EVALUATION OF CELL DIFFERENTIATION EFFICIENCY BY CELL-CELL/CELL-SUBSTRATE ADHESION USING A MICROWELL HAVING CONVERTIBLE CULTURE SURFACE Y. Nakashima, Y. Hikichi, T. Yokokura, Y. Yamamoto, and Y. Nakanishi Kumamoto University, JAPAN

ABSTRACT This paper presents evaluation of cell-cell or cell-substrate adhesion effect on cellular differentiation. A microwell array having convertible culture surface (CCS microwells) is used for limitation of culture area and regulation of the adhesion timing. The CCS microwells are fabricated by photolithography of SU-8 on the cover slip, and formation of alginate film on the microwell bottoms. The alginate 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. This device permits in-situ observation and evaluation of cellular differentiation behavior to conversion of culture surface by optical microscope because this device doesn’t required temperature control and electrochemical operation.

KEYWORDS: Microwell, culture surface conversion, alginate gel, cell differentiation

INTRODUCTION Cell living environments, that is, cellular adhesion to the culture surfaces, hardness of the culture surfaces, and extracellular stimulation such as mechanical or chemical stimulation, are really effective to decide the cell fate such as growth, cell multiplication and differentiation [1]. If the surrounding environment of cells is able to be controlled artificially, cell differentiation and cellular function expression will be controlled. In this research, a microwell array having convertible culture surface (CCS microwells) is fabricated by microfabrication process and an alginate gel coating method [2] in order to evaluate the cell differentiation effect of cell-cell or cell-substrate adhesion. The cell-cell adhesion or cell- substrate adhesion are controlled by the fabricated CCS microwells, and effect of adhesion of each interfacial surfaces on cellular differentiation is evaluated.

MATERIALS AND METHODS The CCS microwells are fabricated by photolithography of SU-8 on the cover slip. The device consists of multiple microwells and each of the microwell bottoms is coated with alginate film (Fig.1(a)). A single spheroid is formed by cell-cell adhesion in CCS microwells before converting the culture surface because cells can not adhere to the culture surface by adhesion inhibition effect of the alginate film (Fig.1(b)). When the culture surface is converted to cell-adhesiveness surface by removing the alginate film, cells adhere to the microwell bottoms (Fig.1(c)). The 85 microwells were arrayed on a cover slip. The diameter and depth of the microwell are 400 ȝm and 200 ȝm, respectively (Fig.2).

EXPERIMENTAL RESULTS The induction of myoblasts differentiation was carried out using the fabricated CCS microwells. When myoblasts were disseminated in the microwells (Fig.3(a)), a single spheroid was formed in each microwell in 36 hours (Fig.3(b)). After the culture surface was converted to cell-adhesive, a portion of the spheroids adhered to the film-free surface (Fig.3(c)). The myoblasts making up spheroids spread out onto the culture surface and cells proceeded to fuse with many adjacent cells by cell-substrate adhesion (Fig.3(d-f)). In these case, mitochondria of myoblasts were stained with fluorescence dye to evaluate cell activity. Cell activity of myoblasts cultured on the device increased substantially as compared with cells cultured on the bare glass surface (Fig.4). In addition, myogenin, a protein responsible for the development of myogenesis, of these differentiated myoblasts was stained by an immunostaining method

th 978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 630 19 International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA to evaluate fusion/differentiation behavior. Fluorescent immunostaining images of fused and differentiated myoblasts after culturing for 244 h are shown in Fig.5. In the case of Fig.5(a-c), myoblasts were two-dimensionally cultured on the bare glass surface, and some cells expressed myogenin.

Figure 1: Schematic of a microwell array Figure 2: Photographs of the fabricated device. having convertible culture surface (CCS microwells).

Figure 3: Sequential time lapse images of Figure 4: Sequential time lapse images of formation of a spheroid, and differentiation to mitochondria activities. (a-c) Fluorescent images myotube cells in the microwell with alginate film. of Fig. 3 (d-f). (d-f) myoblasts cultured on the bare glass plate.

631 However, they did not differentiate effectively. In the case of Fig.5(d-f), myoblasts were three- dimensionally cultured on the convertible culture surface, and the large and long myotubes with many fused myoblasts were more effectively formed than myoblasts cultured on the bare glass surface. We also compared the differences of cellular differentiation behavior on the four kinds of conditions for verification of cell-cell or cell-substratum adhesion effect (Fig.6). We explained that cellular differentiation was able to be guided more efficiently than three kinds of other culture conditions by cell culture on the CCS microwells. These results suggest that cellular adhesion to a surface becomes a potential switch for cellular differentiation.

Figure 5: Fluorescent immunostaining images of Figure 6: Number of forming myotube cells under differentiated myoblasts after 244 h of culture. (a-c) the four type culture conditions. Myoblasts cultured on the bare glass surface. (d-f) Myoblasts cultured on the convertible culture surface.

CONCLUSION In this research, cell-cell or cell-substrate adhesion effect on cell differentiation was evaluated by using CCS microwells. The cell differentiation efficiency was improved by regulating cell-cell and cell- substrate adhesion using the CCS microwells compared with typical two dimensional cell cultures.

ACKNOWLEDGEMENTS This work was partly supported by a fund from the Japan Society for Promotion of Science (JSPS) KAKENHI via the Grant-in-Aid for Challenging Exploratory Research 25630384, the Asahi Glass Foundation. Also, the silicone adhesion bond (KE-44T) was provided by Shin-Etsu Chemical Co., Ltd..

REFERENCES [1] B.E. Reubinoff et. al., “Embryonic Stem Cell Lines from Human Blastocysts: Somatic Differentiation in vitro,” Nature Biotechnology, 18, 399-404, 2000. [2] Y. Nakashima, K. Tsusu, K. Minami, and Y. Nakanishi, “Development of a Cell Surface Conversion Technique Using Alginate Thin Film for Evaluating Effect upon Cellular Differentiation,” Rev. Sci. Instrum., 85, 065004, 2014.

CONTACT * Y. Nakashima ; phone: +81-96-342-3754; [email protected]

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