Arch. Pharm. Res. (2014) 37:96–106 DOI 10.1007/s12272-013-0299-8
REVIEW
Latest status of the clinical and industrial applications of cell sheet engineering and regenerative medicine Mime Egami • Yuji Haraguchi • Tatsuya Shimizu Masayuki Yamato • Teruo Okano
•
Received: 30 September 2013 / Accepted: 26 October 2013 / Published online: 30 November 2013 Ó The Pharmaceutical Society of Korea 2013
Abstract Cell sheet engineering, which allows tissue engineering to be realized without the use of biodegradable scaffolds as an original approach, using a temperatureresponsive intelligent surface, has been applied in regenerative medicine for various tissues, and a number of clinical studies have been already performed for lifethreatening diseases. By using the results and findings obtained from the initial clinical studies, additional investigative clinical studies in several tissues with cell sheet engineering are currently in preparation stage. For treating many patients effectively by cell sheet engineering, an automated system integrating cell culture, cell-sheet fabrication, and layering is essential, and the system should include an advanced three-dimensional suspension cell culture system and an in vitro bioreactor system to scale up the production of cultured cells and fabricate thicker vascularized tissues. In this paper, cell sheet engineering, its clinical application, and further the authors’ challenge to develop innovative cell culture systems under newly legislated regulatory platform in Japan are summarized and discussed.
Abbreviations AFFSAPS Agence franc¸aise de se´curite´ sanitaire des produits de sante´ CAPS Cell Aseptic Processing System CPC Cell Processing Center ECM Extracellular matrix EMA European Medicinal Agency ESD Endoscopic submucosal dissection FDA Food and Drug Administration GMP Good Manufacturing Practice iPSCs Induced pluripotent stem cells KUH Karolinska University Hospital LCST Lower Critical Solution Temperature MHLW Ministry of Health, Labour and Welfare MPA Medical Practitioners Act PAL Pharmaceutical Affairs Law PIPAAm Poly(N-isoproplyacrylamide) PMDA Pharmaceuticals and Medical Devices Agency PAT Process Analytical Technology SOP Standard Operating Procedures 3-D Three-dimension(al)
Keywords Cell sheet engineering Clinical trial Pharmaceutical Affairs Law Regenerative medicine Temperature-responsive culture surface Threedimensional tissue
Introduction
M. Egami (&) Y. Haraguchi T. Shimizu M. Yamato T. Okano Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan e-mail: [email protected]
Recently cell-based regenerative therapy is focused as a most promising method to treat damaged/defective tissues/ organs, and various cell-injection therapies have already been clinically performed (Brittberg et al. 1994; Menasche´ 2008; Atala et al. 2011; Bolli et al. 2011; Makkar et al. 2012). Cell therapy has attractive and infinite potentials. However, the direct injection therapy of dissociated cells has several issues that still remain to be solved. Because
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many injected cells are found to be lost after the injection (Zhang et al. 2001; Suzuki et al. 2004; Hofmann et al. 2005), the target delivery and expected therapeutic effects may be limited. To resolve these problems, tissue engineered methodology have been progressing rapidly as the second generation cell-based regenerative therapy, and the various clinical studies have already started (Langer and Vacanti 1993; Li et al. 1999; Ochi et al. 2002; Wakitani et al. 2002; Moberg et al. 2002; Atala et al. 2006, 2011; Chachques et al. 2007, 2008; Shinoka and Breuer 2008; Iwasa et al. 2009; Lee et al. 2009). Tissue engineering is based on a concept that three dimensional (3-D) materials (polyglycolic acid, collagen gel, fibrin gel, gelatin, etc.) are used as an alternative for extracellular matrix (ECM), and living and functional cells are cultured into the scaffolds. In contrast to the method that uses scaffolds for living cells, the authors’ laboratory has developed an intelligent temperature-responsive culture surface and applied it to a scaffold-free tissue engineered methodology called ‘‘cell sheet engineering’’ (Yamada et al. 1990; Okano et al. 1993; Shimizu T et al. 2009; Haraguchi et al. 2012a). This review describes and explains temperatureresponsive culture surfaces, cell sheet engineering, and its applications to regenerative medicine in addition to the authors’ several challenges to develop innovative cell/tissue culture systems.
Temperature-responsive culture surface and cell sheet Poly(N-isoproplyacrylamide) (PIPAAm) in aqueous solution shows temperature-responsive hydrophilic/hydrophobic alterations (Heskins et al. 1968). The authors’ laboratory has succeeded in the fabrication of PIPAAmgrafted tissue culture surfaces by electron beam irradiation (Yamada et al. 1990; Okano et al. 1993). Nano-scale PIPAAm-grafted layer onto the culture surface also shows reversible temperature-responsive hydrophilic/hydrophobic alterations with an external temperature change, resulting in a thermally modulated cell attachment/detachment (Akiyama et al. 2004; Kumashiro et al. 2010). Therefore, confluently proliferated cells on the surface at 37 °C can be harvested as an intact cell sheet by simply reducing temperature below the lower critical solution temperature (LCST) of PIPAAm molecule (less than 32 °C) (Fig. 1). Since cell sheets can be harvested without protease treatment, the cell–cell junctions and ECM components mediating cell adhesion, which are susceptible to protease treatment, are preserved without damage (Kushida et al. 1999, 2000; Yamato et al. 2001; Nishida et al. 2004a, b; Ohashi et al. 2007). Because recovered cell sheets maintain their cell–cell junction molecules and ECM, 3-D tissue can be made by simply layering cell sheets without the use of
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scaffolds, resulting in biologically functional and communicative cell-dense tissue. For example, 3-D myocardial tissue fabricated by layering cardiac cell sheets has shown electrical and functional coupling and beats spontaneously, synchronously, and macroscopically (Shimizu et al. 2002; Haraguchi et al. 2012a). Various cells can be easily harvested as intact cell sheets using the culture surface, and, importantly, single- or multi-layered cell sheets can be transplanted directly onto the host tissues without suturing (Obokata et al. 2011; Haraguchi et al. 2012a).
