Cell Sheet-Based Tissue Engineering for Organizing Anisotropic Tissue Constructs Produced Using Microfabricated Thermoresponsive Substrates.

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Cell Sheet-Based Tissue Engineering for Organizing Anisotropic Tissue Constructs Produced Using Microfabricated Thermoresponsive Substrates Hironobu Takahashi and Teruo Okano* the recent progress in induced pluripotent stem (iPS) cell technology is going to open up an entirely new era for tissue engineering and regenerative medicine.[8] These advances are expected to provide powerful tools to develop cell-based therapies with the patient's own cells.[9] Moreover, this same technology can be effectively applied to developing human cell-based models for drug discovery and biological studies because patient-specific cells can be used to produce a customized tissue model for a specific disease.[10,11] While iPS cell technology can potentially supply the appropriate cells for a specific disease target, further development of tissue engineering is required to develop the next level of technology to create any kind of tissue, including complex tissues composed of multiple cell types. The specific structure of each kind of tissue in the human body is a key component of its ability to produce the appropriate functionality. Ideally, engineered tissues should be produced in an environment that closely mimics the microstructure of native tissue. Therefore, tissue engineering needs to solve the challenges of creating more complicated tissues and organs.[12] In native tissues, some parts have well-organized orientations of cells and/or extracellular matrixes (ECM).[13–16] Therefore, tissue anisotropy is a critical factor in providing biomimetic functions to engineered tissues. For example, skeletal muscle has a highly oriented structure made of parallel bundles of muscle fibers. The oriented architecture is known to be a key factor for producing the mechanical functions in native skeletal muscle.[17] Also in bone, tendons and ligaments, the specific tissue structures composed of aligned cells, and/or ECMs play important roles in producing mechanical and biological functions.[16,18] In vascular tissue engineering, the 3D oriented layer-by-layer architecture of native vessels must be precisely mimicked to produce the desired function of biomimetic tissues.[19,20] In addition, native myocardial tissue has a complex three-dimensionally organized structure that appears to be a stack of sheet-like tissues of aligned cardiomyocytes oriented in multiple directions transmurally throughout the whole organ, from the epicardium to the endocardium.[15,21] This complex tissue systematically organizes mechanical and electrical functions to produce a unique electrical

In some native tissues, appropriate microstructures, including orientation of the cell/extracellular matrix, provide specific mechanical and biological functions. For example, skeletal muscle is made of oriented myofibers that is responsible for the mechanical function. Native artery and myocardial tissues are organized three-dimensionally by stacking sheet-like tissues of aligned cells. Therefore, to construct any kind of complex tissue, the microstructures of cells such as myotubes, smooth muscle cells, and cardiomyocytes also need to be organized three-dimensionally just as in the native tissues of the body. Cell sheet-based tissue engineering allows the production of scaffoldfree engineered tissues through a layer-by-layer construction technique. Recently, using microfabricated thermoresponsive substrates, aligned cells are being harvested as single continuous cell sheets. The cell sheets act as anisotropic tissue units to build three-dimensional tissue constructs with the appropriate anisotropy. This cell sheet-based technology is straightforward and has the potential to engineer a wide variety of complex tissues. In addition, due to the scaffold-free cell-dense environment, the physical and biological cell–cell interactions of these cell sheet constructs exhibit unique cell behaviors. These advantages will provide important clues to enable the production of well-organized tissues that closely mimic the structure and function of native tissues, required for the future of tissue engineering.

1. Introduction Tissue engineering creates living tissues that can potentially be used for replacement of damaged tissues and organs in regenerative medicine applications.[1,2] Cell-based therapy relies mainly on biodegradable polymeric materials to be used as scaffolds. The most common form of tissue engineering today fabricates three-dimensional scaffolds from natural and synthetic polymers (e.g., collagen, poly(L-lactic-co-glycolic acid)), which provide a range of environments for cell adhesion, proliferation, and differentiation into specific cell phenotypes.[2–7] While this approach is leading current tissue engineering technology,

