Philip J. Lee

Microfluidics-based systems biology

Systems biology seeks to develop a complete understanding of cellular mechanisms by studying the functions of intra- and inter-cellular molecular interactions that trigger and coordinate cellular events. However, the complexity of biological systems causes accurate and precise systems biology experimentation to be a difficult task. Most biological experimentation focuses on highly detailed investigation of a single signaling mechanism, which lacks the throughput necessary to reconstruct the entirety of the biological system, while high-throughput testing often lacks the fidelity and detail necessary to fully comprehend the mechanisms of signal propagation. Systems biology experimentation, however, can benefit greatly from the progress in the development of microfluidic devices. Microfluidics provides the opportunity to study cells effectively on both a single- and multi-cellular level with high-resolution and localized application of experimental conditions with biomimetic physiological conditions. Additionally, the ability to massively array devices on a chip opens the door for high-throughput, high fidelity experimentation to aid in accurate and precise unraveling of the intertwined signaling systems that compose the inner workings of the cell.

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...

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.

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.