (NIH3T3) placed in membrane contact. Integration with lab-on-a-chip technologies offers promising applications for cell-based analytical tools such as drug screening, clinical diagnostics, and soft-state biophysical devices for the study of gap junction protein...

On-chip cell lysis by local hydroxide generation

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

Microfabricated suspensions for electrical connections on the tunable elastomer membrane

Electrical connections through microfabricated suspensions on a pneumatically pumped elastomer membrane were demonstrated. A method to fabricate the suspensions on the elastomer membrane was developed. The elastomer membrane was 1 mm in diameter and 120 μm in thickness. Resistances of the microfabricated suspensions measured across the elastomer membrane were within 1% difference when the membrane’s center deflection ranged from 0 to 100 μm, which corresponded to a numerical aperture change from 0 to 0.2 as well as a 2.6% elongation of the elastomer.

A microfluidic system for dynamic yeast cell imaging

The investigation of cellular processes and gene regulatory networks within living cells requires the development of improved technology for dynamic, single cell imaging. Here, we demonstrate a microfluidic system capable of mechanical trapping of yeast cells with continuous flow and flow switching capability during time-lapse high magnification fluorescence imaging. The novel functionality of the system was validated by observing the response of pheromone-induced expression of GFP in Saccharomyces cerevisiae.

Microfluidic System for Automated Cell-Based Assays

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher-quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long-term continuous perfusion cell culture using a passive gravity-driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained eight independent flow units, each with 104 cells at a flow rate of 50 μL/day (6 min residence time). The cytotoxicity of the anticancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.

Microfluidic Tissue Model for Live Cell Screening

We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue‐like culture and high‐throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 μm wide cell culture pocket, an artificial endothelial barrier with 2 μm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor‐like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for “flow cell” based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow‐through biochemistry. Each fluidically independent tissue unit contained ∼500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti‐cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384‐well dish for up to 5 days of continuous drug exposure.

Microfluidic systems for live cell imaging.

Microfluidic systems provide many advantages for live cell imaging, including improved cell culture micro-environments, control of flows and dynamic exposure profiles, and compatibility with existing high resolution microscopes. Here, we will discuss our approach for design and engineering of microfluidic cell culture environments as well as interfacing with standard laboratory tools and protocols. We focus on an application specific design concept, whereby a shared fabrication process is used to deliver multiple products for different biological applications. As adoption of advanced in vitro models increases, we envision the use of microfluidic cell culture technology to become commonplace.

Alkaline hemolysis fragility is dependent on cell shape: Results from a morphology tracker

The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration‐driven hemolysis or for the relation between cell shape and fragility.

Microfluidic Hepatotoxicity Platform

Microfluidic cell culture technologies offer the ability to create more relevant in vitro environments for preclinical studies using liver hepatocytes. The liver is the critical organ in detoxifying the body of xenobiotics such as biopharmaceuticals and environmental chemicals. Current in vitro screening using isolated hepatocytes are unable to maintain adequate liver-specific activity and are poor predictors of clinical outcomes. We have developed a microfluidic system capable of recreating key aspects of the physiologic hepatocyte environment to provide long-term primary hepatocyte activity for over 28 days. The microfluidic design maintains cells in defined 3D tissue aggregates with continuous perfusion exposure of nutrients from a set of sinusoid channels. The microfluidic plates are arranged on a standard 96-well plate frame and are compatible with existing assay and automation tools.