November 15, 2011 (Vol. 31, No. 20)

Techniques Evolve to Focus on Cells, Tissues, and Whole Organisms

Microfluidics technology development is entering a new phase, according to Andreas Manz, Ph.D., head of research at the Korea Institute of Science and Technology, Saarbrücken, Germany. Now that the initial patents covering the fundamental technology have expired, companies can more easily and cost effectively acquire, develop, and apply microfluidics without the risk of intellectual property infringement. Recent patents have focused on more specialized technology.

Examples of growing industry interest and activity in the microfluidics field include Bio-Rad Laboratories’ recent acquisition of the microfluidic technology startup QuantaLife for $162 million. QuantaLife brought to market its droplet digital PCR (ddPCR) genetic analysis platform.

Sony acquired Micronics at the end of September, which develops point-of-care diagnostic devices for use in disease detection, treatment monitoring, and blood testing. And just weeks earlier, PerkinElmer announced that it planned to acquire Caliper Life Sciences for $600 million. Caliper develops microfluidic-based molecular imaging and detection technologies for the life science research, diagnostics, and environmental markets.

Dr. Manz has been working with microfluidics technology since the early 1990s, developing miniaturized capillary electrophoresis, liquid chromatography, and PCR devices, for example, to achieve higher-throughput chemical analysis, as well as advancing the development of multilayer chip devices for the study of biochemical activity at the single-cell level.

Microfluidics applications in drug discovery have typically focused on miniaturization of bioassays traditionally done in microwell plates to support a variety of DNA-based genomic or proteomic studies. Dr. Manz believes there is growing interest and research on the use of microfluidic devices for cell-based studies.

In contrast to the molecular diagnostics arena—in which “I have seen little that is revolutionary about microfluidics in the sense of obtaining fundamentally new information,” Dr. Manz points to new work from the fields of cell biology and tissue engineering.

The ability to mimic the three-dimensional cellular environment on a microchip and to be able to study rapid cell-signaling events, cell biology, and the changes that may result from exposure to pathogenic stimuli or therapeutic compounds at the tissue or single-cell level, could generate new types of information that have not been obtainable with existing assay strategies.

For diagnostic applications, and particularly diagnostic devices that could be used in Third World regions, field settings, or for emergency response such as in pandemics, Dr. Manz sees a shift away from traditional microfluidics and toward portable, reagent-free, miniaturized equipment that is not chip-based and does not rely on droplet technology.

The emphasis at present is on developing simpler, highly automated devices with small footprints, in which sample loading through data acquisition is achieved in a hands-free manner. He describes, for example, advances in technology such as paper microfluidics, which uses paper-like materials that can be structured and modified to control properties such as porosity, flow, and hydrophobicity.

Dr. Manz describes a fundamental “limitation” of microfluidics technology, which is driven by the commercial interests of instrument manufacturers. At the same time, this limitation is one of the key advantages of microfluidics: its mass-transport properties allow for high-throughput chemical analysis on the surface of a chip—theoretically, throughput up to millions of assays/second.

However, companies that have developed the instruments that are designed to perform microplate-based assays have not embraced the microfluidics technology being developed in academia and are not likely to replace their well-established product lines, in Dr. Manz’ view.

Consider the market for PCR technology, for example. While PCR has continued to advance, the basic approach for performing PCR has not changed substantially, and the cost of the basic instrumentation needed to perform PCR has remained relatively stable, Dr. Manz notes.

Other factors inhibiting broader applicability of microfluidics in bioanalytics is the lack of end-to-end automation, the need for training to achieve optimal results using current microfluidics techniques and devices, the relative complexity of the systems, and the challenges involved in integrating multiple single-function chips into a multifunctional microfluidic system.

Scientists at KIST in Germany are working on a miniaturized PCR system for infectious disease detection based on a virtual reaction chamber (VRC). Four VRCs are shown on a microscope cover slip.

Ali Khademhosseini, Ph.D., associate professor of medicine, Harvard Medical School, is developing microscale and nanoscale technologies for the purpose of generating tissue-engineered organs and controlling cell behavior. His laboratory has developed platforms for gradient microfluidics, microassay microfluidics, and stem cell microfluidics for high-throughput screening of stem cell microenvironments.

Applying microfabrication to biology to develop microscale devices in which stem cells can be manipulated “allows us to create more biomimetic systems that mimic the natural cell environment or the pathological state of a tissue in a very controlled way,” he says.

In Dr. Khademhosseini’s view, one of the main advances in this field over the past decade has been the development of better materials with which cells can interact, such as gel-like materials in which cells can be encapsulated, or degradable materials that cells can remodel.

Another advance has been the introduction of multiple cell types into a microfluidic device to generate complex, miniaturized 3-D structures that mimic natural tissues, tissue structures composed of multiple cell types, and miniaturized multi-organ systems. Growing different cell types in close proximity allows for cell-to-cell communication that may allow the cells to work together to modify and regulate their microenvironment.

A microfluidic system that can support the simultaneous growth of liver and cardiac tissue, for example, each in its own preferred microenvironment, would enable drug-toxicity studies in which the metabolism of a therapeutic compound in the liver could result in the generation of a cardiotoxic bioproduct, and this phenomenon could be identified before the drug is put into humans and advances to clinical trials.