Dr. H. Takahashi, Prof. T. Okano Institute of Advanced Biomedical Engineering and Science Tokyo Women’s Medical University 8–1 Kawada-cho, Shinjuku-ku Tokyo 162–8666, Japan E-mail: [email protected]

DOI: 10.1002/adhm.201500194

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Adv. Healthcare Mater. 2015, 4, 2388–2407

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Hironobu Takahashi received his Ph.D. degree in materials science and engineering from Kyushu University in 2006. He worked at Colorado State University and the University of Utah as a postdoctoral fellow. He currently serves as an assistant professor in the Institute of Advanced Biomedical Engineering and Science at Tokyo Women’s Medical University. His research interest is engineering intelligent materials for biomedical applications including tissue engineering and regenerative medicine.

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propagation in vivo.[5,22] Although a number of tissue engineering researchers demonstrated the potential for tissue-engineered constructs to overcome the current limitations of medical treatments, techniques have yet to be established that can reproduce the sophisticated biomimetic properties of native tissues. Micropatterning approaches can be used to control the behavior and fate of cells such as elongation, differentiation, and cell–cell interaction.[23–29] In particular, numerous materials have been developed and micropatterned with stripes to regulate the cell orientation on the surface.[25,29] However, a technique is required to manipulate the aligned cells for the creation of a 3D tissue construct. For regenerative medicine to be applied to any type of tissue or organ, a reliable method for scaling-up engineered tissues is essential, but is still elusive. Additionally, in the development of cell-based models for drug discovery, some researchers have reported that 3D tissue models are superior to a two-dimensional cell culture model.[30,31] However, the conventional microfabricated substrates make it impossible to release the well-organized cells, even if these materials are very useful to regulate cell orientation. Cell sheet-based tissue engineering promises to overcome this limitation and facilitate the design of 3D oriented tissue structures. Currently, scaffold-based 3D tissues are the most commonly fabricated with tissue engineering; however, few strategies are available for the creation of a 3D oriented architecture.[26,32,33] Moreover, problems such as insufficient cell migration into the scaffold remain unsolved. Since scaffolds occupy some of the space in the engineered tissue, it is difficult to produce a densely packed cell environment. Cell sheet-based tissue engineering is a unique approach to achieve scaffold-free tissue construction. This technology is expected to enable precisely designed complex tissue organized threedimensionally in the near future of tissue engineering. Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)grafted surfaces allow the production of a tissue-like cell monolayer, a “cell sheet”, and Okano and co-workers have developed this unique tissue engineering strategy for application in regenerative medicine (Figure 1).[34,35] Since the cell-dense tissue construct can be harvested intact with complete preservation of the cell–cell junctions and the associated ECM from the intelligent surface, cell sheets can be fabricated without a scaffold, and then transplanted.[36–38] Importantly, since the reserved ECM acts as a glue to adhere tightly to the host tissue, additional treatments such as suturing is not required to apply cell-based therapy. Due to these advantages, the cell sheet engineering has already been applied to human clinical studies targeting the regeneration of cornea,[39] esophagus,[40] heart,[41] periodontal ligament,[42] and cartilage.[43] For example, oral mucosal epithelial cell sheets have been used for corneal reconstruction and esophageal ulceration treatment after endoscopic submucosal dissection in early esophageal cancer patients (Figure 1e).[39,40] Recently, this strategy has been applied for the treatment of middle ear damage during cholesteatoma surgery.[44] These medical approaches are already being applied in human clinical studies. Moreover, cell sheet-based tissue engineering allows the creation of scaffold-free 3D tissues by layering multiple cell sheets (Figure 1f).[45–47] Layered cell sheets stratify tightly because of the preserved ECM underneath individual cell sheets, which can then communicate with each other, both physically and biologically.[48] Previous studies revealed that

