Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin a5b1 ligand scaffolds Zhaohui Li, 1 * Michaela Kreiner, 2 RuAngelie Edrada-Ebel, 2 Zhanfeng Cui, 3 Christopher F. van der Walle, 2 Helen J. Mardon 1 1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom 2 Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom 3 Department of Engineering Science, Institute of Biomedical Engineering, Old Road Campus Research Building, University of Oxford, Headington, Oxford OX3 7DQ, United Kingdom Received 11 January 2011; accepted 25 May 2011 Published online 16 August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33177 Abstract: A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endome- trial stromal cells (EnSCs). EnSCs encapsulated in the func- tional alginate were cultured under perfusion using the TissueFlex V R platform, a multiple parallel microbioreactor sys- tem for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, prolifera- tion, and extracellular matrix (ECM) deposition were deter- mined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetra- meric FN scaffolds under perfusion, associated with a signifi- cant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non- functionalized alginate scaffolds (irrespective of perfused/ static culture) and 3D static culture (irrespective of the scaf- fold). Conjugation of alginate to FN was an absolute require- ment to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate- monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin a5b1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering. V C 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 99A: 211–220, 2011. Key Words: functional alginate, endometrium, tissue engi- neering, fibronectin, perfusion microbioreactor How to cite this article: Li Z, Kreiner M, Edrada-Ebel R, Cui Z, van der Walle CF, Mardon HJ. 2011. Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin a5b1 ligand scaffolds. J Biomed Mater Res Part A 2011:99A:211–220. INTRODUCTION In vitro cell adhesion and proliferation are strongly depend- ent on the cells’ physical environment and consequently the nature of the three dimensional (3D) cell scaffold and the perfusion of media. In particular, the engagement between the cell and its matrix is a critical element in guiding cell behavior and functionality. 1 Integrin cell surface receptors must be able to interact with the surrounding (3D) extracel- lular matrix (ECM) to promote cell adhesion. Numerous materials have been functionalized with ‘‘RGD’’ (arginine– glycine–aspartic acid) the primary cell binding domain of fibronectin (FN) to form biomimetic scaffolds for tissue engineering and application in regenerative medicine. 2 How- ever, the ECM component involved in integrin-mediated cell adhesion is dependent on the integrin subtype. For example, osteoblasts bind collagen via a1b1 and a2b1 integrins, whereas fibroblasts bind FN via a5b1 integrin. 3 Thus, incor- poration of specific integrin ligands into synthetic scaffolds can be used to selectively engage certain cell types and elicit cell-specific responses. This strategy provides a route to generating engineered tissue grafts which have applica- tion in the study of womens’ health and implantation of the embryo, which requires Rac1-dependent endometrial stromal cell migration. 4 *Present address: Zyoxel Ltd., Centre for Innovation & Enterprise, Oxford University Begbroke Science Park, Sandy Lane, Yarnton, Oxford OX5 1PF, United Kingdom Correspondence to: C. F. van der Walle; e-mail: chris.walle@strath.ac.uk or H. J. Mardon; e-mail: helen.mardon@obs-gyn.ox.ac.uk Contract grant sponsor: Biotechnology and Biological Sciences Research Council; contract grant number: BB/D522497/1 Contract grant sponsor: Medical Research Council Milstein Fund V C 2011 WILEY PERIODICALS, INC. 211