Dynamic substrates whose cell adhesiveness changes in response to an external stimulus are useful not only for patterning cells in various geometries but also for inducing cell migration or arraying heterotypic cells. The requirements for such applications are high switching efficiency in cell adhesiveness and long-term persistence of the created cellular patterns. In this study, we prepared a dynamic substrate bearing photocleavable poly(ethylene glycol) (PEG) and examined the effect of the surface PEG density and the charge of cationic base materials on the above-mentioned key requirements. An amino-terminated substrate with a certain amino group density and charge was functionalized with photocleavable PEG5K, with and without subsequent backfilling of photocleavable PEG2K. The PEG chains made the surface non-cell-adhesive, but subsequent near-UV irradiation of the substrate induced photocleavage of the PEG, eventually making the surface cell-adhesive. The substrates were analyzed by atomic force microscopy, contact angle measurements, ellipsometry, and zeta potential measurements, complemented with protein adsorption observations. Although the density of amino group in the base material affected both the grafting efficiency of the backfilling PEG and the electrokinetic potential mainly in the positive range, the latter mainly determined the protein- and cell-repelling abilities of the substrates. Furthermore, varying the surface compositions had almost no effect on the switching efficiency in the early stage of the culture, but it became more significant after culturing cells for a longer time; the cells fouled the nonirradiated PEGylated regions earlier on the surfaces with higher positive zeta potentials. These results indicate that the zeta potential is an essential factor in the long-term persistence of cellular patterns on photoactivatable substrates. This study not only provides a recipe for the development of a dynamic substrate with an adequate time frame but also clarifies how the interfacial nanoarchitectures, composed of the nanometer-scale PEG brushes and charged base materials, affect the biocompatibility.