Teruo Okano is Professor of the Institute of Advanced Biomedical Engineering and Science at Tokyo Women's Medical University (TWMU). He received his Ph.D. degree in polymer chemistry from Waseda University in 1979. After several years as an assistant professor at TWMU, he served as an adjunct professor at the University of Utah. Since 1988, he has been working at TWMU and initiated the present institute in 2001. His research interests involve the use of intelligent biomaterials for research applications in various fields such as tissue engineering and drug delivery system. He has established the new tissue engineering approach “cell sheet engineering” and the technology is currently being applied for regenerative medicine. tightly stacked, multiple cardiomyocyte sheets can communicate electrically, resulting in one synchronized beating throughout the 3D myocardial tissue with millimeter-scale thickness.[49] Therefore, this cell sheet layering technique has the potential for constructing large-scale 3D tissues, without the use of a 3D scaffold. The advantage is that it provides a larger number of therapeutic cells to the patient in the cell sheet transplantation.[50] Cell sheet-based technology is expected to lead the production of sophisticated 3D tissues with complex structures mimicking native tissues. To broaden the applications of regenerative medicine and develop an integrated tissue model for future drug discovery, it has become necessary to construct tissues that are more complex. To accomplish this challenge, a new class of cell sheet engineering needs to be developed to construct any complex tissue with well-organized structures. The combination of microfabricated and thermoresponsive substrates will provide anisotropy within the cell sheets and allow us to utilize them as “anisotropic tissue units” to produce flexible tissue constructs with numerous 3D orientations. Recent progress in cell sheet engineering has shown the potential to produce scaffold-free 3D oriented tissue constructs with complex microstructures mimicking native tissues.



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Figure 1. Cell sheet-based tissue engineering for cell transplantation and 3D tissue construction. a) Schematic illustration of thermally induced cell sheet detachment with preservation of cell–cell junctions and associated ECM. Due to the surface alternation, a cell sheet can be harvested intact simply by lowering culture temperature. b) Phase contrast and c) fluorescence microscopic images of a cell sheet detaching from a PIPAAm-grafted surface. The associated ECM detached with the cell sheet from the surface, which is important for cell sheet transplantation. Fibronectin and cell nuclei were stained with red and blue, respectively. Scale bar: 100 µm. d) Photograph of a cell sheet harvested from a thermoresponsive cell culture dish. The cell sheet shrunk two-dimensionally, but kept the original shape. e) Schematic illustration of cell sheet transplantation for cornea regeneration. Autologous cells were cultured and harvested as a cell sheet for transplantation. Since the associated ECM performs as a glue, a cell sheet can be transplanted on the cornea without any other treatment. f) Cell sheet layering process for construction of multilayered cell sheets. Cell sheets can be stacked tightly simply through the layering process. This strategy is useful to produce cell-dense thick tissues with no scaffold. Reproduced with permission.[38,45] Copyright 2010, American Chemical Society. Copyright 2012, Nature America.

2. Microfabricated Thermoresponsive Substrates Provide Anisotropy 2.1. Thermoresponsive Surfaces for Cell Sheet Fabrication Thermoresponsive cell culture substrate allows the harvest of a single continues cell sheet by using a thermally induced surface alternation. The thermoresponsive polymer PIPAAm has

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been commonly used as one of the most functional polymers for biomedical applications.[51] PIPAAm exhibits thermoresponsive alternation by changing its properties across its lower critical solution temperature (LCST) of 32 °C in aqueous media. Specifically, this polymer exhibits hydrophilic properties due to the hydration of the polymer chain on the surface below the LCST (e.g., 20 °C), while the polymer becomes hydrophobic and the chain conformation changes to a compact globule




2.2. Micropatterning of Thermoresponsive Surfaces to Control Cell Orientation 2.2.1. Microgrooved Thermoresponsive Surface (Groove-Patterned Surface) Micropatterning has been widely used for controlling cell morphology, spatial arrangement, and function by various types of nano/micro-scale guidance, including regulated cell adhesion, topological features.[54,55] A commonly used method in many of these approaches involves the technique known as soft lithography.[27,56] The microtextured polydimethylsiloxane (PDMS) substrate is used as a master mold, and is usually pressed against a polystyrene substrate to provide the topological features at a micro- or nano-scale to the culture substrate. A variety of microgrooved substrates have been used in this process to control cell orientation.[57] For example, skeletal muscle precursor cells, myoblasts, spread with a single orientation on strip-shaped microgrooves.[26,58–60] However, using these substrates the cell morphology can only be controlled in two dimensions. At present, very few studies have been able to achieve 3D cell arrangements.[33,61] By grafting of PIPAAm on topologically patterned surfaces, these substrates can be applied to cell sheet fabrication. To date, some kinds of surface fabrication methods have been developed to provide thermoresponsive