Application of cell sheet engineering to regenerative medicine and the clinical studies Cell sheet-based tissue engineering has been applied and used for the fabrication of various tissues, and the transplantation of cell sheets shows good therapeutic effects in various damaged animal models (Yang et al. 2007; Haraguchi et al. 2012b). The applications of cell-sheet engineering are summarized in Table 1. In addition, a number of clinical studies using this technology have already started (Fig. 2). A first clinical trial for corneal reconstruction for four eyes of four patients with autologous mucosal epithelial cell sheets has resulted in the long-term maintenance of healthy ocular surfaces with the significant restorations of the vision of all patients with corneal limbal epithelial stem cell deficiency (Nishida et al. 2004b). The clinical study for 26 eyes of 25 patients in France with autologous oral mucosal epithelial cell sheets has showed that the cell sheets have been well-tolerated and a safe engineered tissue-product, and confirmed its efficacy on the regeneration of the ocular surface of the patients (Burillon et al. 2012). The result of this study has been presented to both AFFSAPS (a medicinal agency in France) and European Medicinal Agency (EMA) by CellSeed Inc. (Tokyo, Japan), which is a translational venture company from Tokyo Women’s Medical University. Clinical studies using autologous myoblast cell sheets for curing severe heart disease have also started. In the first study, 3 9 108 myoblasts ([90 % purity) are obtained after a 3-week-cultivation from approximately 10 g of the patient’s own skeletal muscle excised from his medial vastus muscle. After being fabricated, circular quadruple-layered myoblast cell sheets with a diameter of 40 mm are transplanted onto five areas by a total of 20 myoblast sheets. The first patient, who suffers from dilated cardiomyopathy and receives cell sheet therapy, is in a good clinical condition including (1) the improvement of cardiac function, (2) no indications of arrhythmia, (3) the discontinuance of a left ventricular assisting device, and (4) no requirement of cardiac-transplantation (Sawa et al. 2012), and has been discharged from the hospital without using the device. Based on
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Fig. 1 The harvest of a cell sheet using a temperatureresponsive culture surface without protease treatment. A Conventional cell-harvest by protease treatment results in the disruption of cell–cell junctions, and extracellular matrix (ECM). B When a temperatureresponsive culture surface is used, those cell structures are preserved, and the cultured cells are harvested as a contiguous and functional cell sheet. The dark and light culture surfaces indicate to be hydrophobic and hydrophilic, respectively
(A) Cell recovery by protease treatment (a conventional method) Cell-cell junction
ECM
Normal culture dish
Protease treatment
(Hydrophobic)
Disruption of cell-cell junctions, and ECM
(B) Cell sheet harvest by temperature-responsive culture surface Cell sheet 20 °C
37 °C
Cell-cell junction
ECM
By lowering temperature
Hydrophobic Temperature-responsive culture surface
Table 1 Fabrication/regeneration of various tissues by cell sheet technology (animal models) Target tissues
References
Corneal epithelial
Nishida et al. (2004a), Hayashida et al. (2005)
Myocardium
Shimizu et al. (2002), Memon et al. (2005), Miyagawa et al. (2005, 2010), Miyahara et al. (2006), Hida et al. (2008), Kobayashi et al. (2008), Sekiya et al. (2009), Matsuura et al. (2009), Hoashi et al. (2009), Sekine et al. (2011), Shudo et al. (2011), Terajima et al. (2012)
Periodontium
Hasegawa et al. (2005), Akizuki et al. (2005), Flores et al. (2008), Iwata et al. (2009), Washio et al. (2010), Tsumanuma et al. (2011), Yoshida et al. (2012)
Esophageal mucosa
Ohki et al. (2006), Takagi et al. (2012), Kanai et al. (2012)
Articular cartilage
Kaneshiro et al. (2006), Sato et al. (2008), Mitani et al. (2009), Ebihara et al. (2012), Kokubo et al. (2013) Kanzaki et al. (2007, 2008)
Lung surfacea Pancreatic islet
Shimizu H et al. (2009, 2013), Saito et al. (2011), Ohashi et al. (2011)
Hepatic tissue
Ohashi et al. (2007), Kano et al. (2008), Kim et al. (2012)
Middle ear mucosa
Yaguchi et al. (2013)
Retinal pigment epithelium
Yaji et al. (2009)
Thyroid gland
Arauchi et al. (2009)
Urothelial tissue
Shiroyanagi et al. (2003, 2004)
Renal tissue
Sekiya et al. (2013)
a
Preventing postoperative lung air-leak
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Hydrophilic Preservation of cell-cell junctions, and ECM
on-going investigative clinical studies at Osaka University (Osaka, Japan), Terumo Co, Ltd. (Tokyo, Japan) is currently sponsoring clinical trials at three institutions including Osaka University in Japan, and the trials are guided by Pharmaceuticals and Medical Devices Agency (PMDA). A cell-sheet clinical study for regenerating esophageal mucosa after oesophageal endoscopic submucosal dissection (ESD) for removing superficial oesophageal neoplasm in nine patients have been also performed (Ohki et al. 2012). The size of resected mucosa ranges from half to fully circumference, and two to eight autologous oral mucosal epithelial cell sheets, which are fabricated from epithelial cells isolated from patients’ oral mucosa tissues, are used. The cell-sheet size is approximately 6 mm in diameter. The cell sheets are successfully transplanted to ulcer surfaces using an endoscope without suturing. Within a median time of 3.5 weeks after transplantation, re-epithelialization occurs completely. No patients experienced dysphagia, stricture, or other complications after the therapy except one patient who has a full circumferential ulceration expanding to the esophagogastric junction. The operative procedure may be used to prevent stricture formation after ESD and improve the quality of life of patients. Under the collaborative research agreement between Tokyo Women’s Medical University and Karolinska University Hospital (KUH) (Stockholm, Sweden), investigative clinical studies to examine the protocol as standardized regenerative medicine for esophagus diseases such as Barrett’s esophagus is being performed at KUH since December 2012. Cell sheet engineering has also been successfully applied to repair and regenerate periodontal and cartilage
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Esophagus
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Corneal epithelium Myocardium
Periodontal tissue
Lung
Fabrication of 3D tissue using cell sheet technology
Cartilage
Temperature-responsive culture surface
Middle ear mucosa Liver
Pancreas
Fig. 2 Fabrication/regeneration of various tissues by cell sheet engineering. Clinical trials using cell sheet engineering have already started in (1) cornea epithelium, (2) myocardium, (3) esophagus, (4) periodontal, and (5) cartilage tissues
tissues in various animal models including large animals as shown in Table 1. The transplantation of layered periodontal ligament-derived cell sheets induces the regeneration of both new bone and cementum connecting with well-oriented collagen fibers in a canine model (Iwata et al. 2009). For the therapy of periodontal disease, a clinical study using autologous periodontal ligament cell sheets has already started from the end of July 2012 (Iwata et al. 2013). Six cases of the cell sheets have been transplanted, and the healing process is found to progress uneventfully. Layered articular chondrocyte sheets maintain the cartilaginous phenotype in vitro and adhere firmly to cartilage tissues after the transplantation (Kaneshiro et al. 2006). The transplanted chondrocyte sheets can act as a barrier against the loss of proteoglycans from damaged cartilage tissue and to protect them from catabolic factors in the knee joint in a rabbit model. In a minipig model, the transplantation of layered chondrocyte sheets shows a good stained matrix and integration with surrounding tissue; indicating sufficient cartilaginous repair and regeneration (Ebihara et al. 2012). A clinical study using cell sheet engineering for cartilage regeneration has also begun in 2011 at Tokai University School of Medicine (Kanagawa, Japan). Furthermore, based on encouraging results in various animal models, additional clinical studies in several tissues such as lung, middle ear etc. using autologous cell sheets are currently being prepared.
