Cell sheet engineering for myocardial tissue reconstruction

Abstract

Myocardial tissue engineering has now emerged as one of the most promising treatments for the patients suffering from severe heart failure. Tissue engineering has currently been based on the technology using three-dimensional (3-D) biodegradable scaffolds as alternatives for extracellular matrix. According to this most popular technique, several types of 3-D myocardial tissues have been successfully engineered by seeding cardiomyocytes into poly(glycolic acid), gelatin, alginate or collagen scaffolds. However, insufficient cell migration into the scaffolds and inflammatory reaction due to scaffold biodegradation remain problems to be solved. In contrast to these technologies, we now propose novel tissue engineering methodology layering cell sheets to construct 3-D functional tissues without any artificial scaffolds. Confluent cells on temperature-responsive culture surfaces can be harvested as a viable contiguous cell sheet only by lowering temperature without any enzymatic digestions. Electrical communications are established between layered cardiomyocyte sheets, resulting in simultaneous beating 3-D myocardial tissues. Layered cardiomyocyte sheets in vivo present long survival, macroscopic pulsation and characteristic structures of native heart tissue. Cell sheet engineering should have enormous potential for fabricating clinically applicable myocardial tissues and should promote tissue engineering research fields.

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.