property to microgrooved substrates.[62,63] For example, PIPAAm is grafted covalently through a “grafting-to” method to a topologically patterned substrate.[63] On the other hand, PIPAAm is also simply grafted in the same process as a commercially available thermoresponsive culture dish. The EB irradiation of the monomer solution on a microgrooved substrate results in nano-scale PIPAAm grafting on the topologically patterned substrate (Figure 2a).[64] In both cases, by adjusting the grafting conditions, the polymer thickness (chain length) can be controlled to detach the cell sheet from the micropatterned surface. Mimicking the tissue organization in a native artery is important to provide biomimetic functions within engineered vascular tissues. The medial layer of the artery wall is composed of multi-layered structures of circumferentially oriented smooth muscle cells (SMCs). This cell arrangement is a key factor to the contractile function in native blood vessels. Therefore, the directionality of SMCs must be considered to accurately mimic the native vessel architecture in an engineered artery.[20,24,65] The alignment of vascular SMCs is regulated on the microgrooved thermoresponsive substrate (e.g., 50 µm wide, 5 µm deep). Furthermore, by lowering the culture temperature, the aligned SMCs can be harvested as a single continuous cell sheet (Figure 2a).[64] This offers a significant advantage in designing a layer-by-layer cell architecture, such as that found in native blood vessels. As described above, multiple cell sheets can be layered to create thick tissue, and it was recently reported that a combination of SMC sheets and an endothelial cell-encapsulating tubular scaffold could be used to construct vascular tissues.[66] To fabricate mature tissue layers with blood vessels, a single SMC sheet harvested from commercially available thermoresponsive culture dish, UpCell dish, can be used to wrap a vascular-mimicking tubular scaffold composed of endothelial cells. Since this technology permits preservation of cell–cell junctions and the associated ECM, by avoiding enzymatic dissociation during manipulation, the cell sheet wrapping provides a biologically and mechanically matured smooth muscle layer to study. In the near future, anisotropic cell sheets composed of aligned SMCs may provide more mature properties in vascular tissue constructs using this combination strategy.[67]

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state above the LCST. Okano and co-workers applied this thermoresponsive behavior of PIPAAm to cell sheet fabrication.[46] Under normal cell culture conditions (37 °C), culture cells can adhere to the PIPAAm-grafted surface. On the other hand, the surface changes to hydrophilic, and the culture surface exhibits a cell-repellent property when the culture temperature is lowered below the PIPAAm's LCST. This alternation triggers the spontaneous detachment of the cells adhering on the PIPAAmgrafted surface.[52] Since cultured cells can be detached from the surface without the use of enzymatic treatments, such as trypsinization, they preserve the very important cell–cell junctions and associated ECM after sheet detachment.[36] In general, the cell–cell junctions are damaged by enzymatic treatment, and in conventional methods, cultured cells are only collected as single cells. Cell sheets can be harvested as a functional cell assembly without any complicated treatment, and are being applied in tissue engineering and regenerative medicine. A thermoresponsive polymer is technically required to be grafted with a nano-scale thickness onto the cell culture substrates, because the thickness directly influences cell adhesion and detachment behavior.[53] Therefore, the thickness (amount grafted) must be adjusted in accordance with the cell type to achieve efficient cell sheet fabrication. Commonly, the polymer is covalently grafted to tissue culture polystyrene (TCPS) dishes by electron-beam (EB) irradiation of the monomer (N-isopropylacrylamide) (IPAAm) solution poured into the dishes. The EB-initiated polymerization results in a nano-scale PIPAAmgel coating on the surface, which provides thermoresponsive property to cell culture dishes. Thermoresponsive cell culture dishes prepared through this method have been used in human clinical studies, and are now commercially available, under the product name UpCell dish (CellSeed, Inc., Tokyo, Japan).