Cell/tissue culture technologies: current and future trends In various laboratories including the author’ laboratory, technically challenging investigations have already been started. At least 1–2 9 109 cells are thought to be necessary to treat the cardiac tissue of one patient after myocardial infarction or to replace b-cells in type 1 diabetics (Zweigerdt et al. 2011). Because two-dimensional culture systems on conventional culture dishes may be unable to produce those enormous numbers of cells due to require culture space and cost, it has been challenged to develop a 3-D cell culture system using several bioreactors to promise to expand cell culture scale to meet the clinical demand described above (Zweigerdt et al. 2011). Recently, the authors’ laboratory has originally developed a suspension cell culture system, which is developed jointly with the authors’ industry partner, ABLE & Biott Co., Ltd. (Tokyo, Japan), to expand and induce the cardiac differentiation of human induced pluripotent stem cells (iPSCs) (Matsuura et al. 2012, Fig. 3). The culture system, which uses several cytokines including activin A, bone morphogenetic protein 4, fibroblast growth factor-2, and vascular endothelial growth factor, and an Wnt-signaling antagonist, IWR-1, combined with the suspension culture apparatus, can produce a large amount of human iPSC-derived cardiomyocytes (6 9 107 cells per a 100-mL bioreactor). A
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Fig. 3 Development of threedimensional cell suspension culture system for pluripotent cells. A Single use bioreactor (100-mL volume) for ‘‘stirring’’ cell suspension culture developed by ABLE & Biott Co., Ltd. B Bio-Jr.8 (ABLE & Biott Co., Ltd.): multistage cell culture apparatus
human cardiac cell sheet, which can beat spontaneously and synchronously, can be successfully engineered by using the differentiated cardiomyocytes, and therefore, the enormous number of human cardiac cell sheets can be fabricated by the system. The system is currently recognized as a breakthrough technology for the large scale expansion of cells at low cost with a high and stable yield. Furthermore, the proliferation of 1 9 109 human iPSCs per a 1,000-mL bioreactor has been succeeded, suggesting that the possibility of sufficient number of cells can be produced to treat a patient suffered from myocardial infarction. The result of joint research project has been also presented at the annual meeting of International Society of Stem Cell Research in June 2013 at Boston. The system is expected to be commercialized in 2014. At the same time, for aiming a clinical usage, human iPSC-derived cardiac cell sheets, which have prepared by other culture system, have been also used in a large animal porcine model, and the safety and feasibility of the therapy are confirmed (Kawamura et al. 2012). Although the in vitro scaling up of bioengineered tissues is known to be limited by diffusion issues due to specifically a lack of vasculature, in vivo engineered 1-mm thick cardiac tissue, which has a well-organized microvascular network and beat synchronously and macroscopically even after resection, has been successfully prepared by a 1-day interval polysurgery procedure with a total of thirty cardiac cell sheets (Shimizu et al. 2006). In vitro fabrication of thicker organ-like functional vascularized 3-D tissues is a long-term goal in the field of tissue engineering. The authors’ laboratory has challenged to the objective by using a coculture system with endothelial cells and two bioreactor systems with perfusable blood vessels, which consist of (1) resected tissue with a connectable artery and vein as a vascular bed, and (2) collagen-based micro-channels (Sekine et al. 2013; Sakaguchi et al. 2013). These systems
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can allow fabricate thicker vascularized cardiac tissues, which can beat spontaneously, synchronously, and macroscopically, in vitro as like ‘‘real heart tissue’’ for overcoming the engineered-tissue thickness limitations described above. Furthermore, after being transplanted into animals for realizing blood vessel anastomoses, the thicker tissues are found to maintain its vascular structure and beat spontaneously for 2 weeks. At present, the authors’ laboratory is applying the systems to other tissues including human iPSC-derived cardiac tissue. The systems can promise significant advances for both preclinical and clinical applications of tissue engineering as well as new in vitro tissue models for evaluating or discovering new drugs. The culture of isolated cells and the preparation of cell sheets, which are used in clinical studies, are manually performed in a clean room system known as Cell Processing Center (CPC). For maintaining the cleanliness of CPC and ensuring the safety and quality of transplanted cell sheets, cell-sheet transplantation materials are generated under strict regulations. In addition to several systems for maintaining the cleanliness of rooms, human handling processes in the CPC are always performed by a pair, namely, one person performs cell-culture procedures including microscopic observation and medium exchange, and another person directs the procedures for avoiding human errors and recording all of the procedures based on Standard Operating Procedures (SOP) following Good Manufacturing Practice (GMP) for the cases of clinical trials, or quasi-GMP for the cases of investigative clinical studies. The production sites and documents are inspected by PMDA regularly. Therefore, the procedure consumes an enormously expensive cost and time. The initial installation cost of GMP-level CPC (floor area: 100 m2) in the authors’ laboratory is more than one million US dollars, and the cost of maintenance is more than 100,000
Latest status of cell sheet engineering and regenerative medicine
US dollars per year (Hannachi et al. 2009). The totally accumulated cost of manipulation in the CPC may discourage the widespread use of cell sheet engineering and the clinical application. Under current regulations, the laboratory has investigated further cost-reduction potentials to use a cell isolator (standardized by ISO 13408-6) in the CPC facility for culturing cells in a closed area, namely Cell Aseptic Processing System (CAPS). Cell culturing cost by CAPS may be reduced to 2/3 of conventional GMP standard culturing cost in CPC. The result of this study has been presented at the Annual Meeting of Japan Society of Regenerative Medicine in 2009 at Tokyo by the authors’ laboratory and CellSeed Inc. for open discussion. Recently, several reports regarding robotic/automatic cell culture systems have been published (Kino-oka and Taya 2009; Kato et al. 2010). The systems requiring no manual manipulation have been significantly advanced and contribute to the improvements of safety and quality of bioengineered tissues, because manual manipulation naturally increases the possibility of human error such as including infectious contamination. The introduction of robotic/automatic systems for sterile cell culture has the advantage of stability and cost-saving for manufacturing qualified bioengineered tissue. It is also widely aware that Food and Drug Administration Office (FDA) in the United States is encouraging manufacturing industry to develop Quality by Design manufacturing method by Process Analytical Technology (PAT), which is automated repeatable manufacturing technology combined with real time remote monitoring function. FDA already recognizes necessity to shift from the current practice of final product test to monitoring PAT for performing the modern quality control of advanced medicinal products along with industry
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and reducing cost for patients [FDA in the United States, October 2010]. Recently, the authors’ laboratory has developed a living-cell isolation device, which can isolate beating cardiomyocytes from heart tissues semi-automatically in an animal model (Shioyama et al. 2013). At present, the laboratory is challenging the introductions of robotic/automatic system in cell isolation, cell culture, cellsheet fabrication, and layering, namely, the establishment of ‘‘tissue factory’’ using cell sheet engineering instead of conventional CPC. Originally patented flexible modular platform (fMP) (Pat. No. JP5051677) in the authors’ ‘‘tissue factory’’ allows each step of cell isolation, culture, sheet fabrication etc. to be maintained by separate automated modules, where the interface of connecting part with various sensors built-in is designed to be standardized. The establishment of the ‘‘tissue factory’’ will give further advancements in cell sheet engineering, tissue engineering, and regenerative medicine, and accelerates both the clinical application and the related regulatory science.
