Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

DOI 10.1002/biot.201300432


Cell sheet engineering for regenerative medicine: Current challenges and strategies Toshiyuki Owaki, Tatsuya Shimizu, Masayuki Yamato and Teruo Okano Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, Tokyo, Japan

Substantial progress made in the areas of stem cell research and regenerative medicine has provided a number of innovative methods to repair or regenerate defective tissues and organs. Although previous studies regarding regenerative medicine, especially those involving induced pluripotent stem cells, have been actively promoted in the past decade, there remain some challenges that need to be addressed in order to enable clinical applications. Designed for use in clinical applications, cell sheet engineering has been developed as a unique, scaffold-free method of cell processing utilizing temperature-responsive cell culture vessels. Clinical studies using cell sheets have shown positive outcomes and will be translated into clinical practice in the near future. However, several challenges stand in the way of the industrialization of cell sheet products and the widespread acceptance of regenerative medicine based on cell sheet engineering. This review describes current strategies geared towards the realization of the regenerative medicine approach.

Received 21 JAN 2014 Revised 04 APR 2014 Accepted 22 MAY 2014

Key words: Cell sheet engineering · Clinical application · Industrialization · Innovative technology

1 Introduction Regenerative medicine is aimed at promoting recovery from disease, injury, or the natural aging process by utilizing highly sophisticated medical technology to repair, replace, or regenerate a patient’s tissues or organs. Regenerative medicine gives birth to a concept contrary to conventional medical principles, and heralds a revolutionary shift from symptomatic treatment to radical treatment. The ability to implement such treatments is dependent on the progress made towards the integration of medicine, science, and engineering crucial to this emerging multidisciplinary field. Regarding the market

Correspondence: Prof. Teruo 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] Abbreviations: CPC, cell processing center; DCM, dilated cardiomyopathy; ESD, endoscopic submucosal dissection; FGF-2, fibroblast growth factor; GMP, good manufacturing practice; LSCD, limbal stem cell deficiency, PDL, periodontal ligament; PIPAAm, poly(N-isoproplyacrylamide); SDF-1, stromal cell-derived factor-1; SOP, standard operating procedure; VEGF, vascular endothelial growth factor


size of regenerative medicine in 2012, the total sales of all cell therapy products was estimated at $900  million for 160 000 patients and the market potential is anticipated to be high due to the active R&D investment into cell therapy products ( While most products on the market are composed of skin or cartilage bioprocessed with somatic cells [1,2], R&D into next-generation products derived from stem cells, such as mesenchymal or embryonic stem cells, has been steadily promoted. Recently, a clinical study into iPSC therapy has been approved by the Ministry of Health, Labour and Welfare in Japan ( This study will be conducted to assess the safety and feasibility of the transplantation of autologous iPSC-derived retinal pigment epithelial cell sheets in patients with age-related macular degeneration. This approval has encouraged clinical investigators studying iPSCs, and subsequent clinical studies for iPSC-derived target cells to treat spinal cord injury [3] or Parkinson’s disease [4] will be conducted within a few years. Many stem cell researchers believe that research findings regarding stem cells will be automatically linked to the realization of regenerative medicine. There is no doubt that these achievements will open the door to breakthrough therapies for patients suffering

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

from refractory diseases, but the requisite technologies for establishing regenerative medicine are as follows: (i) bioprocess technologies applicable to clinical grade products; (ii) optimal transplant procedures for efficient engraftment; (iii) infrastructure improvements such as cell processing facilities; and (iv) storage and shipping for industrialization. Therefore, our research institute has formed a consortium with several industrial companies to find solutions to the bottlenecks currently hampering the realization of the cell sheet engineering-based therapies we have proposed. We have made functional contacts with experts in different fields to gather a variety of leading-edge technologies through partnerships between medicine and engineering, as well as between academia and industry. This review describes our efforts to enable

clinical applications of cell sheet engineering-based therapies using a multidisciplinary approach.

2 Cell sheet engineering and its clinical applications 2.1 Cell sheet engineering It has been reported that the sheet-like cell assembly referred to as a “cell sheet” can be fabricated by several methods. Table 1 shows the outline of various techniques reflecting the current state of cell sheet research. Green et al. first reported a successful expansion of primary human keratinocytes through feeder cell support [5] and

Table 1. Comparison of cell sheets fabricated by different methods.