2.2.2. Micro-Contact Printing on a Thermoresponsive Surface (Protein-Patterned Surface) PIPAAm grafting provides thermoresponsive properties to microfabricated substrates. In the case of microgrooved surfaces, cultured cells are aligned on the surface by the physical guidance cue. On the other hand, an anisotropic cell sheet can also be produced on a biochemically patterned surface prepared through protein patterning on the surfaces.[68] Micro-contact printing is a widely used technique for patterning cell-adhesive proteins on culture surfaces. Many studies have demonstrated that cell shape, pattern, and function can be controlled on a variety of patterned surfaces using patterns of different shape and size, and protein component.[28,69] A microgrooved PDMS mold consisting of lines 50 µm wide and spaced 50 µm apart can spatio-selectively immobilize fibronectin (FN) on



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Figure 2. Microfabricated thermoresponsive substrates to harvest anisotropic cell sheets. a) PIPAAm was grafted via electron beam (EB)-irradiation on a microgrooved substrate prepared by using a microgrooved PDMS stamp. Human aortic SMCs were aligned on the topologically patterned surface, and harvested as an anisotropic cell sheet by lowering culture temperature to 20 °C. b) Fibronectin (FN) was patterned via the micro-contact printing method on a thermoresponsive substrate. Vascular SMCs adhered only on the FN-patterns in serum-free medium and the aligned cells proliferated until reaching confluence with the addition of serum. The aligned SMC sheet was detached from the surface by lowering the temperature. c) Thermoresponsive PIPAAm and hydrophilic PAcMo were grafted through a two-step polymerization process. Through conventional photolithography, the hydrophilic polymer was grafted spatio-selectively on a thermoresponsive polymer surface, resulting in the formation of two kinds of stripe patterns (50 µm/50 µm). Human osteoblasts were aligned on the physicochemical patterns, and reached confluence while maintaining the orientation. By lowering culture temperature, they were harvested as a single continuous cell sheet. Scale bar: 100 µm. Reproduced with permission.[64,68,72] Copyright 2008, Elsevier. Copyright 2009, Wiley-VCH. Copyright 2011, American Chemical Society.

a flat PIPAAm grafted surface, resulting in the formation of stripe-shaped FN micropatterns. The SMCs adhere only to the FN-patterned regions under serum-free culture, and form stripe patterns on the substrate. Importantly, SMCs align on the FN-patterns simply by adjusting to the appropriate pattern width (50 µm). Moreover, by using serum-containing medium, the aligned cells proliferate and migrate outside of the FNpatterns. Once the cells are aligned on the micropatterns with the appropriate pattern width, the aligned cells reach confluence while maintaining their orientation (Figure 2b). Finally, the aligned SMCs form a confluence monolayer and are harvested as an anisotropic cell sheet by lowering the culture

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temperature. Although the harvested cell sheet itself appears to be similar to that produced by a microgrooved thermoresponsive surface, this surface patterning method is relatively simple, compared to the microgroove fabrication method. On the other hand, this micro-contact printing strategy requires a two-step cell culture with serum-free and serum-containing media. Therefore, although SMCs formed an anisotropic cell sheet on both substrates, we may need to be more selective in choosing the appropriate method (physical or chemical pattern) based on the cell types. The best method probably depends on the properties of the cell including adhesion, proliferation, and migration.




PROGRESS REPORT Figure 3. Transferrable anisotropy of a fibroblast cell sheet. a,b) Phase contrast and c,d) fluorescence microscopic images of human dermal fibroblast sheets prepared using a,c) a non-patterned or b,d) patterned thermoresponsive substrate. Actin fibers were stained with AlexaFluor568 (red). e) Photographs of a cell sheet detaching from a non-patterned or micropatterned surface by lowering culture temperature. Whereas a square-shaped cell sheet detaching from a non-patterned surface shrunk while maintaining the original aspect, an anisotropic cell sheet showed a distinctive shrinking rate. f) Phase contrast and fluorescence microscopic images of an anisotropic cell sheet transferred to a normal cell culture surface. Using a gelatin gel-coated plunger, an anisotropic cell sheet can be transferred while maintaining the original orientation. The transferred cell sheet exhibited the original cell alignment even after 7 days of incubation on a normal cell culture dish. Actin fibers and cell nuclei were stained with red and blue, respectively. Reproduced with permission.[73] Copyright 2011, Elsevier.

