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Mashimo Lecture
Cardiac Tissue Reconstruction Based on Cell Sheet Engineering
Teruo Okano
Tokyo Women's Medical University
Tokyo, Japan
  • Cell Sheet Engineering
  • Clinical Application of Engineered Cell Sheets
  • Application for Cardiac Tissue Engineering


  • Cardiac grafts have been constructed using the new technology of cell sheet engineering. This newest generation of tissue engineering's based on the first generation of tissue engineering, which used biodegradable polymers to make scaffolds into which cells were inserted to make tissue in the presence of growth factors. First generation tissue engineering can be used for constructing cartilage, bone, blood vessel, skin and bladder tissue, which are amorphous or simple structures that function physically and have a low blood supply requirement and have large amounts of extracellular matrix (ECM).

    The second generation of tissue engineering is focusing on tissue of the heart, liver, kidney, lung and brain and will require different technology for their construction. Micrometer-scale substructures, such as liver lobule and nephrons, comprise these tissues, which have metabolic functions, a large number of cells, less ECM, and require more blood cells. Using scaffolds does not allow for controlling the balance between ECM and cells. Hence, a new method of tissue reconstruction not based on scaffolds is needed, to avoid the fibrosis that can occur in the heart, liver and kidney due to excess ECM deposition.





    Cell Sheet Engineering


    Cell sheet engineering without scaffolds is an alternative approach to tissue engineering. First, confluent cells sheets are made on the culture dish. The harvesting of these cell sheets allows for making layered, 3-dimensional (3-D) structures.

    Cell sheet construction involves the use of "intelligent biomaterial" that is a temperature responsive culture surface using a temperature responsive polymer Poly(N-isopropylacrylamide) (PIPAAm). The cell sheet is harvested by lowering the temperature. PIPAAm is hydrophilic below 32 degrees Celsius and hydrophobic above this temperature. Irradiation of PIPAAm with an electron beam creates a surface that is slightly hydrophobic and cell adhesive at 37 degrees Celsius, and that become reversibly hydrophilic and non-cell adhesive below 32 degrees Celsius due to rapid hydration and swelling of grafted PIPAAm. This unique surface change provides for spontaneous detachment of cultured cells from the grafted surface by simply changing the temperature. The use of trypsinization or another enzyme disrupts the adhesive proteins and membrane receptor ligand, while these are not disrupted when reducing the temperature. Non-enzymatic cell sheet harvest at 20 degrees Celsius allows recovery of the cell sheet with good preservation of the cell surface junction. Fibronectin, an ECM adhesive protein, completely detaches from the surface. Hence, a good cell sheet maintaining junction proteins can be made, which has one that is sticky for adhesiveness.


    Figure 1. Schematic of two-dimensional cell sheet manipulation.
    Click to enlarge

    Cardiomyocyte cell sheets constructed from chicken embryo beat strongly after being detached from the surface. Two-dimensional cell sheet manipulation involves lowering the temperature to 20 degrees Celsius to allow harvesting the cell sheet, which is then transferred to another dish where the temperature is raised to 37% for several minutes causing the cell sheet to adhere to the new surface after which the support membrane can be removed (Figure 1). Using this system it is possible to maintain the size and shape of cell sheets that are transferred.

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    Clinical Application of Engineered Cell Sheets


    Figure 2. Cultured keratinocyte sheets using 3T3 fibroblasts were made and harvested using the dispase treatment method.
    Click to enlarge

    Figure 3. Schematic of double-layered co-culture.
    Click to enlarge

    Okano and colleagues are making single cell sheets of skin, retina and corneal (epithelial, endothelial) cells; homotypically layered multiple sheets of cardiac muscle; and heterotypically layered multiple sheets of liver, kidney and lung.

    Cultured keratinocyte sheets using 3T3 fibroblasts to be used as an artificial epidermis were made and harvested by the dispase treatment method. However, shortcomings using dispase for harvesting keratinocyte sheets include a low-take ratio on deep wound and susceptibility to infection. The low temperature method maintains the cell junction and basement membrane, while the dispase method does not (Figure 2). Corneal transplantation with epithelial keratinocyte sheets made using the low temperature method has been performed. Polarized retinal pigmented epithelial (RPE) cell sheets with growth factor inserted under the PIPPAm-grafted porous membrane from the chick have been successfully transplanted to the rabbit. Double-layered co-culture tissue provides for prolonged survival of hepatocytes, for at least several months (Figure 3).

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    Application for Cardiac Tissue Engineering


    Figure 4. Electrical communication between the two sheets was synchronized, showing that adhesive proteins maintained the structure and function between layers.
    Click to enlarge

    Figure 5. The electrical stimulation was communicated from one layer to the other.
    Click to enlarge

    The establishment of structural and functional communication between the layered sheets is critical to constructing cardiomyocyte cell sheets. Okano and colleagues constructed primary cultured cardiomyocytes from neonatal rat or chick embryos. Adhesive proteins maintained the structure and function between the two layers and electrical communication between the two sheets was completely synchronized (Figure 4). Beating of the cells was detected. Further, electrical stimulation was transmitted from one sheet to the other (Figure 5). The bilayer sheets adhered closely without delamination. Cross-sectional views showed connexin 43 present in both tissue layers. When the cardiomyocyte sheets were layered on to frame-like collagen membranes to free them from rigid cell surfaces, synchronized movement of both layers occurred. Communication and synchronized movement with four-layer cell sheets on frame-like collagen membranes with macroscopic beating was also achieved. Four layer cardiac grafts were transplanted into dorsal subcutaneous tissue of nude rats. At 3 weeks post-transplantation, beating of the cells was seen on surface electrogram and skin resection revealed continued beating of the cell sheets and formation of new blood vessels. Histological analysis revealed a large amount of neovascularization in the cardiac grafts and connexin 43.

    Cardiac grafts were transplanted into impaired heart due to ischemic heart disease (IHD) or cardiomyopathy (CM), in collaboration with Matsuda and Sawa at Osaka University. Beating layered cardiomyocyte sheets were transplanted onto the impaired heart after myocardial infarction (MI), to determine whether the cell sheet would adhere to the beating heart. Improvement in cardiac performance in infarct rat hearts was seen after transplantation of a 4-layer cardiomyocyte sheet. The ejection fraction in the control animals was 45, and increased to 64 in the transplanted animals.

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