External stimulus

Key material

Cell type (pre-clinical study)

Target tissue (clinical study)

Biochemical Dispase (enzymatic treatment)

Keratinocyte [6], Corneal epithelial cell [77], Oral mucosal epithelial cell [78]

Epidermis [79], Corneal epithelium [80]

Not needed

Amniotic membrane

Oral mucosal epithelial cell [7], Corneal endothelial cell [8], Corneal epithelium [10] Conjunctival epithelial cell [9], Amniotic epithelial cell [81], Keratinocyte [82], PDL cell [83]

Temperature reduction below 32°C

Temperature-responsive polymer (PIPAAm)grafted cell culture vessel

Somatic cell (epithelial): Keratinocyte [13], Corneal epithelial cell [14], Oral mucosal epithelial cell [15], RPE cell [16], Middle ear mucosal cell [17], Urothelial cell [18], Alveolar epithelial cell [19], Tracheal epithelial cell [20]

Corneal epithelium [51], Esophageal mucosa [52]

Somatic cell (non-epithelial): Fibroblast [25], Hepatocyte [26], Pancreatic islet cells [27], Thyroid cell [28], Renal cell [29], Adipocyte cell [30], Smooth muscle cell [31], Synovial cell [32], Anterior cruciate ligament [32], Osteogenic cell [33], Periosteal cell [34], BMSC [35], HUVEC [36], Aortic endothelial cell [37]

Myocardium [53], Periodontium [54], Cartilage [55]

Stem cell or pluripotent stem-derived cell: MSC [25], ADSC [38], Cardiac Stem Cell [39], ESC-derived cardiomyocyte [40], iPSC-derived cardiomyocyte [41]


NIH/3T3 (fibroblast) [42], Keratinocyte [43], RPE cell [44], Cardiomyocyte [45], MSC [46]


Magnetic force

Magnetite (Fe3O4) cationic liposome

Electrochemical polarization

Polyelectrolyte multilayers NIH/3T3 [47] coated on indium tin oxide electrodes

Electrochemically induced pH decrease Hexacyanoferrat (II)induced dissolution

Ferrocyanide, polyelectrolytes

365 nm UV illumination

TiO2 nanodot film-coated quartz

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


MSC [48]


C2C12 (myoblast) [49]


TiO2 nanodot film-coated quartz



Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

harvesting of the cell assembly by dispase, which leads to cell sheet detachment via digestion of several ECM proteins but not cell-junction proteins [6]. The cell sheet graft bioprocessed by this method has been clinically proven as a treatment for deep dermal or full thickness burns [2]. Cell sheet research with an amniotic membrane has been actively conducted in the field of ophthalmology [7–9], an example of the success of regenerative medicine in translational research [10]. In this case, an amniotic membrane acts as an epithelial cell carrier and is collected along with a cell sheet from a cell culture dish followed by directly grafting onto a corneal stroma. These two methodologies predominate fabrication of cell sheets for epithelial regeneration. As an alternative, our group has proposed cell sheet engineering that makes it possible to harvest a cell sheet without the use of proteolytic enzymes or cell scrapers but instead by lowering temperature [11]. This method utilizes the fact that the temperature-responsive polymer (PIPAAm)-immobilized vessel surface becomes hydrophilic below 32°C, resulting in spontaneous cell detachment, whereas cells adhere and proliferate on the vessel at 37°C. This noninvasive cell sheet can be engrafted at the desired transplantation site without suturing the cell sheet because the ECM deposited underneath the cell sheet works as an adhesive agent [12]. Pre-clinical studies demonstrate that cell sheet engineering has been successful with various types of cells: epithelial cells [13–20], non-epithelial cells [21–37], stem cells [25, 38, 39], and pluripotent stemderived cells [40, 41]. These results suggest there is a high potential for cell sheet technologies to contribute to the creation of tissues or organs when combined with other innovative technologies. In recent reports, cell sheet detachment can also be induced by several external stimuli such as magnetic force [42–46], electrochemical polarization [47], pH decrease [48], ionic solution [49], and light [50]. These research results are all based on a common mechanism whereby an external stimulus triggers a change on the culture surface enabling the removal of cultured cells from the culture vessel. Proper use of cell sheet technologies may play an important role in bioprocessing cellular products for regenerative medicine, but not for applications requiring cells sensitive to those particular external stimuli.

