Cell sheet engineering for myocardial tissue reconstruction
Introduction
Recently, alternative treatments for cardiac transplantation have been strongly requested to repair damaged heart tissue, because the utility of heart transplantation is limited by donor shortage. Cell therapy is now considered to be one of the most effective treatments for impaired heart tissue [1], [2]. Direct transplantation of cell suspension has been researched since the early 1990s [3]. In these studies, survival of transplanted cells, integration of native and grafted cells, and improvement of host cardiac function have been reported. It is a critical point how to isolate and expand clinically transplantable myocardial cell source. Autologous myoblast transplantation has been performed clinically and the contraction and viability of grafted myoblasts have been confirmed [4]. Multipotent bone marrow cells or embryonic stem cells have been now aggressively investigated as possible candidates for human implantable myocardial cell source [5], [6], [7], [8].
In direct injection of dissociated cells, it is difficult to control shape, size and location of the grafted cells. Additionally, isolated cell transplantation is not enough for replacing congenital defects. To overcome these problems, research on fabricating three-dimensional (3-D) cardiac grafts by tissue engineering technology has also now begun [9]. Tissue engineering has currently been based on the concepts that 3-D biodegradable scaffolds are useful as alternatives for extracellular matrix (ECM) and that seeded cells reform their native structure in according to scaffold biodegradation [10]. This context has been used for every type of tissue. In myocardial tissue engineering, poly(glycolic acid) (PGA), gelatin and alginate have been used as prefabricated biodegradable scaffolds. Papadaki et al. engineered 3-D cardiac constructs by using PGA scaffolds processed into porous meshes and rotating bioreactors [11]. Li et al. have demonstrated that transplantation of tissue-engineered cardiac grafts using biodegradable gelatin sponges replaced myocardial scar and right ventricular outflow track defect [12], [13]. Furthermore, Leor et al. reported that bioengineered heart grafts using porous alginate scaffolds attenuated left ventricular dilatation and heart function deterioration in myocardial infarction model [14]. As the technique premixing cells and ECM alternatives instead of seeding cells into preformed scaffolds, Zimmermann et al. engineered 3-D heart tissue by gelling the mixture of cardiomyocytes and collagen solution [15], [16]. The construct has allowed direct measurement of isometric contractile force as heart tissue model.
In spite of these desirable results, insufficient cell migration into scaffolds and inflammatory reaction due to scaffold biodegradation remain problems to be solved [13], [14]. In native myocardial tissue, cells are considerably dense (Fig. 1A) in comparison with other tissues including cartilage, vascular, and heart valve, which are cell-sparse tissues and have been successfully engineered by using biodegradable scaffolds (Fig. 1B). Cardiomyocytes are also tightly interconnected with gap junctions, which mediated the reciprocal exchange of small molecules and ions resulting in electrically synchronous beating [17]. In myocardial tissue engineering, biodegradable scaffolds themselves attenuate cell-to-cell connections and scaffold biodegradation leads to fibrous tissues containing excessive amount of ECM, which is shown in pathological states including ischemic heart disease or dilated cardiomyopathy. Investigators are now trying to fabricate more porous structure of biodegradable scaffolds and to develop new techniques seeding more cells into the scaffolds. In particular, structural balance between cells and ECM should be controlled to fabricate native heart-like tissues.
By contrast, we now propose novel tissue engineering methodology that is to construct 3-D functional tissues by layering 2-D cell sheets without any biodegradable alternatives for ECM. To obtain viable cell sheets, we have exploited intelligent culture surfaces, from which cultured cells detach as a cell sheet simply by reducing temperature. In this paper, we present the new technology “cell sheet engineering” and its application to myocardial tissue reconstruction.
Section snippets
Temperature-responsive culture surfaces
Temperature-responsive culture surfaces were developed among the research to control cell adhesion to biomaterials. Cells adhere to culture surfaces via membrane receptors and cell adhesive proteins, including fibronectin, that reside in serum or are secreted from the cells in culture (Fig. 2A). The interaction between adhesive proteins and culture surfaces depends on the wettability of the surface. Normal tissue culture polystyrene (TCPS) dishes are hydrophobic and absorb ECM proteins
Cell sheet engineering
When cells are cultured confluently, they connect to each other via cell-to-cell junction proteins and ECM (Fig. 3A). With enzymatic digestions, these proteins are disrupted and each cell is released separately (Fig. 3B). In the case using PIPAAm-grafted surfaces, cell-to-cell connections are not disrupted and cells are harvested as a contiguous cell sheet by decreasing temperature (Fig. 3C). Furthermore, adhesive proteins underneath cell sheets are also maintained and they play a desirable
Myocardial tissue reconstruction by layering cardiomyocyte sheets [28,30,31]
Cardiomyocytes are tightly interconnected with gap junctions and pulsate simultaneously in native heart tissue. It is also well-known that confluent cultured cardiomyocytes on culture surfaces connect via gap junctions and beat simultaneously [33]. Therefore, in myocardial tissue engineering by layering cell sheets, it is a crucial point whether electrical and morphological communications are established between bilayer cell sheets. Chick embryo or neonatal rat cardiomyocyte sheets released
Future perspectives
Recently, research on myocardial tissue engineering has been accelerated to develop further advanced therapy for severe heart failure. Transplantation of layered cardiomyocyte sheets on the myocardial scar may be more beneficial than that of bioengineered heart tissue including biodegradable scaffolds in the point of scaffold-mediated disadvantages. However, there are several common problems in myocardial tissue engineering. As described in Section 1, myocardial cell sourcing remains a crucial
Acknowledgments
The present work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A) (13308055) and Grant-in-Aid for Encouragement of Young Scientists (13780693). It was also supported in part by the Open Research Grant from the Japan Research Promotion Society for Cardiovascular Diseases.
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