New regulatory framework being formed in Japan Currently in Japan, investigative clinical research is governed by Medical Practitioners Act (MPA), and clinical trial, sponsored by physician or industry, is governed by Pharmaceutical Affairs Law (PAL). Because of separated legal requirements and guidelines, the data quality of investigative clinical study is unable to meet with the minimal requirement of the supplemental data of application documents for obtaining the product marketing approval under PAL. At the same time, the clinical trials of regenerative-medicine product is required to follow drug GMP, which is not intrinsically appropriate for living cell
Fig. 4 Regulatory framework to facilitate the commercialization of regenerative medicine products in Japan. The illustration is the bird’s view of three related laws being legislated in Japan. The illustration was prepared by Ministry of Health, Labour and Welfare (MHLW), and translated by Mime Egami in Aug. 2013
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Fig. 5 Revision of Pharmaceutical Affairs Law (Revision bill, submitted by the Cabinet, passed the Diet on November 20th, 2013) to set up the new category of regenerative medicine and establish its new conditional/ tentative approval system. The illustration was prepared by MHLW, and translated by Mime Egami in Aug. 2013
Fig. 6 Risk-based classification of regenerative medicine is newly designed to legislate the application process of clinical research or the private practice of regenerative medicine. The illustration was prepared by MHLW, and translated by Mime Egami in Aug. 2013. ES cells embryonic stem cells, FIM first-in-man, iPS cells induced pluripotent stem cells, IRB institutional review board
products, or unfeasible to be required for autologous cell cultured products. As the result of immature regulatory framework and the invisibility of regulatory pathway, to date, there are only two products formally approved as medical device in Japan, and four products, which are as drug, are at clinical trial stage, while more than 70 investigative clinical studies are running in Japan. The authors’ laboratory has established joint regulatory-science doctorate program along with Waseda University (Tokyo, Japan) in 2010 for creating regulatory science specialists who can scientifically discuss regulatory convergence into proper
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scientific experiments and self-evident feasible guidelines. Together with the rapid clinical applications of cell sheet engineering, the authors’ laboratory has been encouraging active dialogue to develop new regulatory framework among academia, regulatory agencies, and newly entering industry players. Congressional efforts to establish new promotional framework for regenerative medicine started in 2012 and passed Promotional Act of Regenerative Medicine at Diet in April 2013 (Fig. 4). Cabinet immediately submitted related bills to the Diet to be legislated by 2014, which bills already passed the Diet on November
Latest status of cell sheet engineering and regenerative medicine
20th, 2013. Based on these enacted bills at the Diet, new regulatory framework will be formed by the following two laws. Firstly revised PAL will (1) define regenerative medicine as third category separately from drug or medical devices, which GMP (namely Good Tissue Practice) will be newly designed, and (2) establish conditional approval process after safety review to evaluate efficacy at the bedside before its full approval (Fig. 5). Also newly documented Law of Safety of Regenerative Medicine will (1) first ever legislate the application procedures of both clinical researches and private practices, which are unable to be covered by health insurance, by newly established riskbased classification (Fig. 6) and (2) allow cell culture and processing work to be outsourced from hospital/clinics to industry players by setting up new guidelines of proper risk-sharing outsourcing procedure and preparing a proper authorization and inspection system. Conditional approval system with a visible regulatory pathway, along with qualified cell culture service outsourced to industry, will encourage scientifically qualified regenerative medicine to appear at bedside in a timely manner.
Conclusions Cell sheet engineering has enormous and wide-ranging potentials for the re-construction of various functional tissues including thicker vascularized tissues. In various animal models, the transplantation of cell sheets shows good therapeutic effects and cure potential for chronic diseases. Although various 1st generation cell-sheet clinical studies and therapies have been performed in Japan and in Europe as hospital exemption, there are still many issues that need to be resolved before cell-sheet therapy become a standard treatment at an affordable cost in the World. The authors’ laboratory will initiate further collaborations and active dialogue among researchers with various backgrounds, multi-disciplinary clinicians, industry players, regulatory specialists, and patients for resolving the issues and establish the innovative value chain of regenerative medicine, following our Credo ‘‘Passion for Innovation and Duty to the Patients of Tomorrow’’. Acknowledgments This work was supported by grants from the Global Center of Excellence Program, Multidisciplinary Education and Technology and Research Center for Regenerative Medicine (MERCREM), and Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology ‘‘Cell Sheet Tissue Engineering Center (CSTEC)’’ from the Ministry of Education, Culture, Sports Science, and Technology (MEXST), Japan, the Japan Society for the Promotion of Science (JSPS) through the ‘‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),’’ initiated by the Council for Science and Technology Policy (CSTP).
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REVIEW
Latest status of the clinical and industrial applications of cell sheet engineering and regenerative medicine Mime Egami • Yuji Haraguchi • Tatsuya Shimizu Masayuki Yamato • Teruo Okano
•
Received: 30 September 2013 / Accepted: 26 October 2013 / Published online: 30 November 2013 Ó The Pharmaceutical Society of Korea 2013
Abstract Cell sheet engineering, which allows tissue engineering to be realized without the use of biodegradable scaffolds as an original approach, using a temperatureresponsive intelligent surface, has been applied in regenerative medicine for various tissues, and a number of clinical studies have been already performed for lifethreatening diseases. By using the results and findings obtained from the initial clinical studies, additional investigative clinical studies in several tissues with cell sheet engineering are currently in preparation stage. For treating many patients effectively by cell sheet engineering, an automated system integrating cell culture, cell-sheet fabrication, and layering is essential, and the system should include an advanced three-dimensional suspension cell culture system and an in vitro bioreactor system to scale up the production of cultured cells and fabricate thicker vascularized tissues. In this paper, cell sheet engineering, its clinical application, and further the authors’ challenge to develop innovative cell culture systems under newly legislated regulatory platform in Japan are summarized and discussed.