2.2 Clinical applications for cell sheet engineering Presumably at least a few types of cell sheets will provide cures for refractory patients as well as enable us to create tissues or organs. At the present time, five clinical studies have been conducted transplanting monolayer cell sheets onto cornea [51], esophagus [52], and using triplelayered cell sheets for myocardium [53], peridontium [54], and cartilage [55]. Organ transplantation is the ultimate medical treatment for a patient with organ failure. However, the over-


whelming shortage of donor organs along with transplant rejection make it difficult for patients to undergo transplantation procedures. Regenerative medicine is anticipated as an alternative to organ transplantation and will provide a solution for these two major issues. We have investigated the possibility that cell sheet engineeringbased regenerative medicine can replace organ transplantation to treat cornea [51], and heart [53] problems. Limbal stem cell deficiency (LSCD) is the visual disorder caused by severe trauma or eye diseases such as Stevens-Johnson syndrome, and no effective therapy without limbal allograft transplantation has been established. Cultured epithelial cell sheets derived from oral mucosal tissue have been successfully fabricated on a temperatureresponsive culture vessel. A pre-clinical study with an LSCD rabbit model has demonstrated that this cell sheet graft retained corneal epithelium-like structure, including stem/progenitor-like cells, resulting in not only inhibition of vascularization and inflammation in the cornea but also recovery of the corneal clarity and smoothness. The clinical study where the autologous epithelial cell sheets were transplanted to four  patients with total LSCD confirmed good tolerability and efficacy of the post-grafting. Cell-based myocardial regenerative therapy is also predicted to be a significant treatment for patients with severe heart failure including dilated cardiomyopathy (DCM) and ischemic heart disease. However there still remain some issues to be resolved: (i) suspended cells have poor engraftment to the target cardiac tissue; (ii) needle injection is a highly invasive approach; and (iii) cell death induced by cell injection carries a risk of myocardial infarction. To overcome these issues, the skeletal myoblast sheet derived from medial vastus muscle has been developed as a patch for myocardial regeneration. In a case report [53], 20  autologous myoblast sheets were transplanted to the heart of a patient with DCM who needed LVAD support. This cell sheet transplantation dramatically improved cardiac functions, resulting in no symptoms attributable to heart failure, LVAD weaning, and hospital discharge. As it has been found that the paracrine effect of multiple cytokines (VEGF, HGF, and SDF-1) secreted from myoblast sheets contributes to an improvement of the damaged heart functions and reduction of fibrosis in a rat infarction model [56], the remarkable recovery of the damaged myocardium observed in this clinical study is thought to be based on the same mode of action. The results of these successful clinical trials initiated the procedure to obtain marketing authorization for these cell sheet products [57]. These two products are expected to be launched within a few years as regenerative medicine treatments. Regenerative medicine is promoted as a promising treatment for the reconstruction of tissue defects resulting from resection of cancerous lesions. Endoscopic submucosal dissection (ESD) is a powerful operative procedure to resect superficial early-stage cancers in the gas-

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Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

trointestinal tract with low invasiveness compared to open surgery. However, postoperative inflammation and stenosis are major complications observed after intensive mucosal resection. We attempted novel esophageal regeneration to promote epithelialization of the resected area after ESD for the prevention of postoperative complications. After verifying the safety and efficacy of cultured oral mucosal epithelial cell sheets in a canine model [15], the clinical study was evaluated for the inhibitory effect of esophageal stenosis after ESD by the cultured autologous oral mucosal epithelial cell sheet [52]. No complications, including esophageal stenosis, were observed after the transplantation of the epithelial cell sheets onto the defective site after ESD with the exception of a case of circumferential ESD. It has also been demonstrated that an autologous epidermal cell sheet is feasible for the prevention of esophageal stenosis after ESD, indicating that this methodology enables us to fabricate more autologous cell sheets than ones originating from oral mucosal tissue [58]. Periodontitis is an inflammatory disease characterized by periodontal tissue destruction, and predominates as the main cause of tooth loss in adults. Recently some procedures for periodontal regeneration have been introduced in clinical practice but unfortunately they have failed to fully meet patient’s expectations. It is well-known that periodontal ligament (PDL) tissues include stem cell populations which possibly differentiate into alveolar bone and cementum. Therefore, the clinical study for periodontal regeneration with autologous PDL cell sheets was conducted after pre-clinical studies for safety and proof-of-concept efficacy of PDL cell sheet using animal models [54]. As a protocol, PDL tissue is obtained from a subject having an unwanted tooth such as a wisdom tooth. The PDL cell sheets are fabricated from the cells in the PDL tissue that have colony forming activity, and then the triple-layered cell sheets are implanted in the proximity of the infrabony defect. As of April 4, 2014, no adverse events occurred in nine cases. Both safety and efficacy of implanted cell sheets will be reported in detail within the next few years. The regenerative products for articular cartilage are already available for patients with osteochondral defect, but not osteoarthritis that shows symptoms of immovable joints and pain triggered by repeated abrasion of an articulated surface. This radical treatment for osteoarthritis has not yet been established, and current symptomatic treatments such as non-steroidal anti-inflammatory drugs and injection of hyaluronan are usually conducted to retard disease progression. It has been found that threelayered chondrocyte sheets might be applicable as a curative treatment for this partial thickness defect of articular cartilage [55]. A clinical study is ongoing where autologous chondrocyte sheets are transplanted onto the lesion of early middle-stage osteoarthritis. This scaffoldfree cell transplantation is anticipated to be effective in patients with full-thickness cartilage defects as well as those with partial thickness defects.

