Microcup arrays for the efficient isolation and cloning of cells.

Arrays of transparent, releasable micrometer-scale structures termed "microcups" were created for the purpose of patterning and isolating viable cells from small cell samples. Cells were captured by the microcups without the need for barriers or walls on the intervening substrate. Furthermore, in contrast to prior methods for creating cell arrays with releasable elements, no chemical modification of the substrate was required. Individual microcups were released from the array using a pulsed laser at very low energy. Improvements in microcup design enabled cells in suspension to be loaded into the microcups with greater than 90% efficiency. Cells cultured within the microcups displayed 100% viability and were cultured over 4 days yielding colonies that remained sequestered within the microcups to generate pure clonal populations. Standard microscopic imaging was used to identify cells or colonies of interest, and the microcups containing these cells were then released and collected. Individual target cells isolated in this manner remained viable as demonstrated by clonal expansion of 100% of collected cells. Direct comparisons with cell isolation by fluorescence-activated cell sorting and magnetic-bead-based isolation systems demonstrated that the microcup cell isolation procedure yielded higher purity, yield, and viability than these standard technologies when separating samples with small numbers of cells. The power of this technique was demonstrated by the isolation of hematopoietic stem cells from a human bone marrow aspirate possessing only 4000 total cells.