Macromolecular microstamping with polydimethylsiloxane (PDMS) stamps has been demonstrated to transfer proteins onto glassy substrates for antigen or antibody detection and for cell patterning. For many applications, including neuronal cell patterning, it is important to assure reliable transfer of sufficient quantity of protein. Research has shown that protein transfer is enhanced with the selection of the proper protein-stamp-substrate combination. In addition, detergent studies have shown that detergent-protein complexes detach from surfaces to a greater extent than proteins alone. Therefore, we hypothesized that stamp surface modification (termed here a release layer) can enhance polylysine transfer and benefit cell growth on microstamped substrates. We found unmodified stamps to transfer insufficient polylysine to support good cell survival of hippocampal neurons in a widely used serum-free, reduced-glia cell culture system. However, with modified stamps neuronal growth was reliably good. This enhanced cell growth can be attributed to the increased polylysine transfer due to the release layer rather than increased loading onto the stamp. This enhancement was found to be even greater for two-month old stamps that were stored in water. Furthermore, the physicochemical properties of the release layer can modulate the loading process. Thus, our data supports the conclusions that the release layer: (1) modulates polylysine loading, (2) enhances polylysine transfer, (3) enhances cellular growth on microstamped substrates, and (4) extends the durability (defined as the number of times a stamp can be reused) of PDMS microstamps.