Mesenchymal Stem Cell Interactions with 3D ECM Modules Fabricated via Multiphoton Excited Photochemistry Ping-Jung Su, †,‡ Quyen A. Tran, †,‡ Jimmy J. Fong, § Kevin W. Eliceiri, ‡,§ Brenda M. Ogle, ‡,§,∥ and Paul J. Campagnola* ,‡,§,# ‡ Department of Biomedical Engineering, § Laboratory for Optical and Computational Instrumentation, ∥ Materials Science Program, # Medical Physics Department, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States * S Supporting Information ABSTRACT: To understand complex micro/nanoscale ECM stem cell interactions, reproducible in vitro models are needed that can strictly recapitulate the relative content and spatial arrangement of native tissue. Additionally, whole ECM proteins are required to most accurately reflect native binding dynamics. To address this need, we use multiphoton excited photochemistry to create 3D whole protein constructs or “modules” to study how the ECM governs stem cell migration. The constructs were created from mixtures of BSA/ laminin (LN) and BSA alone, whose comparison afforded studying how the migration dynamics are governed from the combination of morphological and ECM cues. We found that mesenchymal stem cells interacted for significantly longer durations with the BSA/LN constructs than pure BSA, pointing to the importance of binding cues of the LN. Critical to this work was the development of an automated system with feedback based on fluorescence imaging to provide quality control when synthesizing multiple identical constructs. 1. INTRODUCTION Since the isolation and characterization of stem cells from the body, significant effort has been devoted to developing methods to control their behavior, especially differentiation. A prominent direction has been in the form of optimizing mixtures of soluble factors to derive desired lineages. More recently, the extracellular matrix (ECM) has also been shown to be effective in initiating differentiation of stem cells. 1−4 In addition to providing structural support, the ECM directs cell shape, spreading, differentiation, migration, and proliferation, as well as new tissue synthesis, 5−9 by presenting a complex milieu of topographic, mechanical, and biochemical cues to cells. It is now clear that in these functions cells recognize 3D spatial and biochemical domains at the nano/microscale. 10−16 To better study the impact of ECM on stem cell behavior, many technologies have been employed to engineer micro- environments that mimic the native structure. For example, nano/microcontact printing and electrochemical fabrication have been used to modify substrates with micro/nanoscale features to study cell dynamics such as adhesion, spreading, differentiation, and migration. 17,18 The use of synthetic polymers in some of these techniques has reproduced the topography and mechanical properties of the native micro- environment, however, they can be limited in their ability to recapitulate the biochemical cues within the ECM. Additionally, cell-encapsulating hydrogels engineered with bioactive peptides and proteins can provide chemical and biological cues, but are difficult to decorate with sufficient spatial resolution to generate the naturally occurring complex topography. 19 Furthermore, these strategies generally use synthetic peptides or small motifs that cannot provide all the biochemical signals contained in whole proteins. Three-dimensional tissues generated with whole ECM proteins such as stacking cell sheets, decellularized tissues, or collagen gels can provide topographical, mechanical, and biochemical cues. 20−22 However, the undetermined concentration, composition, and lack of spatial control of these microenvironments make separating the role of the ECM, particularly from individual proteins, from the cell behavior a difficult task. In sum, these well-established existing methods do not readily afford the full recapitulation of the complex native ECM microenvironment in a controlled and reproducible manner and there remains a need for continued tool development to achieve this goal. Ultimately, a successful biomimetic approach will both enhance our understanding of stem cell−ECM interactions and also pave the way for therapeutic use of resulting scaffolds for tissue repair. Here we hypothesize that the use of whole protein 3D nano/ microstructured ECM constructs fabricated using multiphoton excited (MPE) photochemistry would provide both the biological and morphological properties necessary to better examine stem cell−ECM interactions. This photochemical process is analogous to two-photon excited fluorescence (TPEF), where the excitation, and here, the fabrication, is confined to the focal volume, resulting in intrinsic 3D capabilities and concurrently affording submicrometer feature sizes. 23−34 This method has advantages over conventional Received: June 21, 2012 Revised: August 8, 2012 Published: August 9, 2012 Article pubs.acs.org/Biomac © 2012 American Chemical Society 2917 dx.doi.org/10.1021/bm300949k | Biomacromolecules 2012, 13, 2917−2925