Abbreviations AFFSAPS Agence franc¸aise de se´curite´ sanitaire des produits de sante´ CAPS Cell Aseptic Processing System CPC Cell Processing Center ECM Extracellular matrix EMA European Medicinal Agency ESD Endoscopic submucosal dissection FDA Food and Drug Administration GMP Good Manufacturing Practice iPSCs Induced pluripotent stem cells KUH Karolinska University Hospital LCST Lower Critical Solution Temperature MHLW Ministry of Health, Labour and Welfare MPA Medical Practitioners Act PAL Pharmaceutical Affairs Law PIPAAm Poly(N-isoproplyacrylamide) PMDA Pharmaceuticals and Medical Devices Agency PAT Process Analytical Technology SOP Standard Operating Procedures 3-D Three-dimension(al)
Keywords Cell sheet engineering Clinical trial Pharmaceutical Affairs Law Regenerative medicine Temperature-responsive culture surface Threedimensional tissue
Introduction
M. Egami (&) Y. Haraguchi T. Shimizu M. Yamato T. Okano Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan e-mail: [email protected]
Recently cell-based regenerative therapy is focused as a most promising method to treat damaged/defective tissues/ organs, and various cell-injection therapies have already been clinically performed (Brittberg et al. 1994; Menasche´ 2008; Atala et al. 2011; Bolli et al. 2011; Makkar et al. 2012). Cell therapy has attractive and infinite potentials. However, the direct injection therapy of dissociated cells has several issues that still remain to be solved. Because
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many injected cells are found to be lost after the injection (Zhang et al. 2001; Suzuki et al. 2004; Hofmann et al. 2005), the target delivery and expected therapeutic effects may be limited. To resolve these problems, tissue engineered methodology have been progressing rapidly as the second generation cell-based regenerative therapy, and the various clinical studies have already started (Langer and Vacanti 1993; Li et al. 1999; Ochi et al. 2002; Wakitani et al. 2002; Moberg et al. 2002; Atala et al. 2006, 2011; Chachques et al. 2007, 2008; Shinoka and Breuer 2008; Iwasa et al. 2009; Lee et al. 2009). Tissue engineering is based on a concept that three dimensional (3-D) materials (polyglycolic acid, collagen gel, fibrin gel, gelatin, etc.) are used as an alternative for extracellular matrix (ECM), and living and functional cells are cultured into the scaffolds. In contrast to the method that uses scaffolds for living cells, the authors’ laboratory has developed an intelligent temperature-responsive culture surface and applied it to a scaffold-free tissue engineered methodology called ‘‘cell sheet engineering’’ (Yamada et al. 1990; Okano et al. 1993; Shimizu T et al. 2009; Haraguchi et al. 2012a). This review describes and explains temperatureresponsive culture surfaces, cell sheet engineering, and its applications to regenerative medicine in addition to the authors’ several challenges to develop innovative cell/tissue culture systems.
Temperature-responsive culture surface and cell sheet Poly(N-isoproplyacrylamide) (PIPAAm) in aqueous solution shows temperature-responsive hydrophilic/hydrophobic alterations (Heskins et al. 1968). The authors’ laboratory has succeeded in the fabrication of PIPAAmgrafted tissue culture surfaces by electron beam irradiation (Yamada et al. 1990; Okano et al. 1993). Nano-scale PIPAAm-grafted layer onto the culture surface also shows reversible temperature-responsive hydrophilic/hydrophobic alterations with an external temperature change, resulting in a thermally modulated cell attachment/detachment (Akiyama et al. 2004; Kumashiro et al. 2010). Therefore, confluently proliferated cells on the surface at 37 °C can be harvested as an intact cell sheet by simply reducing temperature below the lower critical solution temperature (LCST) of PIPAAm molecule (less than 32 °C) (Fig. 1). Since cell sheets can be harvested without protease treatment, the cell–cell junctions and ECM components mediating cell adhesion, which are susceptible to protease treatment, are preserved without damage (Kushida et al. 1999, 2000; Yamato et al. 2001; Nishida et al. 2004a, b; Ohashi et al. 2007). Because recovered cell sheets maintain their cell–cell junction molecules and ECM, 3-D tissue can be made by simply layering cell sheets without the use of
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scaffolds, resulting in biologically functional and communicative cell-dense tissue. For example, 3-D myocardial tissue fabricated by layering cardiac cell sheets has shown electrical and functional coupling and beats spontaneously, synchronously, and macroscopically (Shimizu et al. 2002; Haraguchi et al. 2012a). Various cells can be easily harvested as intact cell sheets using the culture surface, and, importantly, single- or multi-layered cell sheets can be transplanted directly onto the host tissues without suturing (Obokata et al. 2011; Haraguchi et al. 2012a).
Application of cell sheet engineering to regenerative medicine and the clinical studies Cell sheet-based tissue engineering has been applied and used for the fabrication of various tissues, and the transplantation of cell sheets shows good therapeutic effects in various damaged animal models (Yang et al. 2007; Haraguchi et al. 2012b). The applications of cell-sheet engineering are summarized in Table 1. In addition, a number of clinical studies using this technology have already started (Fig. 2). A first clinical trial for corneal reconstruction for four eyes of four patients with autologous mucosal epithelial cell sheets has resulted in the long-term maintenance of healthy ocular surfaces with the significant restorations of the vision of all patients with corneal limbal epithelial stem cell deficiency (Nishida et al. 2004b). The clinical study for 26 eyes of 25 patients in France with autologous oral mucosal epithelial cell sheets has showed that the cell sheets have been well-tolerated and a safe engineered tissue-product, and confirmed its efficacy on the regeneration of the ocular surface of the patients (Burillon et al. 2012). The result of this study has been presented to both AFFSAPS (a medicinal agency in France) and European Medicinal Agency (EMA) by CellSeed Inc. (Tokyo, Japan), which is a translational venture company from Tokyo Women’s Medical University. Clinical studies using autologous myoblast cell sheets for curing severe heart disease have also started. In the first study, 3 9 108 myoblasts ([90 % purity) are obtained after a 3-week-cultivation from approximately 10 g of the patient’s own skeletal muscle excised from his medial vastus muscle. After being fabricated, circular quadruple-layered myoblast cell sheets with a diameter of 40 mm are transplanted onto five areas by a total of 20 myoblast sheets. The first patient, who suffers from dilated cardiomyopathy and receives cell sheet therapy, is in a good clinical condition including (1) the improvement of cardiac function, (2) no indications of arrhythmia, (3) the discontinuance of a left ventricular assisting device, and (4) no requirement of cardiac-transplantation (Sawa et al. 2012), and has been discharged from the hospital without using the device. Based on
