PHENOTYPIC MODULATION OF PLURIPOTENT STEM CELLS (PSCs) INDUCED BY MICROFABRICATION MATERIALS Ken-ichiro Kamei 1 , Yoshikazu Hirai 2 , Yoshihide Makino 2 , Momoko Yoshioka 1 , Li Liu 1 , Minako Nakajima 1 , Qinghua Yuan 1 , Yong Chen 1 and Osamu Tabata 2 1 Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Japan 2 Department of Micro Engineering, Kyoto University, Japan ABSTRACT Microstructured Photoresist (PR) has been applied for cell culture substrate, but their effects on cell behaviors are still unclear. Here, we evaluated the effects of PR substrates on human induced pluripotent stem cells (hiPSCs), which have a great potential for clinical applications. By comparing two types of epoxy based PRs with and without antimony salt, both PRs have affected on hiPSC viability after culture for seven days. KEYWORDS negative photoresists, polydimethylsiloxane, pluripotent stem cell, cellular phenotype, gene expression INTRODUCTION Human induced pluripotent stem cells (hiPSCs) hold a great potential for applications in cell-based therapy, regenerative medicine and drug development/screening. Indeed, hiPSCs can unlimitedly self-renew during culture in vitro without any karyotypical abnormality, and differentiate any kinds of cells in a body. However, there still are concerns of current hiPSC experimental setting consisted with feeder cells, which may release unknown factors resulting low reproducibility for culture and experiments, and cause xenogenic contamination with hPSCs. Thus, it necessitates developing a new hESC/iPSC experimental platform without feeder cells. Polymer MEMS technology has recently offered a simple three-dimensional (3D) fabrication for microfluidic devices, and is advantageous for creating new cellular microenvironmental cues to control stem cell functions [1]. Especially, there is an on-going trend towards applying microfluidics to stem cell biology. Although nano/micro-fabrications and mechanical properties of photoresist have been well-discussed, chemical effects such as a toxicity of antimony salt (an optical polymerization initiator) in photoresists and its derivatives on cell functions are still unknown. Therefore, as a first step in utilizing Bio-MEMS and nano/microfluidic devices for applications in stem cell research, we investigated the effects of these photoresist materials on hPSC phenotypes with multiple assays: cell adhesion, pluripotent status, proliferation, cytotoxicity, cellular heterogeneity and gene expression. With this information, we will be able to modify or develop new PRs that are applicable to stem cell research. EXPERIMENT hiPSC line (253G1) used in this study were cultured on Mitomycin C (MCC)-treated mouse embryonic fibroblast (MEF) cells in hPS medium, consisting of DMEM/F12 medium supplemented with 15% KnockOut Serum Replacement (KSR), 1% non-essential amino acid (NEAA), 0.1 mM β-mercaptoethanol, and 10 ng/mL basic fibroblast growth factor (bFGF). Media was changed daily, and these cells were passaged with StemPRO EZPassage kit (Invitrogen) weekly. First, we examined various surface treatments (i.e., corona treatment, gelatin coating, and Matrigel® coating) for commonly used PRs (TMMR-S2000®; called “S2000”) to facilitate PSC adhesion (Table 1). To facilitate mESC adhesion on PR substrates, we used these surface treatments to prepare PR substrates and used mouse embryonic stem cells (mESCs) to evaluate them. Table 1. Summary of treatments used for PR surfaces to improve cell adhesion Surface treatments Cell adhesion Colony formation Alkaline phosphatase None N.D. N.D. N.D. Gelatin + N.D. N.D. Matrigel +++ +++ +++ Corona + N.D. N.D. Briefly, corona treatment is a surface treatment technique that uses a low temperature discharge plasma to prepare a hydrophilic surface. Gelatin and Matrigel coatings on substrates are commonly used for cell cultures, including PSCs. mESCs could not adhere to a non-treated PR surface because of their hydrophobicity. Corona- and gelatin-treated PR surfaces became hydrophilic, although not sufficiently so for mESC adhesion. Compared with these treatments, Matrigel-coated PR substrates provided for better mESC adhesion. In addition, mESCs grew for seven S2000 Fig. 1 Photomicrograph of mESCs expressing alkaline phosphatase cultured on S2000 coated with Matrigel. 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 28 - November 1, 2012, Okinawa, Japan 978-0-9798064-5-2/μTAS 2012/$20©12CBMS-0001 1063