2.2.3. Micropatterned Polymer Grafting on a Thermoresponsive Surface (Polymer-Patterned Surface) Since the grafted amount and thickness of PIPAAm are important for cell sheet detachment, various kinds of polymer grafting techniques have been applied to precisely control the polymer grafting. In addition to the EB-irradiation method, surface-initiated living radical polymerization processes have mainly been studied in order to prepare thermoresponsive surfaces. Atom transfer radical polymerization (ATRP) and reversible additionfragmentation chain transfer (RAFT) polymerization can be used to induce brush-type polymer grafting on cell culture substrates.[70] Since these grafting approaches allow for the production of polymer brushes with a uniform chain length, the grafting of a thermoresponsive polymer can be controlled precisely by adjusting chain length and graft density.[38,71] On the other hand, these polymerization methods are also useful for micropatterning via two-step polymer grafting. Using this technique, physicochemically patterned thermoresponsive surfaces can be fabricated (Figure 2c).[72] First PIPAAm is grafted via the polymerization process and then hydrophilic poly(N-acryloylmorpholine) (PAcMo) is further grafted onto the surface as the second step. In the second step, PAcMo is grafted spatio-selectively through a conventional photolithography process. As a result, PIPAAm brush domains and block copolymer PIPAAm-b-PAcMo brush domains are successfully patterned (50 µm/50 µm stripes). Due to the presence of the patterned hydrophilic polymer, cells can recognize the difference in cell-to-surface affinity between these two kinds of regions.[73] By adjusting the appropriate width of the stripe pattern, cells are oriented in a direction parallel to the stripe patterns. The mechanism of cell arrangement on this surface is similar to


that on the Protein-patterned surface prepared by micro-contact printing. On the other hand, using the Polymer-patterned substrate, an anisotropic cell sheet can be obtained simply via onepot cell seeding. Human osteoblasts spread in the same direction as the stripe-patterns of the Polymer-patterned substrate and then proliferate while maintaining the same orientation. Consequently, the aligned osteoblasts can be harvested as an anisotropic cell sheet (Figure 2c) (unpublished data). Native bone tissues exhibit a well-organized microstructure composed of the orientated biological apatite and collagen fibers.[74] Therefore, many studies have reported that the native anisotropic structure provides biomimetically the mechanical and biological functions to engineered tissues in bone tissue engineering.[75] Since apatite crystals deposited by osteoblasts show preferential c-axis alignment along osteoblast orientation, cell orientation is a key factor to generate the anisotropic apatite/collagen composites.[76] Cell sheet-based technology has the potential to create 3D bone tissue anisotropy, thus it may provide clues to better understand bone regeneration in native bone tissues and improve tissue construction techniques in bone tissue engineering.

3. Unique Properties of Anisotropic Cell Sheets 3.1. Harvest and Manipulation of Anisotropic Cell Sheets The alignment of normal human dermal fibroblasts (NHDFs) can be regulated by one-pot cell seeding onto the Polymer-patterned substrate.[72,73] The cells align in a direction parallel to the striped patterns; even while they proliferated (Figure 3a,b). Consequently, an anisotropic fibroblast sheet can be produced



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on the surface, and fluorescence imaging revealed that the cytoskeleton formed oriented structures (Figure 3c,d). The oriented organization provides physical anisotropy to the cell sheet. In fact, shrinkage of the cell sheet exhibits the distinctive feature of an anisotropic cell sheet. Generally after detachment from the surface, a cell sheet shrinks two-dimensionally with maintaining the original aspect of the surface. In the case of anisotropic cell sheets, on the other hand, the single cell orientation results in a different shrinkage rate between the vertical and parallel sides of the cell alignment (Figure 3e). This unique behavior indicates that the cell alignment directly contributes to the physical properties of cell sheets. While this unique shrinking suggests that cell sheets composed of aligned cells have anisotropy, anisotropic cell sheets lose their directionality after detachment due to the rearrangement of the cytoskeleton. Therefore, to produce 3D oriented tissue constructs, cell sheets need to be manipulated while maintaining the cell orientation. A manipulation technique using a gelatin gel-coated plunger allows cell sheets to be harvested without shrinkage.[45] Briefly, first the gelatin gel attaches tightly to a cell sheet when the gel is placed on the cell sheet. Following the attachment of the gelatin gel and the cell sheet, the culture temperature is lowered for detaching the sheet and allowing it to be transferred while maintaining the original size, shape and orientation. In fact, the orientation of an anisotropic fibroblast sheet can be maintained after transfer onto a normal TCPS dish (Figure 3f).[73] Interestingly, the cell sheet exhibits the designed orientation for at least 7 days on the non-patterned culture surface. This is probably due to the fact that human dermal fibroblasts prefer to keep their oriented structure even without directionality on culture surface. This behavior is very important to achieve 3D cell arrangement based on cell sheetbased tissue engineering.

3.2. Mechanical Properties of Anisotropic Cell Sheets In some native tissues, the mechanical functions originate from their anisotropic tissue structures.[14,18] For example, native vessel walls have a complex structure composed of circumferentially oriented collagen fibers/cells and lamellar elastin, and the structural organization provides mechanical significance for blood circulation.[77] Therefore, mimicking the architecture must affect the mechanical properties of engineered vascular tissues. Isenberg et al. reported that an anisotropic SMC sheet exhibited mechanical anisotropy similar to that of native vessels in both stiffness and strength.[78] For mechanical testing, a cell sheet fabricated using a microgrooved PDMS substrate was harvested with gelatin gel. The mechanical testing revealed that cell orientation improved the mechanical properties. The failure stress and stiffness were more than 50% greater in the parallel than in the perpendicular direction of cell alignment (Figure 4a). This indicates that cell/ECM orientation is a key factor to engineer biomimetic vascular tissues. Since cell sheets are composed only of cells and ECM, it has advantages over scaffold-based tissue constructs when evaluating the mechanical strength of only the tissues themselves. However, it is difficult to obtain a robust engineered tissue for mechanical testing. Although in this study an estimation was possible by long-term

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culturing of cell sheets, the physical strength of cell sheets may need to be improved for mechanical testing depending on the cell types. Additionally in this study, since the microgrooved substrate has no thermoresponsive properties, the cell sheet cannot be harvested smoothly simply by controlling the temperature. Certainly, the use of thermoresponsive substrates provides a more sophisticated strategy for studying the mechanical properties of cell sheets.

3.3. Biological Property of Anisotropic Cell Sheets The cell alignment influences not only physical (mechanical) functions, but also biological features.[79–81] In some previous studies, which did not use cell sheets, vascular SMCs increased the expression of some contractile proteins, such as smooth muscle myosin heavy chain and smooth muscle α-actin, by regulating their orientation.[79] From this viewpoint, collagen production was expected to be increased by regulating the alignment of collagen-secreting cells such as SMCs and fibroblasts. However, SMC sheets exhibit no significant difference in collagen content between aligned and randomly oriented SMCs (Figure 4b).[78] Similarly, the amount of collagen deposited on fibroblast sheets is independent of cell alignment in the cell sheet (Figure 4c). Interestingly, vascular endothelial growth factor (VEGF) secretion obviously increased by regulating fibroblast orientation (Figure 4d).[73] Although the mechanism has been not yet elucidated, this characteristic is important for the vascularization of engineered tissues, because vascularization is one of the main issues for the construction of large-scale 3D tissues.[82] In many cases, necrosis occurs in thick tissues due to insufficient supply of oxygen and nutrients. Similarly, in native tissues, angiogenic growth factors, such as VEGF, are key mediators to promote vascularization in engineered tissues.[83] Therefore, the increase in VEGF secretion potentially enhances vascularization into engineered tissues. This distinctive property of anisotropic fibroblast sheets may successfully induce a vascular capillary network into engineered tissues composed of cell sheets. On the other hand, the secretion of some other proteins (e.g., transforming growth factor-β1 (TGF-β1)) is not influenced by fibroblast alignment (Figure 4e), and only some specific proteins are enhanced by forming an oriented structure. Additionally, it should be noted that various types of cell sheets might exhibit different biological advantages based on the organizing cell alignment; for example VEGF in fibroblast sheets. This kind of cell sheet must be useful as a supply source of specific proteins in tissue engineering strategy.