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3 Breakthrough in the preparation of clinical-grade cell sheets A cell processing center (CPC) is a facility consisting of a clean room for the aseptic cell process, an examination room for a variety of tests such as the tests for airborne and falling bacteria in the CPC, and a storage facility for cryopreserved cell/tissue with continuously monitored temperature, humidity, and cleanliness. A CPC is indispensable for preparation of cell-based advanced medical products required for cancer immunotherapy or gene therapy, as well as for regenerative medicine. All operations in a CPC must be performed in compliance with good manufacturing practice (GMP) in order to achieve the following major objectives: (i) to minimize human error; (ii) to prevent both contamination and deterioration of product; and (iii) to design a system that assures high quality of the product. Specifically, air-conditioning, differential pressure, and sanitary controls for the aseptic environment, education of operators, validation of instruments in the facility, and annual inspection are the minimum practices necessary to meet the GMP requirement. Moreover, standard operating procedures (SOPs) must be prepared to maintain optimal routine operation in a CPC. For the purpose of manufacturing and quality controls for clinical-grade cell sheets, product master formula must be prepared and all CPC operative notes must be stored. In part due to these considerations, the annual running cost of our CPC is more than $200 000 USD and at most only 40  patients’ cell sheets are produced per annum. Thus this methodology for cell sheet fabrication in the conventional CPC drives the product price high enough to hamper the spread of cell sheet engineering-based therapy. Furthermore, appropriate operations by well prepared SOPs and a product master formula according to GMP guidelines does not completely eliminate the risk of human error or contamination, which would cause deterioration of a cell sheet product and may result in unstable quality. To overcome this serious challenge, it is recommended to introduce an automated system to manufacture cell sheet products. Cell culture in this bioprocess is so simple that the manual procedure can simply be replaced with an automated one. Several technical concepts of automated cell culture system have been reported [59–64] and based on some of the concepts equipment has been designed and made commercially available. Unfortunately those automated cell culture systems are not suitable for our facility because the use of a special culture vessel, called a cell culture insert, during cell sheet processing forces us to customize the system. Therefore, we have begun to develop an automatic apparatus to perform the unique cell culture process required for the manufacture clinical-grade cell sheet according to GMP guidelines. At first, a closed cell culture vessel, called a Cell cartridge, was designed to avoid risk of contamination [Fig. 1A, 65]. The Cell cartridge has two compart-


Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

Figure 1. Automated cell sheet fabrication system. (A) Cell cartridge appearance. (B) Schematic view of Cell cartridge. (C) Automatic cell culture apparatus appearance.

ments for epithelial cells and feeder layer cells separated by a porous membrane on which a temperature-responsive polymer is covalently immobilized (Fig. 1B). The oral mucosal epithelial cell sheets were successfully fabricated with Cell cartridge, which is comparable to ones with our current cell culture insert. A prototype of the automated cell culture apparatus (HITACHI, Ltd.) adjusted to this Cell cartridge has also been constructed including [Fig. 1C, 66]: a centrifugal separator and tip manipulator for seeding cells into Cell cartridges; an incubator and a CO2 gas controller for cell culture; a handling manipulator and a pump for medium change and collection of supernatants; and a CCD camera for microscopic observation of the real-time cell culture situation using computer programs. The epithelial cell sheets fabricated in an automated fashion were robust enough to be engrafted onto ocular surfaces in a rabbit corneal stem cell deficiency model. In a few year we anticipate that this automated cell culture system will become commercially available after the verification of its compliance with safety requirements and feasibility of its utilization by clinical study.

4 Further innovations to create a system for tissue or organ fabrication from cell sheets 4.1 Large-scale production of robust target cells differentiated from pluripotent stem cells Tissue or organ fabrication requires a large number of cells of the desired type; specifically billions of cells must be prepared to fabricate a tissue similar in size to an adult human left ventricle, whereas several million are enough


to complete a cell sheet. A stable and efficient cell expansion system will be demanded in order to provide the quantity of cells required for clinical applications. Cost performance is one of the most essential elements for industrialization and widespread acceptance of cell sheet products. We have designed a 3D cell suspension culture system meeting these requirements [40, 41]. A few improvements to the system were obtained using two types of bioreactors (1 L and 100 mL scales) allowing us to obtain 8  ×  107 cells/100  mL vessel constituting around 80% of cardiomyocytes differentiated by human iPSCs, leading to great savings in both effort and consumable supplies: approximately 80 and 90% reductions of time spent at work and total culture medium consumed, respectively. Furthermore, cell sheets fabricated from cells expanded with this large-scale culture method were comparable to those obtained using common laboratoryscale culturing methods, indicating that this large-scale cell expansion system provides us with enough highquality cells to enable us to fabricate a tissue stably, efficiently, and cost-effectively.

