Despite the rapidly increasing number of publications on the fabrication and use of micro-patterns for cell studies, comparatively little is know about the long-term stability of such patterns under cell culture conditions. Here, we report on the long-term stability of cellular patterns created by three different patterning techniques: selective molecular assembly patterning, micro-contact printing and molecular assembly patterning by lift-off. We demonstrate that although all three techniques were combined with the same background passivation chemistry based on assembly of a PEG-graft copolymer, there are considerable differences in the long-term stability between the three different pattern types under cell culture conditions. Our results suggest that these differences are not cell-dependent but are due to different (substrate-dependent) interactions between the patterned substrate, the passivating molecule and the serum containing cellular medium.