Switching of cell growth/detachment on heparin-functionalized thermoresponsive surface for rapid cell sheet fabrication and manipulation

Abstract

Heparin-functionalized poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surface was designed for the switching of cell growth/detachment, achieved by the regulation of affinity binding between basic fibroblast growth factor (bFGF) and immobilized heparin through the temperature-dependent conformational change of grafted P(IPAAm-co-CIPAAm) chains. At 37 °C, bFGF-bound heparin-thermoresponsive surfaces were able to hold the two- to three-fold number of mouse fibroblast (NIH/3T3) cells than both bFGF-physisorbed surface and PIPAAm surface with soluble bFGF after a 3-day cultivation. Bound bFGF via heparin on shrunken grafted P(IPAAm-co-CIPAAm) chains at 37 °C was able to reinforce the formation and stabilization of bFGF-FGF receptor complex, although the activity of physisorbed bFGF on PIPAAm-grafted surfaces was decreased by non-specific and randomly oriented adsorption. At 20 °C, the cultured NIH/3T3 cell sheet with bFGF detached from heparin-functionalized thermoresponsive surface. The release of bFGF from the surfaces was induced by reducing the affinity binding between bFGF and immobilized-heparin due to increasing the mobility of the swollen grafted P(IPAAm-co-CIPAAm) chains. Therefore, heparin-functionalized thermoresponsive surface was able to enhance cell proliferation, and confluent cells detached themselves as a contiguous cell sheet due to switching cell growth by changing temperature. A cell culture system using this surface is useful for rapid cell sheet fabrication and manipulation.

Introduction

Special attention has been paid to human tissue and organ regenerations, which are achieved by transplanting in vitro engineered three-dimensional (3D) tissues prepared from autologous cells with biodegradable materials as a scaffold [1]. In contrast, our laboratory has pioneered an approach for constructing three-dimensional (3D) tissues without a scaffold from a contiguous cell monolayer (cell sheet) [2], which is prepared on a thermoresponsive cell culture surface where poly(N-isopropylacrylamide) (PIPAAm) [3] exhibiting a lower critical solution temperature (LCST) of around 32 °C was grafted onto tissue culture polystyrene (TCPS). Cultured cells on the thermoresponsive cell-culture surfaces detach themselves as cell sheets by lowering temperature below 20 °C [4], [5]. Cell sheet-based clinical therapies have been successfully applied to the treatments of dilated cardiomyopathy [6], cornea epithelium defect [7], periodontal ligament damage [8], cartilage injury [9], and esophageal ulceration [10], [11].

For the further advancement of cell sheet-based therapy, the development of a cell culture system to reduce a culture period contributes to the rapid fabrication and implantation of cell sheets. In conventional cell culture systems, the frequent dosing of growth factors in medium is required as soluble stimulants for the acceleration of cell growth, because soluble growth factors induce the degradation of signaling molecules to reduce their stimulation [12]. On the other hand, the immobilization of growth factors onto cell culture surfaces suppresses the down-regulation of receptors, resulting in the long-term activation of the intracellular signal transductions [13]. However, protein-immobilization is known to lead to reducing their bioactivities [14] and is difficult to preserve a protein in native state and optimize the orientation for obtaining the adequate interaction with target receptors [15], [16].

For binding biologically active growth factors onto thermoresponsive cell culture surfaces, heparin is covalently tethered onto poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surfaces. Heparin is well known to possess an affinity interaction with various heparin-binding proteins such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), fibronectin (FN), and antithrombin III (AT-III), and plays important roles in regulating the stabilities and activities of these proteins [17]. In particular, heparin has a high affinity with bFGF (dissociation constant < 10–90 nmol/L) [18], [19], [20] and forms a complex of bFGF and FGF receptor (FGFR) [21], [22], which reinforces bFGF-FGFR binding interaction. bFGF molecules easily bound to surface-immobilized heparin on shrunken P(IPAAm-co-CIPAAm) chains at 37 °C, leading to cell growth through the stable formation of bFGF-FGF receptor complex (Fig. 1A). Binding of active bFGF on the surfaces would accelerate cell growth, resulting in the formation of cell sheet. With lowering temperature to 20 °C, bound bFGF molecules to the immobilized-heparin are considered to be released with the cultured cell sheet due to the dynamic motion of heparin accompanied with the swelling of P(IPAAm-co-CIPAAm) chains (Fig. 1B). In previous works, the introduction of bioactive molecules such as peptides and proteins to P(IPAAm-co-CIPAAm)-grafted surfaces [23], [24] not only accelerates cell adhesion [25] and cell growth [26], but also induces the detachment of cells due to reducing the affinity interaction between a receptor on cultured cells and a ligand on the surface by lowering temperature [27]. However, the detachment of cultured cell sheets by regulating the multivalent affinity binding between such as bFGF and immobilized heparin with lowering temperature has hardly been studied.

In this study, the surface design of heparin-functionalized thermoresponsive surfaces was optimized for enhancing cell growth. To elucidate the binding status of bFGF, NIH/3T3 cells were cultured on three different bFGF status types of the surfaces; heparin-mediated bound, physically adsorbed, and soluble forms. Additionally, the detachment of cultured NIH/3T3 cell sheets by low-temperature treatment and the fluorescence immunostaining of the harvested cell sheets to trace the surface-bound bFGF were investigated.