Picking a Needle out of a Haystack

Gel Microdrop System from One Cell Systems can ferret out rare protein secreting cells Imagine, if you will, a single cell suspended in a drop. Now picture that same cell secreting a protein, say an immunoglobulin. Now envision that secreting cell embedded in a matrix that binds the protein as it leaves the cell, and you have the idea of the microdrop hybridoma selection system available from One Cell Systems. A modification of the microdrop technology described in 1991 by James Weaver, Associate Director of the Biomedical Engineering Center, Harvard/MIT, this technique can save weeks of time and tedium in the isolation and stabilization of protein-secreting cells. While microdrops are not a totally new idea, the development of a technology for making small, uniform droplets from a porous material--uniformity is one of the keys to the success of the microdrop assay--combined with a "capture web" of affinity-tagged agarose, it has made this system into a valuable commodity for those interested in isolating rare or high producing cells. Fluorescence profile before (left panel) and after (right panel) enrichment for high secretors. After initial sort, the highest 7% of the population was cultured for three weeks and retested against the original, unsorted cell line. How does it work? The basic concept is simple enough. A population of cells is suspended in agarose under conditions that lead to the envelopment of a single cell per drop. This process can be done with One Cell's CellSys™ Microdrop Maker or manually with common lab devices, such as stirring/heating plates and ice baths. In the case of hybridoma or transfected cell isolation, the gel used to form the microdrops is conjugated to biotin and following the formation of cell-bearing microdrops, streptavidin and biotinylated antibody are added. As these reagents diffuse into the drops, an ELISA-like sandwich is formed totally within the drop. Finally, the addition of a reporter molecule, such as fluoresceinated antibody, allows the detection and isolation of secreting cells by fluorescence-activated cell sorting. Upper Right: Single yeast cell encapsulated in a microdrop. Lower left: Series of photo-micrographs showing the time course of accumulation of antibody by a hybridoma cell. This technology has been applied to the isolation of rare antibody-producing cells from heterogeneous populations of hybridomas (F. Gray et al, Journal of Immunological Methods, 182:155-63, 1995). Unlike conventional flow cytometry, the gel microdrop (GMD) assay sees more than surface antibody, as secreted proteins are trapped within the drop. This feature of the GMD allows not only for the identification of rare producing cells, but also cells producing antibody at varying rates. Glenn Paradis, Research Specialist at the MIT Center for Cancer Research, has been involved with the development of the GMD assay. "The technology is solid... the potential tremendous" says Paradis. Unlike previous methods for isolating high producing cells from populations of hybridomas, the GMD assay can be used to look at protein production over time, according to Paradis, providing more than a single snap shop of the reaction. And with GMD, you can look atmillions of cells in a few hours, while the more typical brute force method of clonal isolation and testing requires a heroic effort to look at just a few hundred. Isolating secreting cells is but one use of the gel microdrop. Any rare cell that bears a marker of some kind can be isolated with the GMD. The effect of compounds on cell growth can be measured with GMD. Even in situ hybridization to isolated chromosomes, nuclei, or cells--referred to as MISH for microdrop in situ hybridization--can be done with GMD. At present, One Cell Systems offers two prepackaged systems: one for clonogenic cytotoxicitiy testing and another for secreting-cell selection. The system for microdrop formation, as well as the reagents needed to perform the assay, can be obtained through a leasing arrangement with the company. For more information, contact One Cell Systems at 617-868-2399 x309 or visit the company web site, www.onecell.com. The author, Laura De Francesco can be reached at ldefrancesco@the-scientist.com.

Picking a Needle out of a Haystack

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