Thermo‐Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets

Abstract

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. [1] 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. [2] 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 [3] and these surfaces commonly have very low bioactivity. [4] 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 [5] and serious leakage of the biomolecules, [4, 6] 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. [7] 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. [8] We herein report a novel system for harvesting cell sheets which r...

Molecular imprinting: perspectives and applications

“…Lightly cross-linked polymer gels can undergo reversible swelling and shrinking in response to environmental temperature changes, called thermo-responsive gels, which can be used as smart materials for drug delivery, tissue engineering, and cell encapsulation in biochemistry. [251][252][253][254][255][256] This type of thermo-sensitive recognition is very similar to the recognition of proteins in natural systems. Thus, thermo-responsive gels with biocompatibility could have potential applications in the design of protein-imprinted polymer matrices.…”

“…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. 256 Unlike the above reported PNIPAAm-based MIPs, which demonstrated thermo-sensitive properties simply because of the hydrophilicity/hydrophobicity of PNIPAAm responding to changes in temperature, new thermo-responsive MIPs have been developed recently. For example, Aburto et al 258 synthesized a dibenzothiophene sulfone (DBTS)-imprinted chitosan hydrogel (IH DBTS ) by cross-linking chitosan with glutaraldehyde in the presence of DBTS as the template.…”

Switchable phase transition behavior of thermoresponsive substrates for cell sheet engineering

“…Up to now, many types of cells such as bovine aortic endothelial cells, L929 mouse fibroblasts, NIH/3 T3 mouse fibroblasts, and MC3T3‐EI mouse calvarial osteoblasts have been detached from PNIPAAm‐modified thermoresponsive surfaces. Because of structural properties variation in PNIPAAm‐modified surfaces constructed by different fabrication techniques, the efficacy of cell detachment significantly alters from case to case.…”

Polypropylene non-woven supported fibronectin molecular imprinted calcium alginate/polyacrylamide hydrogel film for cell adhesion

“…Pan et al [9] 29 employed a molecular imprinting methodology to introduce the 30 cell-adhesive peptide RGDS onto a thermo-responsive cell culture 31 substrate, which was innovatively used as a highly efficient novel 32 system for harvesting cell sheets. Fukazawa et al [10] proposed a 33 new molecular imprinting procedure for the purpose of cell 34 capture using the cell-adhesive protein fibronectin (FN) as the 35 template.…”