Preparation of nano-patterned substrates via Dip-pen Nanolithography for stem cell applications S. Oberhansl */** , A. Lagunas */** , E. Martinez */** , H. Jamil *** and J. Samitier */**/**** * Nanobioengineering Group, IBEC, Baldiri Reixac 10-12, 08028 Barcelona, Spain ** Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain *** Nanoink Inc. NanoInk Inc., 8025 Lamon Ave, Skokie, IL 60077, USA **** Department of Electronics, University of Barcelona, c/ Martí i Franquès 1, 08028 Barcelona, Spain ABSTRACT Micro- and nano-scale patterns are known to have a high impact on cell adhesion and are very interesting for stem cell research since they represent highly defined and reproducible systems which might be beneficial for obtaining a homogeneous stem cell culture. Homogeneity is a basic requirement for subsequent therapeutic application in the field of regenerative medicine. In this work, we are using nano-patterned substrates created with Dip-pen Nanolithography (DPN) together with the immobilization of BMP-2 in order to achieve this highly controlled environment for stem cell differentiation experiments. Keywords: Dip-pen Nanolithography, stem cell differentiation, nano-patterning, BMP-2 1 INTRODUCTION In the past two decades, the emerging nanotechnologies gave access to the field of nanomedicine, which amongst other include approaches to biosensing [1], basic cell biology, tissue engineering and regenerative medicine in order to treat various diseases [2]. The role of micro- and nano-technology in these areas is evident since already existing fabrication technologies could not provide access to nanoscale structures. The reason for this is in the body’s cells: cell adhesion is a crucial process for anchorage dependent cells. In vitro experiments have proved that, in order to be viable, spread and proliferate, these cells need to attach to adhesive surfaces [3]. The cell receptors that play a role in attachment and signal transduction are at the micro- and nano-length scale themselves [4], as well as the intracellular proteins that enable the cells to exert forces onto the substrate [5]. In literature, there are many examples of cells being influenced by chemical or topographical cues as micro- or nano-features, wherein the cell alignment, polarization, migration, proliferation and gene expression is altered [6]. Recent studies have shown that the geometrical order of the micro- or nano-pattern is important [7] and controls cell proliferation and even differentiation. Given these novel tools, it is evident to not only apply them in somatic cell culture but moreover take advantage of them in stem cell research, focussing on their potential ability to generate or maintain a population of cells with homogeneous phenotype, which is the ultimate challenge when working in the area of stem cell differentiation, in order to fully take advantage of their therapeutic potential. Stem cells have been a hot topic in the last few years and there were many publications and press releases about their promising application in regenerative medicine [8]. They are undeveloped cells and posses the ability of self renewal, differentiation and tissue regeneration. There are two different types of human stem cells – embryonic stem cells (ESC) and adult stem cells. Latter ones can be obtained from different adult cell sources like bone marrow, umbilical cord blood, adipose and neural tissue. They are called multipotent and are not able to differentiate into any type of cell, but only into several phenotypes. A well known cell line are the mesenchymal stem cells (MSC) which upon exposing to appropriate stimuli can be differentiated into either osteoblasts, myoblasts or neurons [9]. The advantage of this cell type (non-ESC) is that they can be obtained from adult tissue and could be used in stem cell therapy (re-implantation of stem cells in an individual after in-vitro differentiation). ESCs are obtained from the embryo and are called pluripotent since they can differentiate into every cell type found in the body [10]. Although ESCs are the ideal model for regenerative medicine, they are very controversial considering the ethical issue of extraction of cells from embryos. Opposing the theory of potential benefits of stem cells, in practice there are still many unsolved problems concerning effective stem cell culture. Like every other cell type, stem cells are sensitive to many different signalling cues coming from their microenvironment [11, 12]. Therefore, a high amount of control over these cues is necessary in order to achieve homogeneous cell populations. Homogeneity can be influenced, among other things, by the administration of so called soluble exogenous stimuli (biochemical factors, often presented as media supplements). The interactions of cells with these soluble factors, as well as with extra-cellular matrix (ECM) proteins, are usually studied by multiwell plate assays. This NSTI-Nanotech 2010, www.nsti.org, ISBN 978-1-4398-3402-2 Vol. 2, 2010 206