4.2 Unique technologies for fabrication of functional 3D tissue from cell sheets Among the difficulties in fabricating functional 3D tissue from cell sheets, the two major challenges to be overcome are to perfectly layer a number of cell sheets and to form a well-organized vascular network into the multilayer cell sheet. As a result of several trials to resolve the former challenge, we have adopted a novel approach to stack cell sheets in layers with a cell sheet layering manipulator [67, Fig. 2A]. First of all, this manipulator is coated with hydro-

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Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

gel and a cell sheet placed on the contact surface, the manipulator is then incubated at 20°C and the cell sheet adheres to the hydrogel. The manipulator can then pick up the cell sheet and place it on top of another cell sheet. This transfer of the developing layer of cell sheets onto a new cell sheet is repeated until a multilayered cell sheet of the desired thickness is achieved. Finally, the multilayered cell sheet is harvested using warm medium which rinses the hydrogel out of the manipulator. In practice this methodology permits 10 sheets of spontaneously-beating cardiomyocyte to be layered. Use of this manipulator requires some skill and knowledge in order to successfully stack the cell sheets into layers. With this in mind we recently developed an automatic apparatus which can stack several cell sheets in a reproducible fashion without requiring an operator with skill and knowledge [68]. This automatic apparatus was designed to be compact enough to be put in a safety cabinet, and the manipulator built into the apparatus has been modified on several occasions until reproducibility was attained. At the present time, this automatic process has succeeded in the five-layer human skeletal muscle myoblast sheets without requiring the delamination used under the optimized condition for manually stacking those cell sheets, suggesting that such an automatic apparatus can contribute to the mass production of high quality multilayered cell sheets in the clinical settings. The other challenge to be addressed is the development of technologies to form a well organized vascular network in the multilayer cell sheet. Conceptually the engineering of a multilayer cell sheet allows us to fabricate a cell-dense construct structurally-similar to native tissues. In practice, however, the layering of multiple cell sheets does not always result in the thickness of a neonatal rat cardiomyocyte sheet construct. Thickness of the

construct transplanted into the dorsal subcutaneous tissue of a nude rat has become a constant form for a more than four-layered construct where thickness increases linearly up to a three-layered construct, implying that this methodology provides us with a tissue only 80  μm in thickness. Histological analysis revealed that stacking of more than four  layers of cell sheets induced necrosis inside the transplanted graft, assumedly because of hypoxia, nutrient insufficiency, and waste accumulation caused by fragile vasculature [21]. Therefore, we hypothesized that neovascularization within the multilayered cell sheets might help to supply oxygen and nutrients to the inside of the transplanted graft. We simultaneously initiated basic research on how to form a vascular network during construction of 3D transplantable graft from cell sheets in order to scientifically clarify the mechanism and manipulate the formation. As a result of those research endeavors [69–72], we have found that formation of an endothelial cell network within a cardiac cell sheet could be controlled by changing the ratio of endothelial cells present in the cell sheets, and the graft-derived blood vessels rapidly invade the triple layered construct after transplantation into rat dorsal subcutaneous tissue forming functional connections with host vasculature [69]. Based on those findings, we developed a unique fabrication of 3D and cell-dense graft with functional vessels which we named multistep transplantation procedure for vascularized graft. This detailed process is shown in Fig. 2B and is described as follows: (i) triple-layered cell sheets co-cultured with endothelial cells are transplanted in vivo as a primary graft; (ii) successful engraftment of the construct onto the transplantation site drives formation of vascular networks between the construct and host; (iii) the secondary triple-layered construct is overlaid on the primary graft; (iv) the transplanted construct is connect-

Figure 2. Unique technologies for 3 D tissue fabrication by layering cell sheets. (A) Stamping technology to stack cell sheets in layers with the manipulator. (B) Vascular network formation inside of the stacked cell sheets.

