Evaluation of cell-cell or cell-substrate adhesion effect on cellular differentiation using a microwell array having convertible culture surface

Abstract

In this paper, effects of cell-cell adhesion or cell-substrate adhesion on cellular differentiation are evaluated using a microwell array having convertible culture surface (CCS microwell array). The CCS microwell array is used for limitation of culture area and regulation of the adhesion timing. The CCS microwell array is fabricated by photolithography of SU-8 photoresist on the cover slip, and coating of the alginate thin film on the microwell bottoms. The alginate thin film is used as a material that inhibits adhesion of cells. It uses an alginate film that is not toxic to cells and can be removed by ethylendiaminetetraacetate (EDTA), which is calcium chelator, at arbitrary timing during cell culture. A single spheroid is formed by cell-cell adhesion in microwells because cells can't adhere to the culture surface by adhesion inhibition effect of the alginate thin film. When the culture surface is converted to cell-adhesiveness surface by removing alginate thin film, cells adhere to the microwell bottoms. The transition of the diameter of spheroid cultured in the CCS microwell array was evaluated by observation of an inverted microscope. Also, cellular differentiation behavior under four kinds of conditions was compared for verification of cell-cell or cell-substrate adhesion effect. This result shows the efficiency of induction of cellular differentiation was increased by cell culture in the CCS microwell array compared with three kinds of other culture conditions.