Researchers from the University of California, Irvine have devised a microfluidic chamber that may aid in the study of axons from living central nervous system cells. Their research has attracted the attention of a small California-based technology-licensing company, which plans to create a start-up company based on the technology.
If successfully brought to market, the platform could provide academic and pharmaceutical researchers with a more efficient way to isolate the axonal regions of live CNS neurons in cell culture, and therefore a way to screen for compounds with regenerative potential for afflictions such as spinal cord injury and Alzheimer's disease.
As detailed in the August issue of Nature Methods, the UC-Irvine scientists used photolithography to microfabricate a prototype of a microfluidics-based cell-culture chamber with a molded polymer on top of it. The chamber is divided into two sections — a somal side and an axonal side — divided by a wall with consecutive, equally spaced micron-scale openings.
Primary rat CNS neurons are cultured in the somal side, and within three to four days, axonal growth is guided through the micron-scale openings into the axonal side. As stated in the paper, "a volume difference between the somal side and axonal side allows chemical microenvironments to be isolated to axons for over 20 hours owing to the high fluidic resistance of the microgrooves."
Furthermore, the cell chamber is optically clear; thus, the researchers were able to isolate individual axons using standard microscopy, selectively injure them through micro-incision, and, in a proof-of-principle experiment, and treat the axons with a combination of neurotrophins known to induce axonal regeneration. Within a week, the treated axons demonstrated a significant increase in branching, growth, and growth cone density.
"If you can put a cell body in one side of the chamber and let it grow in the right direction, you have an instant result that may take you an hour when before it took you months."
According to the UC-Irvine researchers, in vitro models — namely Campenot chambers — do exist for studying axonal biology in the peripheral nervous system, but not the central nervous system.
Campenot chamber assays are "very difficult to work with," Noo Li Jeon, professor at the UC-Irvine engineering school and corresponding author on the paper, told CBA News. "It's a similar assay, but it takes a long time to set up — maybe a couple of days to set up 20 assays, and only maybe 10 would work on a really good day," Jeon said. "There are some labs that work with it, but it really requires a lot of skill, and usually in a lab, only one person can do it reliably.
"But ours is easier to set up the assay, it's reproducible, and you can replicate it," Jeon added.
The current prototype of the UC-Irvine chamber contains 120 microgrooves, each with the ability to isolate one or a few axons. The researchers said that the plan would be to increase this number, as well as to adapt the chamber to well-plate format for higher throughput studies.
In fact, that is just what Tustin, Calif.-based technology-licensing firm Invinium plans to do as part of its bid to create a start-up firm to commercialize the assay tool. Invinium founder Richard Herstone told CBA News that his company, which licenses a wide variety of technologies across several industries, is currently negotiating a technology-transfer license with UC-Irvine, which owns the rights to the platform.
"We have had numerous meetings — they want us to do it, I want to do it — we've been trading documents back and forth, and the patents are pending, so nothing can be done," Herstone said. "So we'll have it tied up once the patents are issued, but at the same time, we can create a company with patents pending, and that's what we're looking to do right now."
Herstone said he became involved in the technology mainly because of his association with one of the Nature Methods paper's co-authors, Carl Cotman, a professor of neurology at UC-Irvine. "We are definitely doing a start-up around this technology," he said. "That is a given already — we have already circled our resources for it," he said. "Right now, we're in our own investigative stages of scalability, manufacturing procedures, and how we can get this into the marketplace on a worldwide basis in a cost-effective manner."
Although he proclaimed himself a layman, Herstone said he views the technology from one angle: "What has the historical process been to do the test that you're trying to do, and what did it cost you to do it?" he said.
"Before, it was taking graduate students and finding out where one cell body begins and axon ends, when you've got millions of cell bodies in there," Herstone continued. "So it could take days, weeks, or months, and you'll never figure out the results of a test.
"Here, if you can put a cell body in one side of the chamber and let it grow in the right direction, you have an instant result that may take you an hour when before it took you months," he added.
Many companies have already been established based on microfluidics technology for various biological research applications, but microfluidics has been slow to mesh with cellular analysis. Several academic research groups, however, have also recently been toying with the idea of start-up companies based on the use of microfluidics to analyze difficult-to-study cell types.
These include researchers at Trinity College Dublin, which last year formed start-up Cellix to commercialize a microfluidics platform designed to mimic animal capillaries (CBA News, 8/10/2004); University of Alberta professor Jed Harrison, who co-founded Advanced Integrated Microsystems to commercialize microfluidics tools for cellular toxicity studies (CBA News, 6/6/2005); and UC-Berkeley bioengineering professor Luke Lee, who hopes to create a start-up based on his microfluidic-based cell-culture array (CBA News, 1/18/2005).
The UC-Irvine researchers and Invinium are hoping to fill a niche that is yet to be occupied with the neuronal culture assay tool.
Besides imaging, the platform is expected to be useful for biochemical analyses of CNS axons, such as isolation of mRNA, subsequent PCR studies, and Western blot analysis — all of which the researchers either demonstrated in the paper or have done preliminary work on.
"You can do all sorts of imaging like DIC, high-resolution confocal, two-photon, and those kinds of things," said Anne Taylor, lead author on the paper and a graduate student in Jeon's lab. "But it can also be used to biochemically analyze the axons, as well as the response to injury in the soma. In the paper, we show a proof-of-principle experiment, where after injuring the axon, we see the immediate early gene expression of c-fos rapidly induced. So it's not just imaging, but that is a main component."
— Ben Butkus ([email protected])