Microfabricated Arrays for Splitting and Assay of Clonal Colonies

摘要

A microfabricated platform was developed for highly parallel and efficient colony picking, splitting, and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies, creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 μm diameter and 100 μm length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78-92%. To demonstrate the technique's utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only 5 man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34 man hours expended over 47 days. These data support the considerable reduction in time, manpower, and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches.