Microwell arrays that have many micro-sized cavities on the device have been employed to form multicellular spheroids. However, methods to efficiently harvest the constructed spheroids from the microwell arrays have not been thoroughly investigated. We evaluated the effects of poly(N-isopropylacrylamide) (PNIPAAm) for constructing and harvesting spheroids from microwell arrays. Microwell arrays were coated with ethanol containing 1%, 2.5%, 5%, or 10% PNIPAAm by a solvent-casting method and then dried. Spheroids formed using the coated microwell arrays were harvested. Highly efficient and rapid recovery of NIH3T3 mouse fibroblast spheroids were achieved for the 5% and 10% coated wells (93.2% +/- 1.6% and 93.6% +/- 1.1% at 60 s, respectively), whereas recovery was not efficient for 0%, 1%, and 2.5% coated wells (0.2% +/- 0.2%, 1.1% +/- 0.6%, and 7.8% +/- 4.0% at 60 s, respectively). Because PNIPAAm is a thermoresponsive polymer that exhibits a lower critical solution temperature (LCST) of 32 degrees C, we examined the effects of temperature on the recovery rate. The recovery rates at 4 degrees C (below LCST) were equivalent to or higher than those at 37 degrees C (above LCST) for all four cell types examined. Functional assessment suggests that the PNIPAAm microwell arrays are not toxic to the formed spheroids. The PNIPAAm microwell array developed in the present study will be useful for constructing and harvesting spheroids. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.