Rapid cell sheet detachment using spin-coated pNIPAAm films retained on surfaces by an aminopropyltriethoxysilane network Nikul G. Patel a , John P. Cavicchia a , Ge Zhang a,⇑ , Bi-min Zhang Newby b,⇑ a Department of Biomedical Engineering, The University of Akron, Akron, OH 44325-0302, USA b Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906, USA article info Article history: Received 30 September 2011 Received in revised form 8 March 2012 Accepted 20 March 2012 Available online 1 April 2012 Keywords: Rapid cell sheet detachment Thermally responsive material Poly(N-isopropylacrylamide) (pNIPAAm) Aminopropyltriethoxysilane (APTES) Human mesenchcymal stem cells abstract The ability to harvest cell sheets grown on thermoresponsive polymers, such as poly(N-isopropylacryl- amide) (pNIPAAm), has been widely studied for use in tissue engineering applications. pNIPAAm is of special interest because of the phase change that it undergoes in a physiologically relevant temperature range. Two primary approaches have been adopted to graft pNIPAAm chains covalently onto tissue cul- ture polystyrene dishes: electron beam irradiation and plasma polymerization. These approaches often involve non-easily accessible (e.g. e-beam) facilities and complicated procedures that have hindered most tissue culture laboratories in adopting this technology for their specific applications. In this study, we developed a simple and cost-effective approach to create thermoresponsive surfaces using commercially available pNIPAAm. Using a simple spin-coating technique, thermoresponsive thin films were deposited on glass slides or silicon wafers using pNIPAAm blended with a small amount of 3-aminopropyltriethoxy- silane (APTES), which enhances the retention of pNIPAAm on the surface. We found that the thermore- sponsive films created using our method support cell attachment and proliferation without additional adhesive proteins as well as cell sheet detachment within minutes. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction The use of thermoresponsive polymers (TRPs), especially poly(N-isopropylacrylamide) (pNIPAAm) and its co-polymers, has attracted considerable attention in the area of cell sheet engineer- ing [1–3]. For cell sheets formed on TRPs, a simple change of temperature allows cells to spontaneously detach from these sur- faces. Compared to proteolytic enzymes (e.g. trypsin) or mechanical agitations, using TRPs to harvest confluent cell sheets minimizes damage to cells and their excreted extracellular matrix (ECM), thus preserving their biological functions [4]. Tissue engineering con- structs based on cell sheets allow increased cell–cell interactions and eliminate the risk of immunogenic materials present in scaf- folds. Furthermore, cell sheets harvested from TRPs can be pat- terned and assembled together to mimic the microarchitecture of native tissue, which is crucial for functional tissue regeneration. TRPs have the ability to respond to a change in temperature and can be classified into two main types: TRPs possessing a lower crit- ical solution temperature (LCST) and TRPs possessing an upper critical solution temperature (UCST). pNIPAAm is one of the most commonly used TPRs that presents an LCST. Other polymers with thermoresponsive properties include poly(N,N-diethylacrylamide) (PDEAAm), poly(N-vinlycaprolactam) (PVCL), poly[2-(dimethyl- amino)ethyl methacrylate] (PDMAEMA) and poly(ethylene oxide) (PEO) [5]. Among all the TRPs, pNIPAAm is of special interest in bioengineering applications because of the phase change that it undergoes in a physiologically relevant temperature range. It has a lower critical solution temperature (LCST) of 32 °C in water. The polymer chains reside in a collapsed hydrophobic state above the LCST and in an extended hydrophilic state below LCST [6]. In most studies, a grafted pNIPAAm thin layer (<30 nm in thickness) or a combination of adhesive proteins with bulk pNIPAAm allows cell attachment above LCST and detachment when cooled below LCST. Two primary approaches have been adopted to covalently graft pNIPAAm chains onto tissue culture polystyrene dishes. One meth- od uses electron beam irradiation [7] and the other uses plasma polymerization [8–10]. Although the covalently grafted pNIPAAm surface is a major achievement in cell sheet engineering, grafting pNIPAAm chains to form an optimal layer for cell seeding involves non-easily accessible (e.g. e-beam) facilities and complex fabrica- tion methods. The use of commercially available thermoresponsive dishes is impractical for long-term tissue culture and large-scale investigation because of the cost of these dishes (over US$20 for one 35 mm dish at the time of writing [11]). Therefore, a simpler and more efficient approach to fabricate thermoresponsive sur- faces for cell sheet harvesting is desired. 1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.03.031 ⇑ Corresponding authors. Tel.: +1 330 972 2510; fax: +1 330 972 5856. E-mail address: bimin@uakron.edu (B.-m. Zhang Newby). Acta Biomaterialia 8 (2012) 2559–2567 Contents lists available at SciVerse ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat