title: Laser-based directed release of array elements for efficient collection into targeted microwells.
abstract: A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Dobes
authors.forename: Rahul
authors.initials: R
authors.lastname: Dhopeshwarkar
authors.forename: W Hampton
authors.initials: WH
authors.lastname: Henley
authors.forename: J Michael
authors.initials: JM
authors.lastname: Ramsey
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D046888:Tissue Array Analysis; D014162:Transfection
chemical_list: D004129:Dimethylpolysiloxanes; D049452:Green Fluorescent Proteins; C013830:baysilon