Micropallet arrays: A novel microtechnology for profiling and recovery of tumor cellular elements.

摘要

The evolving appreciation that cellular elements within a tumor play critical roles in defining its biology is driving efforts to intensively study single viable cells of defined phenotypes from within these heterogeneous tissues. The determination of cellular profiles and study of individual rare cells obtained from tissues, including tumors, is highly likely to provide tremendous benefit to patients with a wide range of pathophysiologic processes. The inability to isolate relevant cellular subsets from a tumor is one critical barrier to accomplishing this goal.;The micropallet array is a recently developed technology for the analysis of adherent cell populations and enables the isolation and recovery of single adherent cells. Micropallet arrays consist of hundreds of thousands of transparent microscale polymer pedestals ("micropallets") uniformly arrayed on a glass microscope slide. Each micropallet holds a single adherent cell in culture and can be selectively recovered from the array at any point in time with minimal perturbation to the cell. We made significant advancements to this technology and created a platform capable of rapid identification, isolation, and evaluation of rare cells from a tumor biopsy specimen.;These advancements include: establishment of methods to coat micropallet arrays with a variety of extracellular matrix components and evaluation of each coating's ability to capture cells, specifically of the types expected to be present within a tumor; development of multichannel immunofluorescent labeling and imaging techniques for use with micropallet arrays, which are necessary to characterize cell populations and identify rare cells via panels of cell surface markers; invention of a new laser-based method for release of individual micropallets that is integrated with the same system used for fluorescent imaging, greatly improving ease of use and throughput capabilities of the overall system; and finally, creation of a magnetic micropallet array and accompanying magnetic collection system for single micropallet manipulation, which also dramatically improves throughput capabilities, and furthermore, enables genomic analyses of singly recovered cells. These individual advancements to the micropallet arrays technology coalesce to produce a powerful platform capable of analyzing the cellular makeup of small tumor biopsy specimens and identifying and recovering individual rare cells for further analysis.