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Fig. 1 The harvest of a cell sheet using a temperatureresponsive culture surface without protease treatment. A Conventional cell-harvest by protease treatment results in the disruption of cell–cell junctions, and extracellular matrix (ECM). B When a temperatureresponsive culture surface is used, those cell structures are preserved, and the cultured cells are harvested as a contiguous and functional cell sheet. The dark and light culture surfaces indicate to be hydrophobic and hydrophilic, respectively
(A) Cell recovery by protease treatment (a conventional method) Cell-cell junction
ECM
Normal culture dish
Protease treatment
(Hydrophobic)
Disruption of cell-cell junctions, and ECM
(B) Cell sheet harvest by temperature-responsive culture surface Cell sheet 20 °C
37 °C
Cell-cell junction
ECM
By lowering temperature
Hydrophobic Temperature-responsive culture surface
Table 1 Fabrication/regeneration of various tissues by cell sheet technology (animal models) Target tissues
References
Corneal epithelial
Nishida et al. (2004a), Hayashida et al. (2005)
Myocardium
Shimizu et al. (2002), Memon et al. (2005), Miyagawa et al. (2005, 2010), Miyahara et al. (2006), Hida et al. (2008), Kobayashi et al. (2008), Sekiya et al. (2009), Matsuura et al. (2009), Hoashi et al. (2009), Sekine et al. (2011), Shudo et al. (2011), Terajima et al. (2012)
Periodontium
Hasegawa et al. (2005), Akizuki et al. (2005), Flores et al. (2008), Iwata et al. (2009), Washio et al. (2010), Tsumanuma et al. (2011), Yoshida et al. (2012)
Esophageal mucosa
Ohki et al. (2006), Takagi et al. (2012), Kanai et al. (2012)
Articular cartilage
Kaneshiro et al. (2006), Sato et al. (2008), Mitani et al. (2009), Ebihara et al. (2012), Kokubo et al. (2013) Kanzaki et al. (2007, 2008)
Lung surfacea Pancreatic islet
Shimizu H et al. (2009, 2013), Saito et al. (2011), Ohashi et al. (2011)
Hepatic tissue
Ohashi et al. (2007), Kano et al. (2008), Kim et al. (2012)
Middle ear mucosa
Yaguchi et al. (2013)
Retinal pigment epithelium
Yaji et al. (2009)
Thyroid gland
Arauchi et al. (2009)
Urothelial tissue
Shiroyanagi et al. (2003, 2004)
Renal tissue
Sekiya et al. (2013)
a
Preventing postoperative lung air-leak
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Hydrophilic Preservation of cell-cell junctions, and ECM
on-going investigative clinical studies at Osaka University (Osaka, Japan), Terumo Co, Ltd. (Tokyo, Japan) is currently sponsoring clinical trials at three institutions including Osaka University in Japan, and the trials are guided by Pharmaceuticals and Medical Devices Agency (PMDA). A cell-sheet clinical study for regenerating esophageal mucosa after oesophageal endoscopic submucosal dissection (ESD) for removing superficial oesophageal neoplasm in nine patients have been also performed (Ohki et al. 2012). The size of resected mucosa ranges from half to fully circumference, and two to eight autologous oral mucosal epithelial cell sheets, which are fabricated from epithelial cells isolated from patients’ oral mucosa tissues, are used. The cell-sheet size is approximately 6 mm in diameter. The cell sheets are successfully transplanted to ulcer surfaces using an endoscope without suturing. Within a median time of 3.5 weeks after transplantation, re-epithelialization occurs completely. No patients experienced dysphagia, stricture, or other complications after the therapy except one patient who has a full circumferential ulceration expanding to the esophagogastric junction. The operative procedure may be used to prevent stricture formation after ESD and improve the quality of life of patients. Under the collaborative research agreement between Tokyo Women’s Medical University and Karolinska University Hospital (KUH) (Stockholm, Sweden), investigative clinical studies to examine the protocol as standardized regenerative medicine for esophagus diseases such as Barrett’s esophagus is being performed at KUH since December 2012. Cell sheet engineering has also been successfully applied to repair and regenerate periodontal and cartilage
Latest status of cell sheet engineering and regenerative medicine
Esophagus
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Corneal epithelium Myocardium
Periodontal tissue
Lung
Fabrication of 3D tissue using cell sheet technology
Cartilage
Temperature-responsive culture surface
Middle ear mucosa Liver
Pancreas
Fig. 2 Fabrication/regeneration of various tissues by cell sheet engineering. Clinical trials using cell sheet engineering have already started in (1) cornea epithelium, (2) myocardium, (3) esophagus, (4) periodontal, and (5) cartilage tissues
tissues in various animal models including large animals as shown in Table 1. The transplantation of layered periodontal ligament-derived cell sheets induces the regeneration of both new bone and cementum connecting with well-oriented collagen fibers in a canine model (Iwata et al. 2009). For the therapy of periodontal disease, a clinical study using autologous periodontal ligament cell sheets has already started from the end of July 2012 (Iwata et al. 2013). Six cases of the cell sheets have been transplanted, and the healing process is found to progress uneventfully. Layered articular chondrocyte sheets maintain the cartilaginous phenotype in vitro and adhere firmly to cartilage tissues after the transplantation (Kaneshiro et al. 2006). The transplanted chondrocyte sheets can act as a barrier against the loss of proteoglycans from damaged cartilage tissue and to protect them from catabolic factors in the knee joint in a rabbit model. In a minipig model, the transplantation of layered chondrocyte sheets shows a good stained matrix and integration with surrounding tissue; indicating sufficient cartilaginous repair and regeneration (Ebihara et al. 2012). A clinical study using cell sheet engineering for cartilage regeneration has also begun in 2011 at Tokai University School of Medicine (Kanagawa, Japan). Furthermore, based on encouraging results in various animal models, additional clinical studies in several tissues such as lung, middle ear etc. using autologous cell sheets are currently being prepared.
