Abstract
Cell spheroid formation is necessary to develop three-dimensional (3D) cellular environments that provide appropriate cell-cell and cell-matrix interactions similar to in vivo environments without additional substrates. Although some methods including stirring culture, low adhesion plate culture, hanging drop, and microfluidics are used to construct cell spheroids, there is no method to fulfill all of the mass production of uniform spheroids, simple media change, and easy retrievability. Here, bulk poly(N-isopropylacrylamide) (PNIPAAm) hydrogel substrate (PHS) was used to fabricate, culture, and retrieve cell spheroids. Adipose-derived stem cells (ASCs) were cultured on bulk PHS to form spheroids. ASCs formed cell spheroids directly on substrates without additional manipulation. These spheroids adhered to the semi-adhesive substrate, while the spheroids fabricated using the nonadhesive surface method floated without getting fixed to the surface. Bulk PHS stiffness was evaluated using the compressive test (compressive modulus: 153±11kPa). A poly(ethylene glycol) (PEG) hydrogel microwell pattern was created on PHS to control the spheroid size, forming uniform ASC spheroids between 100 and 150μm in diameter on 200 and 300μm well-patterned substrates. Cell-cell interactions in the resulting ASC spheroids were evaluated based on fibronectin and laminin expression; fluorescence intensities of fibronectin- and laminin-immunostained images of ASC spheroids were 10.9 and 7.3 times higher than those of ASCs cultured on the tissue culture plate, respectively. ASC spheroids were detached following incubation at 4°C for 10min (retrieval efficiency: 74±19%). Retrieved spheroid cell viability was over 97.5%. The PEG hydrogel microwell-patterned PHS is a convenient spheroid fabrication and retrieval platform that can increase cell spheroid usage.
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