Dr. Khademhosseini describes his group’s recent work to develop imaging technology capable of capturing real-time images in a wide field of view to document and quantify changes in cell- and tissue-based systems. His lab has integrated an inexpensive webcam-like imager into microscale devices.

If cardiac cells are grown in these devices, for example, the webcam can detect and monitor the beating of the cardiac cells and detect changes in the rate of beating or other characteristics of the cells in response to the introduction of a chemical or other stimulus.

Overall, the laboratory’s efforts to integrate cells and microfluidic systems are focused in three main areas: studying how different microenvironments and chemical stimuli affect the growth and differentiation of embryonic stem cells in a controlled system; analyzing the behavior of cells in microfluidic systems; and developing new microfluidic chips that can advance the technology and increase the ability to control cell behavior.

Dr. Khademhosseini identifies several key trends in the field, including a move toward reduced complexity and decreased cost in the microfabrication of microfluidic systems. For example, the use of paper and the material of choice to generate pattern structures in microfluidic devices for point-of-care diagnostic applications is becoming increasingly popular.

Another trend relates to cell-based applications and the focus on creating 3-D artificial tissues suitable for transplantation. Whereas most fabrication technologies are based on 2-D methods, generating individual layers that can then be stacked to create modular 3-D systems, emerging technologies such as 3-D printing will make it possible to create microfluidic devices in three dimensions with precision down to the single-micron scale.

Dr. Khademhosseini sees market value for both modular, off-the-shelf microfluidic devices produced at large scale for universal types of applications, and individually fabricated 3-D structures developed from a patient’s cells that are grown in a matrix and physiological setting that mimics the cells’ natural microenvironment and the body’s metabolic and pharmacokinetic properties, which could help advance the development of therapeutic strategies for personalized medicine.

A PDMS microfluidic device for generation and dilution of two-dimensional combinatorial solution mixtures, integrated with a well array for cell storage and culture [Harvard Medical School]

Probing Disease Processes

The scale of microfluidic channels—tens to hundreds of microns—lends itself to the study of whole organisms, if that organism is a nematode, and specifically, C. elegans, which is a commonly used model organism to study development, physiology, and disease processes.

Work published by a team of researchers at McMaster University has shown that worms contained in microchannels and exposed to an alternating current (AC) electric field can be immobilized. Similarly, the introduction of a direct current (DC) electric field stimulus results in directed movement of the worms along the microfluidic channel, quite predictably toward the negative end of the channel.

Bhagwati Gupta, Ph.D., associate professor of biology, explains how the team is using a combination of AC and DC electrical fields as a control mechanism to confine and manipulate worms, a technique called microfluidic electrotaxis. They are applying this technique to study movement-related disorders, such as Parkinson disease, exposing the worms to neurotoxic stimuli and screening compound libraries to identify potential drug candidates.

The facts that C. elegans will move in a predictable manner in response to an electric field, that this movement is controlled by neurons, and that neuronal defects can disrupt normal electrotaxis, make this a valuable strategy for studying the neural basis of behavior.

Furthermore, “neuronal circuits are fairly well understood in worms, allowing us to focus on specific kinds of neuronal activity and degeneration involved in movement,” says Dr. Gupta.

Parkinson disease is caused by a loss of cells that produce the neurotransmitter dopamine. C. elegans has only eight dopamine-producing cells, and only six of those appear to be involved in signaling in the brain, making this a relatively simple biological system. Techniques are in development to knock out the dopamine-producing activity of individual cells, says Dr. Gupta, but at present, one of the challenges is that the introduction of neurotoxins or the use of gene-knockdown approaches affects all of these cells.

The group at McMaster is employing microfluidic technology to study the effects of neurotoxins on dopamine function and the movement of individual organisms.

“Our assay system is up to 10 times more sensitive than conventional C. elegans assays,” he says. One aspect of their research is to understand which cells actually perceive the electrical field stimulus. Are they the dopamine-producing cells themselves or another cell type that then signals the dopamine-producing cells to release the neurotransmitter?

“We are now developing different kinds of microfluidic devices to look at neuronal activity at the single-cell level,” says Dr. Gupta. The goal is to enable faster and more accurate measurements—at the millisecond level—to improve the sensitivity of detecting neuronal responses.

Another area of technology development is focused on identifying better and less costly imaging options. Additional challenges include the need for more automation to minimize the amount of hands-on time still needed to prepare the worms and load them into the microchannels, as well as improved electrical sensors and detectors that would be less costly yet as powerful as conventional CCD cameras and could be mounted on the microfluidic devices.

New types of software are also needed that can integrate tasks and data collection across a series of linked microfluidic chips, each of which performs one or more dedicated tasks.

A schematic view of the microfluidics setup for an electrotaxis experiment is shown on the top. Worms are introduced into the device by a syringe pump. The electric field is set up using a power supply and the response of the worm is observed and recorded. The bottom left drawing shows a detailed view of the device. The bottom right images are the snapshots of a worm moving in the channel in response to a DC field (arrow marked E) at two different time points. The upward arrow marks the position of the worm. [McMaster University]

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