In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.