Cell sheet technology is a novel approach to preparing and harvesting monolayer cell sheets by using poly(N-isopropylacrylamide)(PNIPAAm)-modified surfaces as thermoresponsive cell culture substrates. At lower temperatures the cultured cells detach spontaneously as the surface of the substrate changes from hydrophobic to hydrophilic. In this way, an intact cell monolayer can be harvested non-invasively together with its underlying extracellular matrix (ECM). As a frequently used way to achieve scaffold-free tissue engineering, cell sheet technology holds great promise in cell-based regenerative medicine. Considering the limited availability of autologous cells and the timeliness requirement of clinical treatments, current attempts at improving the efficiency of thermo-responsive cell sheet harvest systems face a dilemma. On the one hand, the cell culture substrate must be very cell-adhesive to markedly promote rapid adhesion and proliferation of the limited autologous cells for a timely therapy. On the other hand, the same substrate should become very cell-repulsive after the formation of a confluent cell monolayer such that the mature cell sheet can be rapidly released without hurting the cells and their underlying ECM. Specifically, simple regulation of the chemical composition or the topography of the surface can only either promote cell adhesion or accelerate cell detachment and these surfaces commonly have very low bioactivity. Introducing cell-adhesive biomolecules by means of covalent binding or physical adsorption can improve the surface bioactivity but result in the inevitable deceleration of cell detachment and serious leakage of the biomolecules, respectively. Thus far, no single method without additional auxiliary means can effectively enhance cell adhesion during culture as well as facilitate the rapid harvest of cell sheets. To conquer this long-standing problem, we conceive of introducing cell-adhesive biomolecules to a thermo-responsive cell culture substrate in a reversible way and modulating them through temperature-dependent interactions. In this case, biomolecules can be stably immobilized on the substrate at cell culture temperature (37 8C), while they can be released as the temperature drops (e.g., 20 8C), thus facilitating both the initial cell adhesion and the final detachment of the cell sheet. With this strategy in mind, we find that the reversible interaction known as “specific binding” in noncovalent molecular imprinting is very appealing. As is well known, polymeric receptors with tailor-made recognition sites and “specific binding” properties comparable with those of natural receptors can be easily prepared by molecular imprinting. More importantly, molecularly imprinted polymers (MIPs) containing thermo-responsive recognition sites (i.e., the sites with temperature-dependent interactions between MIPs and targeted molecules) can be readily obtained using PNIPAAm-based materials. We herein report a novel system for harvesting cell sheets which relies on a PNIPAAm-based MIP hydrogel layer with thermo-responsive affinity toward specific biomolecules (Scheme 1). The commonly used cell-adhesive peptide ArgGly-Asp-Ser (RGDS) was chosen as the target biomolecule to demonstrate the proof-of-principle of our strategy. In our design, the thermo-responsive recognition sites in the MIPs were the tactic used to achieve temperature-dependent interactions between RGDS molecules and cell culture substrate. Specifically, besides the temperature-induced change of the surface wettability, the thermo-responsive recognition sites on the MIP hydrogel layer also resulted in the stable recognition and binding of RGDS at 37 8C and the triggered release of RGDS as the temperature was decreased. In contrast to the introduction of biomolecules by means of covalent binding or physical absorption, the thermo-responsive affinity of the MIPs toward RGDS not only significantly promotes cell adhesion during cell culture (37 8C) but also facilitates the detachment of cell sheets at low temperature (20 8C). To the best of our knowledge, although MIPs have exhibited some expanded bioapplications with the emergence of various biomolecule (e.g., proteins or peptides) imprinted polymers, this study is the first demonstration of molecular imprinting as a methodology to biofunctionalize thermoresponsive cell culture substrates to harvest cell sheets for potential biomedical applications. To achieve the best affinity between RGDS and the MIP matrix during cell culture, the imprinting process was performed by redox-initiated polymerization at 37 8C in [*] Dr. G. Pan, Q. Guo, Prof. Dr. H. Yang, Prof. Dr. B. Li Department of Orthopaedics The First Affiliated Hospital of Soochow University 188 Shizi Street, Suzhou, Jiangsu 215006 (China) and Orthopaedic Institute, Soochow University 708 Renmin Road, Suzhou, Jiangsu 215007 (China) E-mail: yueer@suda.edu.cn binli@suda.edu.cn
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Open Access
May 18, 2024
Bogdan-Cezar Iacob + 5
