Masayuki Yamato, Tokyo Women's Medical University
Tissue engineering was originally proposed in 1980’s by the collaboration of a chemist, R. Langer and medical doctors, J.P. Vacanti and C.A. Vacanti. As demonstrated in reconstruction of cartilage tissues that is world-famous human ears on mice, the key technology is the use of biodegradable polymer scaffolds for cell seeding preformed in the target tissue shapes. By the combination of preformed biodegradable polymer scaffolds and specific cell types, various tissues including cartilage, bone, blood vessel, and urinary bladder were reconstructed, although the therapeutic use has been very limited. Here, we present another tissue engineering without biodegradable polymer scaffolds, named “cell sheet engineering”.
2. Temperature-responsive culture surfaces
Conventionally, proteolytic enzymes such as trypsin and dispase are used in cell harvest. These enzymes degrade cell adhesion molecules and deposited extracellular matrix (ECM) to detach cultured cells. But at the same time, cell-cell junctional proteins as well as membrane proteins such as ion channels and growth factor receptors are often degraded. In order to solve this problem, we first developed temperature-responsive culture dishes. A temperature-responsive polymer, poly(N-isopropylacrylamide), is covalently immobilized on dish surfaces. The grafted polymer thickness is tailored around 20 nm by utilizing electron beam irradiation-initiated radical polymerization to achieve temperature-responsive cell adhesion/detachment control. The dish surfaces are relatively hydrophobic at 37°C similarly to commercially available tissue culture dishes, but changes to hydrophilic below 32°C. Various cell types adhere, spread, and proliferate on the surfaces at 37°C. Only by reducing temperature, cells are spontaneously lifted up from the surfaces without the need for trypsin. Confluently cultured cells are recovered as a single contiguous cell sheet with intact cell-cell junctions. Harvested viable cell sheets can be transferred to other surfaces of culture dishes in vitro or tissue surfaces in vivo since the ECM associated with the basal side of cell sheets shows adhesion. The harvested cell sheets can be stratified to reconstruct thicker or complex tissue architectures such as cardiac muscle.
3. Clinical applications
We have succeeded in the clinical applications for the regeneration of cornea, heart and esophagus. For the corneal epithelium and esophageal mucosa, patients’ own oral mucosal epithelial cells are utilized. From a small biopsy, epithelial cells including its stem/progenitor cells are isolated and subjected to fabrication of transplantable epithelial cell sheets. In the case of treatment of esophageal lumen, cell sheets are transplanted by endoscopy. For the treatment of severe heart failure such as dilative cardiomyopathy, myoblasts are isolated from patients’ own leg muscles. Autologous myoblast sheets are transplanted onto damaged heart, and then remodel cardiac muscles by secreting humoral factors such as growth factors, cytokines, and matrix metalloproteinase.
Nanotechnology-based cell sheet engineering is a unique approach to regenerative functional tissues and should prove the fundamental potency in its clinical applications.
1. J. Yang, M. Yamato, K. Nishida, Y. Hayashida, T Shimizu, A. Kikuchi, Y. Tano and T. Okano, “Corneal epithelial stem cell delivery using cell sheet engineering: Not lost in transplantation”, Journal of Drug Targeting, 14, 471-482 (2006).
2. K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, K. Yamamoto, E. Adachi, S. Nagai, A. Kikuchi, N. Maeda, H. Watanabe, T. Okano and Y. Tano, “Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium”, N. Engl. J. Med., 351, 1187-1196 (2004).
3. T. Shimizu, H. Sekine, J. Yang, Y. Isoi, M. Yamato, A. Kikuchi, E. Kobayashi and T. Okano, “Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues”, FASEB J., 20, 708-710 (2006).
4. H. Hata, G. Matsumiya, S. Miyagawa, H. Kondo, N. Kawaguchi, N. Matsuura, T. Shimizu, T. Okano, H. Matsuda Hikaru and Y. Sawa, “Grafted skeletal myoblast sheets attenuate myocardial remodeling in pacing-induced canine heart failure model”, J. Thorac. Cardiovasc. Surg., 132, 918-924 (2006).
5. Y. Miyahara, N. Nagaya, M. Kataoka, B. Yanagawa, K. Tanaka, H. Hao, K. Ishino, H. Ishida, T. Shimizu, K. Kangawa, S. Sano, T. Okano, S. Kitamura and H. Mori, “Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction”, Nature Med., 12, 459-465 (2006).
6. T. Ohki, M. Yamato, D. Murakami, R. Takagi, J. Yang, H. Namiki, T. Okano, K. Takasaki, “Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model”, Gut, 55, 1704-1710 (2006).
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