Biomaterials 23 (2002) 561–567 Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture Masayuki Yamato, Chie Konno, Mika Utsumi, Akihiko Kikuchi, Teruo Okano* Institute of Biomedical Engineering, Tokyo Women’s Medical University, Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, Japan Received 24 January 2001; accepted 3 April 2001 Abstract Tissue engineering constructs that effectively duplicate natural tissue function must also maintain tissue architectural and organization features, particularly the integration of multiple cell types preserving distinct, integrated phenotypes. Cell–cell communication and biochemical cross-talk have been shown to be essential for the maintenance of differentiated cell functions in tissues and organs. Current limitations of cell-culture hinder progress in understanding the features and dynamics of heterotypic cell communication pathways critical to developing more sophisticated or effective tissue-engineered devices. We describe a method to conveniently electron-beam pattern cell culture surfaces with thermo-responsive polymer chemistry that exploits changes in cell- polymer adhesive interactions over a temperature window amenable for high-throughput cell culture. Cells seeded on these patterned surfaces at 201C adhere only to surface areas lacking thermo-responsive grafting chemistry: grafted domains at 201C are hydrophilic and non-cell adhesive. The culture temperature is then increased to 371C, collapsing the hydrated grafted chemistry. A second cell type is added to the culture and adheres only to these exposed relatively hydrophobic grafted patterns. Both cell types can then be effectively co-cultured at 371C under multiple conditions. Long-term cell pattern fidelity and differentiated cell functions characteristic of each co-planar cell type are observed. This method is simple and has few limitations, compared with other existing co-culture methods. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Surface patterning; Electron beam irradiation; Hepatocyte; Poly(N-isopropylacrylamide); Cell culture; Co-culture 1. Introduction We have previously reported our methods for controlling cell-surface adhesion by exploiting cell culture temperature and surface-grafted temperature- responsive polymers [1–4]. Using electron beam irradia- tion, poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted as a thin layer onto commercial tissue culture grade polystyrene (TCPS) dishes. Above this polymer’s lower critical solution temperature (LCST, 321C), dehydrated PIPAAm-grafted surfaces exhibit properties similar to TCPS, facilitating cell adhesion and culture. Various cells including hepato- cytes, endothelial cells, fibroblasts, keratinocytes, epithe- lial cells, macrophages, and microglial cells adhere and readily proliferate on these grafted surfaces [5]. By simply reducing the culture temperature, PIPAAm- grafted culture surfaces rapidly hydrate, exhibiting hydrophilicity similar to that of hydrated, non-adhesive poly(ethylene glycol)-grafted surfaces [6]. Cell adhesion is thus blocked on PIPAAm-grafted surfaces below the LSCT. Therefore, cells adhered on PIPAAm-grafted surfaces spontaneously lift by reducing culture tempera- ture below the LCST without any need for trypsin or EDTA [2]. This cell recovery method is so non-invasive that differentiated cells such as hepatocytes and en- dothelial cells retain higher differentiated functions compared to trypsin-harvested cells [1]. Electron beam radiation, conveniently masked by various materials including glass, metals, and plastics including polyethylene, can be used to pattern grafted PIPAAm on culture surfaces, yielding localized tem- perature-responsive surface regions in various size regimes and geometries (Fig. 1) [7,8]. We previously utilized patterned grafted surfaces to successfully produce a heterotypic cell co-culture of primary hepatocytes and endothelial cells (Method 1 in Fig. 2) [8]. In this earlier preliminary study, PIPAAm was *Corresponding author. Tel: +81-3-3353-8111; fax: +81-3-3359- 6046. E-mail address: tokano@lab.twmu.ac.jp (T. Okano). 0142-9612/02/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII:S0142-9612(01)00138-7