Biomaterials 24 (2003) 2863–2870 A modified microstamping technique enhances polylysine transfer and neuronal cell patterning John C. Chang a, *, Gregory J. Brewer b , Bruce C. Wheeler a a Department of Electrical and Computer Engineering, Beckman Institute, University of Illinois at Urbana-Champaign, 405 N. Mathews Ave., Urbana IL 61801, USA b Deparments of Neurology and Medical Microbiology and Immunology, Southern Illinois University School of Medicine, PO Box 19626, Springfield IL 62794-9626, USA Received 23 September 2002; accepted 20 February 2003 Abstract 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. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Patterning; PDMS; Hippocampal; Microstamping; Surfactant 1. Introduction Over the last decade, several groups have promoted microstamping as an ideal technique for transferring small and large molecules to pattern cells in culture. In using small molecules to pattern cells, both alkanethiols and alkylsilanes have been transferred onto substrates with patterned polydimethylsiloxane (PDMS) stamps. The small molecules are first absorbed into the PDMS polymer and then diffuse to the substrate when the substrate is in contact with the stamp. Alkanethiols have been patterned with PDMS stamps by Singhvi et al. to control hepatocyte growth on stamped substrates [1]. The preferential growth is achieved presumably because the alkanethiols form a hydrophobic interface to adsorb more protein than the surrounding gold which is hydrophilic [2]. Alternatively, St. John patterned astro- glia by stamping aminosilane, a positively charged molecule to attract cells, onto silicon surfaces [3]. Larger macromolecules can also be stamped to induce patterned growth of neurons. Branch et al. first patterned cells by directly stamping polylysine onto activated glass surfaces [4], and then demonstrated high- quality, long-term patterning of hippocampal neurons by attaching polyethyleneglycol (PEG) to the back- ground regions [5]. Similarly, James et al. adsorbed polylysine from microstamps to guide neuronal growth on electrode arrays [6]; other scientists have also used microstamps to pattern macromolecules on other substrates [7–10]. *Corresponding author. Tel.: +1-217-244-2692. E-mail addresses: jcchang@uiuc.edu (J.C. Chang), gbrewer@ siumed.edu (G.J. Brewer), bwheeler@uiuc.edu (B.C. Wheeler). 0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00116-9