**1. Introduction**

34 Advances in Unconventional Lithography

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Regeneration of the central nervous system (CNS), where proliferative potency is limited, is one of the most important research themes in neuroscience and neuroengineering. Pluripotent stem cell lines have attracted broad attentions as an important model system for regenerating damaged brain. The important key for realizing regenerative medicine using pluripotent stem cells is to induce undifferentiated cell into objective cells with high efficiency and reproducibility. Although the methods to induce embryonic stem (ES) cells into specific neuronal subtype with pharmacological treatment have been proposed, however, the handling is difficult and thus the efficiency rate of objective cells is low (Barberi et al., 2003). The main reason for low differentiation rate of pluripotent stem cells is difficulty in precise control of interaction between pharmacological treatment and cell signalling. To overcome these problems, development of alternative methods for precise control of cell differentiation processes is required.

During cell differentiation, both endogenous factors, such as cell-cell signal transmission, and exogenous factors, such as pharmacological application, play important roles. Micro and soft lithography-based surface modification of culture substrate enabled to control stem-cell-aggregation (EB; Embryoid body) sizes and therefore to increase cell differentiation efficiency through promotion of cell-cell signal interactions (Karp et al., 2007; Wang et al., 2009). Particularly, Park et al. reported that small-size EBs of mouse ES cells (100 ~ 200 m of diameter) tended to differentiate into ectoderm and large-size EB (500 m of diameter) into mesoderm (Park et al., 2007). These reports suggested that manipulation of EB size and shape could affect endogenous factors of stem cell EBs and thus was an important approach for precise control of differentiation processes. Another important technique for regulating cell differentiation is applying physical stimulation. It was reported that electrical or magnetic stimulation could induce cellular and molecular responses and affected the gene expressions during differentiation (Kimura et al., 1998; Piacentini et al., 2008). Particularly, Yamada et al. reported that applying electrical stimulation induced mouse ES cells efficiently into ectoderm cells (Yamada et al., 2007). These reports suggested

Toward the Precise Control of Cell Differentiation Processes by Using Micro and Soft Lithography 37

each microcavity of the electrode substrates. To our experience, initial plating of 500 cells resulted in a EB of 200 m diameter and 4000 cells in a EB of 500 m diameter at next day of plating. The microcavity-array electrode substrates were previously coated with MPC (2-methacryloyloxyethyl phosphorylcholine) polymer (Lipidure CM5206E, NOF corp.) to prevent nonspecific cell adhesion (Ishihara et al., 1999) and to localize EBs into each

Fig. 1. Schematic of procedures for fabricating the microcavity-array electrode substrate

Fig. 2. Schematic diagram of the experimental system. Size-controlled EBs were

simultaneously stimulated through the microfabricated ITO substrates.

microcavity.

using SU-8 negative photoresist.

that applying physical stimulation could affect exogenous factors and also differentiation processes of stem cells. Although the experimental methods to regulate the endogenous or exogenous signalling have been proposed as above, increase in differentiation efficiency is inadequate for regeneration therapy, and combination of the two approaches is not reported.

Based on these problems, we have tried to propose a precise control method for cell differentiation by combining EB size treatment and electrical stimulation technique. To do this, we developed microcavity-array device with embedded electrodes (Takayama et al., 2009). Uniform-sized EBs of P19 cells were prepared and aligned in the electrode substrate, and were stimulated uniformly and simultaneously. However, difficulty in handling EBs and limited number of stimulating EBs hindered further analysis. In this chapter, we describe an alternative method to stimulate more number of EBs based on soft-lithography technique and recent attempt to analyse the effects of EB size treatment and electrical stimulation on the gene expression of P19 cells.
