This chapter focuses on the use of micro-sized cell culture substrates fabricated by micro electro mechanical systems (MEMS) technologies in the field of three-dimensional (3D) cellular tissue constructions. First, we provide a brief overview of MEMS-based tissue engineering methods, and explain why the micro-sized cell-laden substrates are important. Second, we explain the fabrication processes of the micro-sized cell-laden substrates. The fabrication of micro-sized substrates is described by focusing on their materials, structures, cell culture processes, and handling. Third, their applications for single cell handling is described by providing information on observation, collection, and arrangement. Finally, assembly of 3D cellular constructs is explained by pick-and-place assembly, self-assembly, and self-folding methods. Key advantages of the micro-sized substrates are the ability of manipulating various types of adherent cells and also spatial control of cells in 3D cellular tissues. The single cell handling ability facilitates the cell-cell interaction analysis in the field of pharmacology, and 3D cellular assembly ability will open the route for fabrication of spatially-controlled heterogeneous 3D cellular tissues in the regenerative medicine field.