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Biotechnology Journal

Biotechnol. J. 2014, 9, 904–914

ed with host through vasculature; (v) this vascular network supplies oxygen and nutrients to multilayer cell sheet-derived graft for the survival; and (vi) this procedure is repeatedly conducted until the desired thickness of the graft is achieved. In practice we have succeeded in creating approximately 1 mm of myocardium-like tissue with well-organized microvessels using 10 transplantations of triple-layered cardiac cell sheets (30 sheets in total) [21]. Moreover, surgically accessible artery and vein connecting to a multilayer graft permitted graft perfusion through those vessels and ectopic transplantation of the graft. Thus, a combination approach with cell sheet engineering and this multistep transplantation procedure leads to a solution to enable the fabrication of a 3D tissue with functional blood vessels previously unobtainable through conventional approaches. Given the clinical application of tissue or organ regeneration, in vitro fabrication of functional 3D tissue will be required. To satisfy this demand, we substituted two types of in vitro vascular bed: femoral muscle tissue with a connectable artery and vein [73]; and collagen-gel fabricated with embedded microchannels [74] where triplelayered cell sheets were repeatedly transplanted. Each vascular bed was perfused with culture medium in the bioreactor system to set up the optimized condition. Both bioreactors monitored several factors such as oxygen concentration and pH. When triple-layered cardiac cell sheets co-cultured with endothelial cells were transferred onto each vascular bed, endothelial cells migrated to both vascular beds for the formation of a perfusable vascular network followed by the medium supply to graft. The migration of endothelial cells from the triple-layered cell sheets to those vascular beds, supporting the formation of a vascular network, was promoted by angiogenesis factors such as FGF-2 or VEGF. The overlaying of the triple-layered cell sheets onto those vascular beds has increased their thickness depending on the number of overlays. As of now, four-fold overlaying, namely, 12-layered cell sheets have been achieved by both vascular beds. Moreover, the vascularized cardiac cell sheet construct with an artery and vein survived in the nude rat anastomosed by those blood vessels. Concretely speaking, the six-layer tissue graft had spontaneously beaten in association with a perfusable vascular network since the protruding femoral artery and vein of the graft were reconnected to the carotid artery and the jugular vein of a nude rat [74]. These research achievements have demonstrated that both in vitro fabrication systems are feasible for formation of viable and 3D cardiac tissue with a vascular network. This innovative technology to bioengineer a cell sheet-derived construct structurally similar to native tissues has a potential impact on clinical applications for regenerative medicine. As is the case with stacking technology, this 3D tissue fabrication will also be automatically performed for clinical applications in the future. This platform technology to fabricate cardiac tissue from the triple-layered


cardiac cell sheets should be applicable to the creation of other 3D tissues with a well-organized vascular network.

4.3 Automated manufacturing system for clinical-grade tissue For an autologous cell therapy product released on the market, it is thought that the manufacture cycle established for bioprocessing will be as shown in Fig. 3. In our case, a specimen collected from a patient in hospital is shipped to the CPC where each operation should be correctly performed under SOPs – cell isolation, primary and expansion cultures, cell sheet fabrication, and stacking by cell sheet-layering manipulator. Then the final product is shipped back to the hospital and returned to the patient. For the purpose of cell sheet manufacturing automation, we constructed the prototype system named “T-factory” (Fig. 4) which facilitates this manufacture cycle for cell sheet products [75]. This system has three features: (i) specialization of operation by compact isolator; (ii) multiple manufacturing lines in parallel by fMP; and (iii) an integrated product management system. Firstly, the compact isolator specialized for individual aseptic processing, specifically, cell isolation/primary culture, cell expansion culture, cell seeding/medium replacement, and cell sheet formation/layering, has been built as a module. This design permits downsizing, and the reduction of contamination risks because operators do not need to invade the sterile environment for cell sheet processing. Secondly, we have introduced the concept of fMP that an individual module can be aseptically attached to, or removed from, a transfer module to customize the manufacturing process for a cell sheet product on an as-needed basis. A cell sheet intermediate in the individual module is transferred to another module for the next step of processing through the transfer module acting as a hub. The standardization of an interface connected between modules allows us to install the module developed with new types of cell processing. This concept achieves multiple manufacturing lines in parallel followed by an efficient and mass production of cell sheets, leading to significant time- and cost-savings. Moreover, removal of a module from fMP contributes not only to the manufacture of a wide variety of cell sheet products in small quantities but also to limiting the expansion of contamination. The concept of fMP allows new companies to participate in this T-factory project whenever it is needed because the developed machines can connect to the standardized interface to be involved in the T-factory system, providing an advantage to enable us to rapidly introduce innovative technologies related to efficient manufacturing and high quality for cell sheet products. Thirdly, a system integrated from starting material to final product is required for GMP-complying manufacturing. All the manufacturing processes in each isolator are regulated by the software customized for this quality control, e.g. production sched-

