Glenn Walker

A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia.

Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the hosts vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells.

A Review of Automated Microinjection Systems for Single Cells in the Embryogenesis Stage

Modern genetics research has resulted in significant advances in cell in vitro microinjection systems. Such systems provide biological and medical practitioners with high volume cell throughput and statistically relevant data. This paper provides the reader with a comprehensive review of the major research technologies used in automated cell microinjection and of their individual subsystems. Microinjection subsystems reviewed include machine vision, nonvision sensors and user interface (input), cell modeling, piercing mechanisms and injection control loop (control), cell holder and manipulator, and microinjection (output). The interdisciplinary technologies reviewed for microinjection sensing, automation, and control include microfluidic actuation, optical field actuation (optical trapping and optical guidance), electrical field actuation (electrorotation, electrowetting, and dielectrophoresis), and ultrasonic vibration. The survey concludes that research into automated microinjection systems will focus on reducing the scale of microinjection systems and developing appropriate controllers.

Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies.

Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices – RealBio® and QuasiVivo® (QV) – and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental conditions for long-term hepatocyte cell culture using the QV bioreactor. This system facilitated the use of low system volumes coupled with higher flow rates. This design supports cellular respiration by increasing oxygen concentrations in the vicinity of the cells and facilitates long-term kinetic studies of low clearance test compounds. These two goals were achieved while simultaneously keeping the shear stress experienced by the cells within acceptable limits.

Biocompatibility of Tygon® tubing in microfluidic cell culture

Growth of the MDA-MB-231 breast cancer cell line in microfluidic channels was inhibited when culture media was delivered to the channels via microbore Tygon® tubing. Culture media incubated within this tubing also inhibited growth of these cells in conventional 96-well plates. These detrimental effects were not due to depletion of critical nutrients due to adsorption of media components onto the tubing surface. A pH change was also ruled out as a cause. Nuclear magnetic resonance spectroscopy of the cell growth media before and after incubation in the tubing confirmed no detectable loss of media components but did detect the presence of additional unidentified signals in the aliphatic region of the spectrum. These results indicate leaching of a chemical species from microbore Tygon® tubing that can affect cell growth in microfluidic devices.

Microfluidics: Pensioning off pipettes

A microfluidic device design that allows a nanolitre droplet to be trapped and sequentially diluted without the need for any moving parts opens up new possibilities in high-throughput screening.

Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate

Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po2 levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po2 ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions.

Microfluidic Aqueous Two-Phase Systems

Aqueous Two-Phase Systems (ATPS) are an established technology that have been used to separate out biologically important particles such as biomolecules, organelles, and whole cells. ATPS are formed by mixing polymers such as polyethylene glycol (PEG) and dextran (Dex) at sufficiently high concentrations such that two immiscible phases are formed. Traditional macroscale ATPS are performed in test tubes and require relatively large reagent volumes and are limited to a vertical configuration where the interface lies perpendicular to the direction of gravity. Settling becomes problematic for larger particles and the long diffusion distances mean that separations require significant time. Recent advances in microfluidics systems allow novel configurations of ATPS that are impossible with traditional techniques. Examples are nanoliter ATPS droplets or parallel streams of ATPS which enable new applications and improvements over traditional separations. Microscale ATPS can separate particles in seconds instead of hours using only microliters of reagent.

Expanding the introduction of microfluidics through a problem-based laboratory course to multiple engineering disciplines at five universities

Microfluidics is a multidisciplinary field that deals with the behavior and precise control of microliter and nanoliter volumes of fluids. While microfluidics has transformed many areas of engineering and applied sciences, minimal effort has been directed at transferring microfluidics research to the undergraduate curricula. Addressing this need, the University of Cincinnati has developed an undergraduate laboratory course to introduce students to microfluidics device development that is currently being expanded to four other universities with the assistance of an NSF CCLI Phase II grant (DUE-0814911). This course has been taught at University of Cincinnati four times, the University of Illinois at Chicago three times previously and the University of Utah, Louisiana State University, and North Carolina State University once. This paper will discuss course evaluation and the issues encountered in offering the course at five diverse educational institutions. Some of the issues included course structure (quarters vs. semesters), student background knowledge (engineering majors), and domain expertise level (graduate or undergraduate). The initial success of the pilot program and expansion to other universities is encouraging as course materials are adapted and more engineering students are introduced to advanced multidisciplinary research topics using microfluidics.