Eukaryotic cell sorting is a ubiquitous technique used for biological applications ranging from pharmaceutical drug discovery to cancer research. Evaluating and differentiating cells based on their features, whether they be intracellular or extracellular, has revealed that cellular systems are highly complex, possessing varying behavior even among cells typically classified within the same groups. While there have been massive improvements in characterization capabilities for single cells achieved through use of inventions such as the fluorescence activated cell sorter, there has yet to be an equally impactful technology developed for analyzing clonally expanded cell colonies. Being able to analyze and isolate cell colonies is necessary for cancer and stem cell research where the observed behavior of cells in colonies is just as important, if not more so than when they are in their single cell forms. There is currently a need for new analysis and sorting systems that enable researchers to conduct more in depth studies of cell colonies as well as increase the throughput of colony collection protocols which are typically time consuming and manually intensive.;Micropallet arrays are a powerful technology specifically designed for high precision sorting of adherent mammalian cells. This technology, composed of thousands of photolithographically fabricated microscopic structures, has enabled researchers to study, identify, and collect individual cells from within vastly heterogeneous samples. Furthermore, micropallet arrays have been an ideal sorting technology for characterizing the natural morphology of single adherent cells preserved by enabling continuous cellular contact with the pallets throughout the entirety of analytical studies. In some cases, rare cells such as cancer stem cells with an occurrence rate as low as 0.01% have been successfully identified and collected from within heterogeneous cell samples prepared from breast cancer tumor biopsies and seeded to the arrays. While being a powerful sorting technology for single cells, micropallet array technology has just recently been expanded upon for meaningful cell colony research.;In this work, we describe a novel micropallet array design specifically created for efficient, high throughput cell colony sorting. A new approach for simple, consistent large scale micropallet ejection was achieved by utilizing an innovative pallet formation substrate consisting of ultra-thin gold coated microscope slides. These semi-transparent slides maintained the optical clarity necessary for in depth cellular analysis on the pallet surfaces while also acting as laser absorption layers for the creation of heat generated vapor microbubbles. The formation of the bubbles generated sufficient force to lift off targeted pallets with little to no damage to the cell colonies adhered to the pallet surfaces, a result previously shown to be difficult. In addition to enabling consistent pallet ejection, the conductive gold layers also acted as seed layers for the electroformation of ferromagnetic nickel structures integrated into the micropallet arrays. With creative pallet designs, the ferromagnetic structures were fabricated within every pallet, making individual pallet manipulation via magnetic probe possible. This complete system with simplified pallet ejection and collection was successfully utilized to perform highly efficient cell colony sorting studies at rates not possible with conventional methods.
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