By David F. Salisbury
Published: August XX, 2008
A t first glance, the core of the multi-trap nanophysiometer (MTNP) looks like nothing more than two or three glass microscope slides stacked together. But a closer look shows an intricate pattern of tiny channels and traps designed to hold hundreds or thousands of individual cells for scientific study.
The new device, which has been developed by researchers at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), consists of hair-sized channels molded in a translucent polymer glued onto the surface of a glass coverslip. A shoebox-sized pump pushes special liquid media used to keep cells alive through the microscopic network. It is one of the first microfluidic devices that has been used to study the behavior of individual cells in the immune system.
In a typical configuration, each channel opens up into a triangular chamber filled with hundreds of tiny, three-sided wells small enough to trap individual cells. When cells are injected into the liquid upstream of the tiny traps, they are carried into the chamber where they bounce like pinballs among the microscopic buckets until they are trapped in one of the wells or are carried out of the chamber.
Once they are caught in a trap, the cells are held there by a microcurrent that is created by the fluid flowing out of openings in the bottom of the wells. The cells have the ability to escape from the traps, but only a small percentage make the effort under normal conditions.
The MTNP is made from the silicon-based organic polymer, known as PDMS, which is used to make soft contact lenses. The system of channels and traps is created by an inexpensive molding process that is easy to customize for specific applications. For example, trap dimensions can be changed to catch cells of different sizes, and channels can be added for injecting additional chemical compounds. The device is so easy to modify for new applications that it can be done by individual researchers.
The nanophysiometer is mounted on an optical microscope equipped with an incubator that keeps the cells at a comfortable temperature. A broad spectrum light source, optical filters and a digital camera are attached to record the behavior of the cells through the glass coverslip. This allows the researchers to use the large repertoire of fluorescent tags that biological scientists have developed to detect a wide range of cell behavior, including T-cell activation. Unfortunately, the light is damaging to cells, so the researchers limit themselves to taking one image every 30 seconds.
According to VIIBRE director John Wikswo, the MTNP has several characteristics that will help scientists discover important new information about how living things work at the molecular level. It gives scientists the ability to:
Study the behavior of normal, healthy human cells. Human cells are relatively difficult to grow outside of the body, even with the addition of special nutrients and growth factors. For most experiments, researchers use "immortalized” cells that have been specially modified so they grow continually in tissue cultures, but their biological responses may differ from those of their normal counterparts. By simply controlling the conditions in the nanophysiometer, the VIIBRE researchers have shown that they can keep normal cells alive and healthy for at least 24 hours. Carrying nutrients and other chemicals to the cells with microcurrents creates an environment that is much closer to what the cells experience in the body than that of cell cultures. So the device allows the researchers to study cells in a more natural state.
Observe the individual behavior of large numbers of cells, particular mobile cells like those in the immune system. "These are the naïve cells that would normally be wandering around the bottom of the dish in a cell culture: If you stick them down to study them, you change them into a different state,” says Wikswo. Standard tissue culture techniques reveal the mass behavior of millions of cells. As a result, variations in behavior of individual cells tend to be lost.
Record how the behavior of individual cells changes over time. Currently, the major method for analyzing the physical and chemical characteristics of single cells is fluorescent-activated cell sorting (FACS). This uses a $100,000 instrument called a flow cytometer to produce a focused stream of fluid that passes through beams of laser light. A number of detectors, aimed at points where the stream and laser beams meet, provide information about the state of the cells carried past. This efficiently provides data about millions of cells at a time, but it cannot follow changes in the state of individual cells over time. "The nanophysiometer allows us to study cell dynamics, which is something that FACS analysis cannot do,” says Wikswo.
Detect even the faintest chemical signals that individual cells rely upon to coordinate their activities. Nanophysiometer chambers have a total volume of about 1 percent of a typical cell culture dish. As a result, chemical signals from individual cells are not diluted beyond the point where other cells can recognize them. The VIIBRE researchers are collaborating with John McLean, assistant professor of chemistry, to combine the nanophysiometer with an instrument called an ion-mobility mass spectrometer that can identify these chemical factors despite the minute quantities involved.
Control the cell's micro-environment with unprecedented precision and change that environment rapidly. "You can load the device with cells and then expose them to a drug or toxin all at the same time, and then you can remove or replace the toxin very rapidly,” says Kevin Seale, assistant professor of the practice of biomedical engineering, who has helped develop the nanophysiometer. This ability should be useful in studying the adverse side effects of environmental toxins, new drugs and chemotherapeutic agents at the level of the single cell.
IIn addition to the study of cell signaling among immune cells, the VIIBRE researchers argue that the multi-trap nanophysiometer has a number of possible research and clinical applications. These include:
Figuring out why the immune systems fails to prevent tumors from forming in breast cancer;
Developing a method for detecting HIV infection at an early stage that is low-cost, compact, disposable, low-power and easy to operate, and therefore suitable for use in developing countries;
Studying the regulatory pathways that govern stem-cell differentiation and self renewal. Environmental cues that stem cells receive in the body play a dominant role in determining their fate. According to Shannon Faley at the University of Glasgow the MTNP provides a platform that is allowing researchers to begin to identify the cell interactions that are most influential in stem cell development, an ability that can help scientists attempting to use stem cells as therapy for hard-to-treat illnesses;
Providing a means for simultaneously analyzing how thousands of individual cells respond to particular agents, a feature important for high-throughput drug discovery. This is possible because the device is easily expandable;
Studying the interactions of different cell types. The device's large number of small traps can be filled with a mixture of cells, allowing researchers to observe a wide range of cell-cell combinations in a single device. For example, scientists think that these interactions may cause the failure of chemotherapy in some breast cancers and the MTNP provides a powerful new means to study this effect.