A novel method combining microfluidic channels and holey poly(dimethylsiloxane) PDMS membranes is presented for patterning multiple types of cells on a large variety of substrate, including substrate bearing micro- and nanometer-scale structures. Different kinds of cell are patterned on diverse flat substrates composed of various materials. Further, different cells are patterned on substrate with microgrooved substrate, and the influence of cell–cell and cell–substrate interactions about cell group behaviors is shown. This method provides a new approach for studying many processes in vitro caused by cell–cell and/or cell–substrate interactions. In addition, the method is straightforward, easy to access, convenient, and may be useful for a broad set of biological studies.