Laser-based directed release of array elements for efficient collection into targeted microwells

• Published in: Analyst (London) Vol. 138; no. 3; pp. 831 - 838
• Main Authors: Dobes, Nicholas C, Dhopeshwarkar, Rahul, Henley, W Hampton, Ramsey, J Michael, Sims, Christopher E, Allbritton, Nancy L
• Format: Journal Article
• Language: English
• Published: England 01.01.2013
• Subjects: Arrays; Cell Line, Tumor; Cell Separation; Cell Survival; Cloning; Collection; Dimethylpolysiloxanes - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Lasers; Pallets; Retrieval; Signatures; Sorting; Tissue Array Analysis; Transfection
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c2an36342a

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%.