A fundamental need in almost all areas of biomedical research is the ability to separate single or small groups of cells from within a heterogeneous population. In order to obtain a living cell possessing a desired characteristic, individual cells or homogeneous groups of cells within the population must be analyzed followed by identification and isolation of the target cell(s). Most live-cell separation methods require that cells be dispersed into a single-cell suspension. Unfortunately, many crucial studies of cell biology cannot be performed in this manner. In the current application, an interdisciplinary team is collaborating to address the technical challenge of analyzing, sorting and collecting viable cells from a mixed population while they remain adherent to their growth surface. Easily implemented fabrication techniques and chemical surface modifications will be used to produce large arrays of releasable cell """"""""pallets"""""""" for the analysis, release and collection of single cells or colonies. Adherent cells will be cultured on the array, and the array will be analyzed using standard methods from image cytometry. Pallets containing single cells or small cell colonies will be individually released using a pulsed laser and collected for further analysis or expansion of the cells. Several applications for this system will be demonstrated. The early and rapid establishment of stably transfected cell lines based on the ability to identify and collect fluorescent microcolonies of cells expressing a red fluorescent protein fused with a key signal transduction enzyme will be performed. The value of selection based on morphology will be shown by cloning pure populations of cells transformed by an oncogenic retroviral protein. In addition, the approach enables living cells to be collected based on their dynamic characteristics, a selection criterion not feasible with current techniques. This capability will be used to select single cells based on siRNA perturbation of their calcium response or protein translocation. The expected benefits of this separation strategy are maintenance of cell viability, reduction in time and manipulation of the selected cells, and a broader set of cell attributes available for cell selection. The applications which involve basic cell biology, tumorigenesis, and signaling mechanisms in diabetes mellitus will demonstrate that the novel separation system should be widely applicable to the biomedical sciences. ? ? ?
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