Cell/tissue culture technologies: current and future trends In various laboratories including the author’ laboratory, technically challenging investigations have already been started. At least 1–2 9 109 cells are thought to be necessary to treat the cardiac tissue of one patient after myocardial infarction or to replace b-cells in type 1 diabetics (Zweigerdt et al. 2011). Because two-dimensional culture systems on conventional culture dishes may be unable to produce those enormous numbers of cells due to require culture space and cost, it has been challenged to develop a 3-D cell culture system using several bioreactors to promise to expand cell culture scale to meet the clinical demand described above (Zweigerdt et al. 2011). Recently, the authors’ laboratory has originally developed a suspension cell culture system, which is developed jointly with the authors’ industry partner, ABLE & Biott Co., Ltd. (Tokyo, Japan), to expand and induce the cardiac differentiation of human induced pluripotent stem cells (iPSCs) (Matsuura et al. 2012, Fig. 3). The culture system, which uses several cytokines including activin A, bone morphogenetic protein 4, fibroblast growth factor-2, and vascular endothelial growth factor, and an Wnt-signaling antagonist, IWR-1, combined with the suspension culture apparatus, can produce a large amount of human iPSC-derived cardiomyocytes (6 9 107 cells per a 100-mL bioreactor). A
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Fig. 3 Development of threedimensional cell suspension culture system for pluripotent cells. A Single use bioreactor (100-mL volume) for ‘‘stirring’’ cell suspension culture developed by ABLE & Biott Co., Ltd. B Bio-Jr.8 (ABLE & Biott Co., Ltd.): multistage cell culture apparatus
human cardiac cell sheet, which can beat spontaneously and synchronously, can be successfully engineered by using the differentiated cardiomyocytes, and therefore, the enormous number of human cardiac cell sheets can be fabricated by the system. The system is currently recognized as a breakthrough technology for the large scale expansion of cells at low cost with a high and stable yield. Furthermore, the proliferation of 1 9 109 human iPSCs per a 1,000-mL bioreactor has been succeeded, suggesting that the possibility of sufficient number of cells can be produced to treat a patient suffered from myocardial infarction. The result of joint research project has been also presented at the annual meeting of International Society of Stem Cell Research in June 2013 at Boston. The system is expected to be commercialized in 2014. At the same time, for aiming a clinical usage, human iPSC-derived cardiac cell sheets, which have prepared by other culture system, have been also used in a large animal porcine model, and the safety and feasibility of the therapy are confirmed (Kawamura et al. 2012). Although the in vitro scaling up of bioengineered tissues is known to be limited by diffusion issues due to specifically a lack of vasculature, in vivo engineered 1-mm thick cardiac tissue, which has a well-organized microvascular network and beat synchronously and macroscopically even after resection, has been successfully prepared by a 1-day interval polysurgery procedure with a total of thirty cardiac cell sheets (Shimizu et al. 2006). In vitro fabrication of thicker organ-like functional vascularized 3-D tissues is a long-term goal in the field of tissue engineering. The authors’ laboratory has challenged to the objective by using a coculture system with endothelial cells and two bioreactor systems with perfusable blood vessels, which consist of (1) resected tissue with a connectable artery and vein as a vascular bed, and (2) collagen-based micro-channels (Sekine et al. 2013; Sakaguchi et al. 2013). These systems
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can allow fabricate thicker vascularized cardiac tissues, which can beat spontaneously, synchronously, and macroscopically, in vitro as like ‘‘real heart tissue’’ for overcoming the engineered-tissue thickness limitations described above. Furthermore, after being transplanted into animals for realizing blood vessel anastomoses, the thicker tissues are found to maintain its vascular structure and beat spontaneously for 2 weeks. At present, the authors’ laboratory is applying the systems to other tissues including human iPSC-derived cardiac tissue. The systems can promise significant advances for both preclinical and clinical applications of tissue engineering as well as new in vitro tissue models for evaluating or discovering new drugs. The culture of isolated cells and the preparation of cell sheets, which are used in clinical studies, are manually performed in a clean room system known as Cell Processing Center (CPC). For maintaining the cleanliness of CPC and ensuring the safety and quality of transplanted cell sheets, cell-sheet transplantation materials are generated under strict regulations. In addition to several systems for maintaining the cleanliness of rooms, human handling processes in the CPC are always performed by a pair, namely, one person performs cell-culture procedures including microscopic observation and medium exchange, and another person directs the procedures for avoiding human errors and recording all of the procedures based on Standard Operating Procedures (SOP) following Good Manufacturing Practice (GMP) for the cases of clinical trials, or quasi-GMP for the cases of investigative clinical studies. The production sites and documents are inspected by PMDA regularly. Therefore, the procedure consumes an enormously expensive cost and time. The initial installation cost of GMP-level CPC (floor area: 100 m2) in the authors’ laboratory is more than one million US dollars, and the cost of maintenance is more than 100,000
Latest status of cell sheet engineering and regenerative medicine
US dollars per year (Hannachi et al. 2009). The totally accumulated cost of manipulation in the CPC may discourage the widespread use of cell sheet engineering and the clinical application. Under current regulations, the laboratory has investigated further cost-reduction potentials to use a cell isolator (standardized by ISO 13408-6) in the CPC facility for culturing cells in a closed area, namely Cell Aseptic Processing System (CAPS). Cell culturing cost by CAPS may be reduced to 2/3 of conventional GMP standard culturing cost in CPC. The result of this study has been presented at the Annual Meeting of Japan Society of Regenerative Medicine in 2009 at Tokyo by the authors’ laboratory and CellSeed Inc. for open discussion. Recently, several reports regarding robotic/automatic cell culture systems have been published (Kino-oka and Taya 2009; Kato et al. 2010). The systems requiring no manual manipulation have been significantly advanced and contribute to the improvements of safety and quality of bioengineered tissues, because manual manipulation naturally increases the possibility of human error such as including infectious contamination. The introduction of robotic/automatic systems for sterile cell culture has the advantage of stability and cost-saving for manufacturing qualified bioengineered tissue. It is also widely aware that Food and Drug Administration Office (FDA) in the United States is encouraging manufacturing industry to develop Quality by Design manufacturing method by Process Analytical Technology (PAT), which is automated repeatable manufacturing technology combined with real time remote monitoring function. FDA already recognizes necessity to shift from the current practice of final product test to monitoring PAT for performing the modern quality control of advanced medicinal products along with industry
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and reducing cost for patients [FDA in the United States, October 2010]. Recently, the authors’ laboratory has developed a living-cell isolation device, which can isolate beating cardiomyocytes from heart tissues semi-automatically in an animal model (Shioyama et al. 2013). At present, the laboratory is challenging the introductions of robotic/automatic system in cell isolation, cell culture, cellsheet fabrication, and layering, namely, the establishment of ‘‘tissue factory’’ using cell sheet engineering instead of conventional CPC. Originally patented flexible modular platform (fMP) (Pat. No. JP5051677) in the authors’ ‘‘tissue factory’’ allows each step of cell isolation, culture, sheet fabrication etc. to be maintained by separate automated modules, where the interface of connecting part with various sensors built-in is designed to be standardized. The establishment of the ‘‘tissue factory’’ will give further advancements in cell sheet engineering, tissue engineering, and regenerative medicine, and accelerates both the clinical application and the related regulatory science.
