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Japanese Researchers Develop Cell Microarray; Tech Could Facilitate Monoclonal Ab Screens

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Japanese scientists have developed a single-cell microarray chip and demonstrated how it can be used to screen for intracellular calcium in immune cells and possibly isolate antigen-specific lymphocytes for monoclonal antibody production.

The technology could provide a higher-throughput method for developing highly specific monoclonal antibodies as medicines, and has the potential to enable high-throughput screening of ligand-stimulated cells of any type.

The microchip could also compete with a similar cellular array technology being marketed by nascent biotech Molecular Cytomics.

A proof-of-principle research paper describing the chip, published in the Dec. 15 issue of Analytical Chemistry, was authored by scientists from the Toyama Medical and Pharmaceutical University, Toyama New Industry Organization, and Japan Advanced Institute of Science and Technology, all in Japan. Eiichi Tamiya of JAIST is the corresponding author.

As detailed in the paper, the scientists designed a polystyrene microarray chip containing more than 30,000 microchambers, each capable of accommodating only single cells. They subsequently added lymphocytes, including B-cells, which had been derived from mouse spleens or human blood and labeled with Fluo-4-AM fluorescent calcium indicator dye.

After individual cells "settled" into the bottom of each well, the researchers scanned the microarray chip with a Hitachi laser-based microarray scanner, stimulated the cells with antibodies, and then re-scanned to determine which cells responded with intracellular calcium increases.


The microchip could also compete with a similar cellular array technology being marketed by nascent biotech Molecular Cytomics.

Lastly, the researchers were able to retrieve target cells that had responded above a particular fluorescence threshold by using a micromanipulator system coupled with a Zeiss microscope. They subsequently recovered DNA from target cells in an attempt to isolate and analyze antibody cDNA, which can then be used, in theory, to develop antigen-specific monoclonal antibodies.

Currently, one of the most widely used methods for isolating cells in this manner is flow cytometery. "However, it is quite difficult to screen and identify the minor cell population of antigen-specific single B-cells … with a flow cytometer because fluorescent signals of cells are buried in the noise signals of nonspecific cells," the authors wrote.

"The single-cell microarray platform developed in this study could successfully screen and detect the low frequency of antigen-specific single B-cells using a single chip in one run," they added. "In addition, it also analyzes the same single cells before and after stimulation with antigen, which a flow cytometer could not."

While the new technology might compete indirectly with flow cytometers in this specific application area, it might also compete more directly with another nascent cellular array technology in terms of other drug-discovery screening.

The single-cell microarray technology is quite similar — especially in its design — to the Optical LiveCell Array currently being marketed by Newton, Mass.-based Molecular Cytomics. That technology is a disposable microscope slide that contains up to 10,000 individual micron-scale wells, each of which is large enough to hold only individual cells.

However, Molecular Cytomics is marketing the Optical LiveCell Array for use primarily in the burgeoning high-content screening market. Because it is optically clear, it is compatible with essentially any imaging platform on the market.

"I think the main difference is the way the arrays are made," Israel Biran, chief operating officer of Molecular Cytomics, told CBA News. "Our concept is imaging. We believe that the information you get from imaging, from actually being able to see and record the morphology of the cells, as well as the fluorescence, is the key issue."

Another difference, Biran pointed out, is the incorporation of microfluidics channels in the Optical LiveCell Array, which allows compounds and other chemicals to be added to the cells without disrupting them.

The single-cell microarray developed by Tamiya and colleagues was used with a standard microarray scanner lacking sub-cellular resolution, and therefore has only been used thus far in assays where a "yes/no" fluorescence signal is desired, such as the calcium dye indicator screen.

It is unclear at this point whether the Japanese scientists will attempt to incorporate imaging into their technology, or whether they will explore other potential application areas. Tamiya did not respond to questions in time for the publication of this article.

In the paper, the scientists did pinpoint specific improvements that could be made to the technology, specifically better instrumentation.

"The assay we have adopted — measurement of increase in the intracellular calcium after antigenic stimulus using Fluo-4 — hardly lasts for a few minutes," the researchers wrote. "During this short period of time, the microarray scanner could scan the analyzable area of 30,000 to 40,000 microchambers.

"This number of microchambers is sufficient to screen and identify as little as 0.1 percent of antigen-specific single cells in a total B-cell population, which is the limitation of [a] flow cytometer," the researchers continued. "The ultimate potential of this … microarray can be realized by improving the assay system and the scanning speed of the microarray scanner for detecting [the] total number of microchambers on the microarray chip."

Biran said that Molecular Cytomics' product could likely be used for the type of "yes/no" screen described by the Japanese researchers, but that it would be limiting the ability of the Optical LiveCell Array.

"The question is how available this technology would be to researchers, and the ease with which you can use it and extract data in a high-throughput and straightforward manner," Biran said. He agreed that the new technology would be limited in the information one can get by the detection method used.

"Our technology is compatible with basically any instrument," he added. "But it's a very interesting technology. It's definitely along the same lines as our technology, and designed to address the same needs."

— Ben Butkus ([email protected])

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