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Biotechnol. J. 2014, 9, 904–914

Figure 3. Schematic of the manufacturing cycle to supply patients with autologous cell sheets. Every clinical-grade cell sheet is fabricated through the following common processing pathway at Cell processing center: cell isolation from patient’s tiny amount of tissue; primary and/or expansion culture; cell seeding on temperature-responsive culture vessel where a cell sheet is formed.

ule, materials control, monitoring for aseptic environment and cell sheet processing. All operations are digitally recorded at all times, dispensing with both complicated paperwork regarding its operation and storage of large quantities of paper. Hereby, T-factory will be able to overcome those challenges present in manual manipulation in the current CPC, that is to say, risk of contamination, human error, low productivity, unstable quality, and the high cost of CPC maintenance, as the most critical issues. Furthermore, this system is more consistent with GMP guidelines than conventional operations in the CPC. A shift from manual production to automation by T-factory introduces the next generation of manufacturing process for clinical-grade cell processing products as used as grafts in the present clinical studies. Although the cell-sheet-processing technologies introduced into T-factory can obviously be applied to organ creations as well, the fabrication of vascularized tissue grafts as described above (Section 4.2) is still in a development stage and much needs to be improved prior to clinical application. If additional innovative technologies produce considerable improvements and help to overcome the current challenges, we will certainly achieve an integrated automation system which deserves to be named the “organ factory”.

5 Other barriers hampering realization of regenerative medicine in the future In this review, we describe our research efforts regarding clinical applications for cell sheet engineering-based regenerative medicine. The next barrier to the application of cell sheet regenerative medicine will be regulatory issues. In general, most of the products for regenerative medicine have been developed based on cutting-edge research findings. These cell-based products have unique properties – a wide variety of formulations, bioprocessings, mode of actions, unstable quality, and difficult storage. In addition, they are too innovative to be simply categorized as medical drugs or devices. As such assessment by regulatory agencies is complicated, it is expected to take much effort and a long time in order for market authorization as a medicinal product to be granted. Therefore regulatory science, encouraging the social harmonization of medical innovation, will play a prominent role in the realization of the applications of regenerative medicine. It can be said that regulatory science aims at adjusting the scientific and technological research achievements to desirable forms for both people and society [76]. Smooth pathways to pharmaceutical approval for cutting-edge products in accord with this concept of reg-

Figure 4. Prototype of T-Factory and flexible modular platform. Various types of compact isolator are currently under development: (A) module for cell sheet layering; (B) cell seeding / medium replacement; (C) CO2 incubation; (D) the central module that transfers cell sheet intermediates between modules during the manufacture of cell sheets; and (E) the isolator where starting materials are manually processed before subjected to an automatic manufacture.

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ulatory science requires scientific review and assessment based on the latest findings. Development of human resources should also be essential for optimal regulatory review. Furthermore, the review process of the approved products deserve to be investigated as an important theme of regulatory science for future reviews and assessments. Thus, the significance of regulatory science will be increasingly recognized. In conclusion, the realization of regenerative medicine requires the presence of multidisciplinary integration – collaboration among medicine, science and engineering, and cooperation among government, industry and academia. To this end, in addition to a variety of stem cell studies, other key players including engineers, clinicians, industrial workers and regulatory agencies must actively participate during the various phases, keeping in contact with players in different fields, and affirming a common recognition of truly patient-oriented medical care. We are confident that this collective intelligence and active trialand-error approach will pave an innovative way to realize the promise of the field of regenerative medicine.

Teruo Okano is currently the Vice President and Professor of Tokyo Women’s Medical University in Tokyo, Japan, and Director of the Institute of Advanced Biomedical Engineering and Science at the same university. He is also an Adjunct Professor at the University of Utah, USA. Currently he is the President of the Japanese Society for Regenerative Medicine. He is the author of more than 650 peer-reviewed journal articles as well as over 250 books and book chapters. He received the Leona Esaki prize (2005) and the Emperor’s Medal with Purple Ribbon (National Meritorious Achievement Award) (2009).

Toshiyuki Owaki is an Assistant Professor at the Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Japan. He was granted the degree of

This work was supported by the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from the Japan Society for the Promotion of Science, and the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems ‘Cell Sheet Tissue Engineering Center (CSTEC)’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Conflict of interest: Teruo Okano is a director of the board of CellSeed Inc., a cell sheet regenerative medicine company licensing technologies and patents from Tokyo Women’s Medical University. Teruo Okano, Masayuki Yamato, and Tatsuya Shimizu are stake holders of CellSeed Inc. Tokyo Women’s Medical University is collaborating with CellSeed Inc., NIHON KOHDEN CORPORATION, HITACHI, Ltd., SHIBUYA KOGYO Co., Ltd., TERUMO CORPORATION, and Asahi Kasei Corporation, and receiving research funds from them.