New regulatory framework being formed in Japan Currently in Japan, investigative clinical research is governed by Medical Practitioners Act (MPA), and clinical trial, sponsored by physician or industry, is governed by Pharmaceutical Affairs Law (PAL). Because of separated legal requirements and guidelines, the data quality of investigative clinical study is unable to meet with the minimal requirement of the supplemental data of application documents for obtaining the product marketing approval under PAL. At the same time, the clinical trials of regenerative-medicine product is required to follow drug GMP, which is not intrinsically appropriate for living cell
Fig. 4 Regulatory framework to facilitate the commercialization of regenerative medicine products in Japan. The illustration is the bird’s view of three related laws being legislated in Japan. The illustration was prepared by Ministry of Health, Labour and Welfare (MHLW), and translated by Mime Egami in Aug. 2013
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Fig. 5 Revision of Pharmaceutical Affairs Law (Revision bill, submitted by the Cabinet, passed the Diet on November 20th, 2013) to set up the new category of regenerative medicine and establish its new conditional/ tentative approval system. The illustration was prepared by MHLW, and translated by Mime Egami in Aug. 2013
Fig. 6 Risk-based classification of regenerative medicine is newly designed to legislate the application process of clinical research or the private practice of regenerative medicine. The illustration was prepared by MHLW, and translated by Mime Egami in Aug. 2013. ES cells embryonic stem cells, FIM first-in-man, iPS cells induced pluripotent stem cells, IRB institutional review board
products, or unfeasible to be required for autologous cell cultured products. As the result of immature regulatory framework and the invisibility of regulatory pathway, to date, there are only two products formally approved as medical device in Japan, and four products, which are as drug, are at clinical trial stage, while more than 70 investigative clinical studies are running in Japan. The authors’ laboratory has established joint regulatory-science doctorate program along with Waseda University (Tokyo, Japan) in 2010 for creating regulatory science specialists who can scientifically discuss regulatory convergence into proper
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scientific experiments and self-evident feasible guidelines. Together with the rapid clinical applications of cell sheet engineering, the authors’ laboratory has been encouraging active dialogue to develop new regulatory framework among academia, regulatory agencies, and newly entering industry players. Congressional efforts to establish new promotional framework for regenerative medicine started in 2012 and passed Promotional Act of Regenerative Medicine at Diet in April 2013 (Fig. 4). Cabinet immediately submitted related bills to the Diet to be legislated by 2014, which bills already passed the Diet on November
Latest status of cell sheet engineering and regenerative medicine
20th, 2013. Based on these enacted bills at the Diet, new regulatory framework will be formed by the following two laws. Firstly revised PAL will (1) define regenerative medicine as third category separately from drug or medical devices, which GMP (namely Good Tissue Practice) will be newly designed, and (2) establish conditional approval process after safety review to evaluate efficacy at the bedside before its full approval (Fig. 5). Also newly documented Law of Safety of Regenerative Medicine will (1) first ever legislate the application procedures of both clinical researches and private practices, which are unable to be covered by health insurance, by newly established riskbased classification (Fig. 6) and (2) allow cell culture and processing work to be outsourced from hospital/clinics to industry players by setting up new guidelines of proper risk-sharing outsourcing procedure and preparing a proper authorization and inspection system. Conditional approval system with a visible regulatory pathway, along with qualified cell culture service outsourced to industry, will encourage scientifically qualified regenerative medicine to appear at bedside in a timely manner.
Conclusions Cell sheet engineering has enormous and wide-ranging potentials for the re-construction of various functional tissues including thicker vascularized tissues. In various animal models, the transplantation of cell sheets shows good therapeutic effects and cure potential for chronic diseases. Although various 1st generation cell-sheet clinical studies and therapies have been performed in Japan and in Europe as hospital exemption, there are still many issues that need to be resolved before cell-sheet therapy become a standard treatment at an affordable cost in the World. The authors’ laboratory will initiate further collaborations and active dialogue among researchers with various backgrounds, multi-disciplinary clinicians, industry players, regulatory specialists, and patients for resolving the issues and establish the innovative value chain of regenerative medicine, following our Credo ‘‘Passion for Innovation and Duty to the Patients of Tomorrow’’. Acknowledgments This work was supported by grants from the Global Center of Excellence Program, Multidisciplinary Education and Technology and Research Center for Regenerative Medicine (MERCREM), and Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology ‘‘Cell Sheet Tissue Engineering Center (CSTEC)’’ from the Ministry of Education, Culture, Sports Science, and Technology (MEXST), Japan, the Japan Society for the Promotion of Science (JSPS) through the ‘‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),’’ initiated by the Council for Science and Technology Policy (CSTP).
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References Akiyama, Y., A. Kikuchi, M. Yamato, and T. Okano. 2004. Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control. Langmuir 20: 5506–5511. Akizuki, T., S. Oda, M. Komaki, H. Tsuchioka, N. Kawakatsu, A. Kikuchi, M. Yamato, T. Okano, and I. Ishikawa. 2005. Application of periodontal ligament cell sheet for periodontal regeneration: A pilot study in beagle dogs. Journal of Periodontal Research 40: 245–251. Arauchi, A., T. Shimizu, M. Yamato, T. Obara, and T. Okano. 2009. Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models. Tissue Engineering Part A 15: 3943–3949. Atala, A., S.B. Bauer, S. Soker, J.J. Yoo, and A.B. Retik. 2006. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 367: 1241–1246. Atala, A., R. Lanza, J.A. Thomson, and R. Nerem. 2011. Principles of regenerative medicine, 2nd ed. New York: Academic Press. Bolli, R., A.R. Chugh, D. D’Amario, J.H. Loughran, M.F. Stoddard, S. Ikram, G.M. Beache, S.G. Wagner, A. Leri, T. Hosoda, F. Sanada, J.B. Elmore, P. Goichberg, D. Cappetta, N.K. Solankhi, I. Fahsah, D.G. Rokosh, M.S. Slaughter, J. Kajstura, and P. Anversa. 2011. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): Initial results of a randomised phase 1 trial. Lancet 378: 1847–1857. Brittberg, M., A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson. 1994. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New England Journal of Medicine 331: 889–895. Burillon, C., L. Huot, V. Justin, S. Nataf, F. Chapuis, E. Decullier, and O. Damour. 2012. Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) transplantation for the treatment of corneal limbal epithelial stem cell deficiency. Investigative Ophthalmology & Visual Science 53: 1325–1331. Chachques, J.C., J.C. Trainini, N. Lago, M. Cortes-Morichetti, O. Schussler, and A. Carpentier. 2008. Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM trial): Clinical feasibility study. Annals of Thoracic Surgery 85: 901–908. Chachques, J.C., J.C. Trainini, N. Lago, O.H. Masoli, J.L. Barisani, M. Cortes-Morichetti, O. Schussler, and A. Carpentier. 2007. Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM clinical trial): One year follow-up. Cell Transplantation 16: 927–934. Ebihara, G., M. Sato, M. Yamato, G. Mitani, T. Kutsuna, T. Nagai, S. Ito, T. Ukai, M. Kobayashi, M. Kokubo, T. Okano, and J. Mochida. 2012. Cartilage repair in transplanted scaffold-free chondrocyte sheets using a minipig model. Biomaterials 33: 3846–3851. Flores, M.G., M. Hasegawa, M. Yamato, R. Takagi, T. Okano, and I. Ishikawa. 2008. Cementum–periodontal ligament complex regeneration using the cell sheet technique. Journal of Periodontal Research 43: 364–371. Hannachi, I.E., M. Yamato, and T. Okano. 2009. Cell sheet technology and cell patterning for biofabrication. Biofabrication 1: 022002. Haraguchi, Y., T. Shimizu, T. Sasagawa, H. Sekine, K. Sakaguchi, T. Kikuchi, W. Sekine, S. Sekiya, M. Yamato, M. Umezu, and T. Okano. 2012a. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nature Protocols 7: 850–858. Haraguchi, Y., T. Shimizu, M. Yamato, and T. Okano. 2012b. Scaffold-free tissue engineering using cell sheet technology. RSC Advances 2: 2184–2190.
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