Masayuki Yamato

Treatment of oesophageal ulcerations using endoscopic transplantation of tissue engineered autologous oral mucosal epithelial cell sheets in a canine model

Background: With the recent development of endoscopic submucosal dissection (ESD), large oesophageal cancers can be removed with a single procedure, with few limits on the resectable range. However, after aggressive ESD, a major complication that arises is postoperative inflammation and stenosis that can considerably affect the patient’s quality of life. Aims: To examine a novel treatment combining ESD and the endoscopic transplantation of tissue-engineered cell sheets created using autologous oral mucosal epithelial cells, in a clinically relevant large animal model. Methods: Oral mucosal epithelial cells, harvested from beagle dogs, were cultured under normal conditions at 37°C, on temperature-responsive dishes. After ESD (5 cm in length, 180° in range), cell sheets were harvested by a simple reduction in temperature to 20°C, and transplanted by endoscopy. Results: The transplanted cell sheets were able to adhere to and survive on the underlying muscle layers in the ulcer sites, providing an intact, stratified epithelium. Four weeks after surgery, complete wound healing, with no observable stenosis, was seen in the animals receiving autologous cell sheet transplantation. By contrast, noticeable fibrin mesh and host inflammation, consistent with the intermediate stages of wound healing, were observed in the control animals that received only ESD. Conclusions: These findings in a clinically relevant canine model show the effectiveness of a novel combined endoscopic approach for the potential treatment of oesophageal cancers that can effectively enhance wound healing and possibly prevent postoperative oesophageal stenosis.

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet-Based Regenerative Therapy and 3D Tissue Fabrication

This chapter describes the concept of cell sheet engineering for the creation of transplantable cellular tissues and organs. In contrast to scaffold-based tissue engineering, cell sheet engineering facilitates the reconstruction of scaffold-free, cell-dense tissues. Cell sheets were harvested by changing the temperature of thermoresponsive cell culture surfaces modified with poly(N-isopropylacrylamide) (PIPAAm) with a thickness on the nanometer scale. The transplantation of 2D cell sheet tissues has been used in clinical settings. Although 3D tissues were formed simply by layering 2D cell sheets, issues related to vascularization within 3D tissues and the large-scale production of cells must be addressed to create thick and large 3D tissues and organs.

Sociocytology Illuminated by Reconstructing Functional Tissue with Cell Sheet Based Technology

To create cell-dense functional tissues, the authors have developed a new class of tissue reconstructing technology, called “cell-sheet engineering”. In contrast to biodegradable scaffold-based three-dimensional (3D) tissue engineering, cell sheet-based bioassembler technology can reconstruct cell-dense tissues with extracellular matrix (ECM). For further mimicking living tissues and organs such as the liver and myotube, novel 3D bioassembler technologies are required for controlling the micro-scaled alignment of cells. This chapter summarizes the reconstruction of functional tissues with cell-sheet based technologies including (1) functional liver tissues using co-culture system of hepatocyte/nonparenchymal cells, (2) micro-patterned co-culture by microcontact printing systems with fine alignment, and (3) multi-layered oriented myotube tissues using anisotropic cell sheets and gelatin gel coated devices. Eventually, the fabrications of functional tissues and organs with bioassembler technology would lead to illuminating “sociocytology”.

Cell Scooper: A Device for the Rapid Transfer of Living Cell Sheet

In this study, we developed a device that could easily, rapidly, and completely transfer cell sheets from one material to another or transplant cell sheets onto the dorsal subcutaneous tissues of rats without leaving residual cells. Because the manipulation is as simple as pipetting, technical expertise is not required to transfer cell sheets very rapidly (the transfer time was 3.7 ± 1.6 s) using the device compared with that of a conventional method using a pipette (430 ± 180 s). After transfer by the device, C2C12 skeletal myoblast sheets showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor and stromal-derived factor-1α, indicating transfer without cell damage. Cardiac cell sheets after transfer showed spontaneous and synchronous beating, indicating intact cell-cell junctions and ion channel proteins on the cell opsurface. In addition, the device allowed us to transfer C2C12 cell sheets onto soft, rugged and curved surfaces such as human hands. Furthermore, cardiac cell sheets adhered rapidly and tightly onto the dorsal subcutaneous tissues of rats. This transfer/transplantation device may be a powerful tool in cell sheet-based tissue engineering and regenerative medicine.

Principles of Cell Sheet Technology

Tissue engineering has given several successes in primary clinical trials of regenerative medicine using biodegradable scaffolds. In contrast to approaches using scaffolds, we have developed novel approaches that use culture surfaces covalently immobilized with the temperature-responsive polymer poly(N-isopropylacrylamide) that allows for controlled attachment and detachment of living cells by simple temperature changes. Using fabricated cell sheets harvested from temperature-responsive surfaces including deposited extracellular matrices, we have established cell-sheet technology to fabricate functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, periodontal disease, cartilage disease, and cardiac failure. Cell-sheet technology therefore provides a novel alternative for regenerative medicine approaches that require the reconstruction of functional tissue structures.

Quantitative evaluation system for tissue-engineered corneal epithelial cell sheets using image-based technology

Abstract nBackground: Tissue-engineered corneal epithelial cell sheets have been applied for treating ocular surface diseases. It is essential to establish a standardized validation system for fabricated cell sheets

Cell-Based Therapy for Cardiovascular Injury

Cardiovascular injury is a major cause of morbidity and mortality and a major public health problem especially in developed countries. Various therapies for cardiovascular injury are researched actively and have been performed clinically. Recently, a cell-based therapy appears and is focused as an alternative therapy for cardiovascular injury. Scaffold-based and cell sheet-based tissue engineering contribute to the enhancement of cell transplanting efficiency, resulting in the induction of effective therapy. These cell-based regenerative therapies have promising and enormous possibilities for curing cardiovascular injury, and the clinical trials have been started. This chapter summarizes cell-based therapies including (1) cell injection therapy and (2) scaffold-based and (3) cell sheet-based tissue engineering. In addition, cell sources are also discussed.