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Doctor of Pharmacology by Tokyo University of Science in 2006. He has engaged in bio-research including drug discovery research utilizing cell and molecular biological approaches. He is currently the project manager for cell sheet clinical studies. He also dedicates efforts to infrastructure improvement encouraging the spread of cell sheet-based therapy using all his skills. His prime concern providing safe and effective regenerative medicine to many patients without delay.

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ISSN 1860-6768 · BJIOAM 9 (7) 863–990 (2014) · Vol. 9 · July 2014

Systems & Synthetic Biology · Nanobiotech · Medicine


Stem cells and regenerative medicine

Stem cell therapy Microfluidics culture Serum-free medium

Special Issue: Stem cells and regenerative issue. This is the second special issue of Biotechnology Journal in collaboration with the Asian Federation of Biotechnology and is edited by Prof. Byung-Soo Kim and Prof. Jiandong Ding. The cover shows a fluorescence image of a rat eye tissue section. Transplanted neural stem cells expressing GFP (green) have integrated into the host retina. Light-detecting photoreceptors (rods and cones) of the host eye are stained red and all cell nuclei are stained blue. The image is from the article by Jisun Oh et al.

Biotechnology Journal – list of articles published in the July 2014 issue. Editorial: Scientific and engineering progress in stem cell and regenerative medicine research Byung-Soo Kim and Jiandong Ding

Research Article Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways

Review Induced pluripotent stem cells for modeling of pediatric neurological disorders

Ahmed Abdal Dayem, BongWoo Kim, Sangiliyandi Gurunathan, Hye Yeon Choi, Gwangmo Yang, Subbroto Kumar Saha, Dawoon Han, Jihae Han, Kyeongseok Kim, Jin-Hoi Kim, and Ssang-Goo Cho

Jiho Jang, Zhejiu Quan, Yunjin J. Yum, Hyo Sook Song, Seonyeol Paek and Hoon-Chul Kang Review Stem cell therapy and cellular engineering for treatment of neuronal dysfunction in Huntington’s disease Kyung-Ah Choi, Insik Hwang, Hang-soo Park, Seung-Ick Oh, Seongman Kang and Sunghoi Hong Review Preserving human cells for regenerative, reproductive, and transfusion medicine Waseem Asghar, Rami El Assal, Hadi Shafiee, Raymond M. Anchan and Utkan Demirci Research Article Angiogenic/osteogenic response of BMMSCs on bonederived scaffold: Effect of hypoxia and role of PI3K/Aktmediated VEGF-VEGFR pathway Yi Zhou, Xiaoxu Guan, Mengfei Yu, Xinhua Wang, Wenyuan Zhu, Chaowei Wang, Mengliu Yu and Huiming Wang Research Article S-Fms signalobody enhances myeloid cell growth and migration Masahiro Kawahara, Azusa Hitomi and Teruyuki Nagamune

Review Cell sheet engineering for regenerative medicine: Current challenges and strategies

Research Article A serum-free medium developed for in vitro expansion of murine intestinal stem cells

Toshiyuki Owaki, Tatsuya Shimizu, Masayuki Yamato and Teruo Okano

Mahmoud S. Mohamed, Yun Chen and Chao-Ling Yao Mini-Review Antibody approaches to prepare clinically transplantable cells from human embryonic stem cells: Identification of human embryonic stem cell surface markers by monoclonal antibodies Hong Seo Choi, Won-Tae Kim and Chun Jeih Ryu Research Article Multipotent adult hippocampal progenitor cells maintained as neurospheres favor differentiation toward glial lineages Jisun Oh, Gabrielle J. Daniels, Lawrence S. Chiou, Eun-Ah Ye, Yong-Seob Jeong and Donald S. Sakaguchi Research article Detachably assembled microfluidic device for perfusion culture and post-culture analysis of a spheroid array Yusuke Sakai, Koji Hattori, Fumiki Yanagawa, Shinji Sugiura, Toshiyuki Kanamori and Kohji Nakazawa Research Article The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells Xue Cao, Kenneth Y. C. Kwek, Jerry K. Y. Chan, Qingfeng Chen and Mayasari Lim

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Cell sheet engineering for regenerative medicine: current challenges and strategies.

Substantial progress made in the areas of stem cell research and regenerative medicine has provided a number of innovative methods to repair or regene...
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