4. Arrangement of 3D Cell Orientation through Layering Anisotropic Cell Sheets 4.1. Cell Sheet Layering Technique for Thick Tissue Constructs Whereas 3D scaffolds are useful to construct large-scale tissues, scaffold-free tissue construction methods provide numerous advantages to produce biomimetic thick tissues.[84] Importantly, and unlike conventional methods, a cell-dense 3D tissue structure fabricated by cell sheet layering does not require a 3D




PROGRESS REPORT Figure 4. Mechanical and biological properties of anisotropic cell sheets. a) Mechanical properties of bovine aortic SMC sheets harvested from nonpatterned and patterned substrates. Failure stress and stiffness were significantly increased by regulating cell alignment. b) Biological properties of SMC sheets showed no difference in the secretion of elastin and collagen. This indicates that the cell orientation affected the mechanical features independent of the amount of proteins. c–e) Biological characteristics of anisotropic fibroblast sheets. c,e) The amount of secreted collagen and TGF-β1 were not different between cell sheets composed of randomly oriented cells (Random) and aligned cells (Aligned). d) The secretion of VEGF was remarkably increased by regulating fibroblast alignment in a cell sheet. Reproduced with permission.[73,78] Copyright 2011, Elsevier.

scaffold.[45] Simply by layering multiple cell sheets they are able to connect with each other both physically and biologically to perform as a single communicative thick tissue. For example, previous studies demonstrated that two-layered cardiomyocyte sheets quickly synchronized their beatings due to the formation of functional gap junctions.[49] Furthermore, when multilayered cardiomyocyte sheets were transplanted subcutaneously to nude rats, the engineered tissue pulsated for a long period (more than one year) independently from the host electrocardiogram.[47] That is to say, the implanted cardiomyocyte sheets performed very similarly to native myocardium. To date, there have not been any clinical trials using human cardiomyocytes. However, the recent progress in iPS cell technology is providing an attractive potential for regenerative therapy using autologous cardiomyocytes.[11] Recently, Matsuura et al. developed a large-scale culture system for the expansion and cardiac differentiation of human iPS cells.[85] In that system, human iPS cell-derived cardiomyocytes are now available for the production of human cardiomyocyte sheets.[35,86] With this advance, tissue engineering research has moved one step closer towards human heart regeneration. It is expected that various kinds of


cell sheets will be prepared using a patient's own cells, and that this cell sheet layering technique will be able to use many different human cell sources to construct any human 3D tissue in the near future.

4.2. Cell Sheet Engineering for Oriented Muscle Tissue Constructs 4.2.1. Anisotropic Myoblast Sheet for Muscle Tissue Engineering In the field of the cell sheet-based regenerative therapy, muscle precursor cells, skeletal muscle myoblasts, have been used in a clinical trial of myocardial tissue repair. Sawa and co-workers have established a cell sheet-based therapy using skeletal muscle myoblast sheets made of the patient's own myoblasts, and achieved a significant improvement in damaged cardiac functions.[41] In this regenerative medicine approach, myoblast sheets are used as an appropriate cell source for secretion of therapeutic cytokines to the target site.[2] On the other hand, in skeletal muscle tissue engineering, myoblasts are the essential



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Figure 5. Cell sheet engineering using anisotropic myoblast sheets for the arrangement of cell orientation. a) Phase contrast and b) fluorescence microscopic images of an anisotropic myoblast sheet. Desmin-positive human skeletal muscle myoblasts were aligned on the micropatterned thermoresponsive substrate (the Polymer-patterned substrate). c) Confocal microscopic image of three-layered myoblast sheets prepared using a gelatin gel-coated plunger. Actin fibers showed a single orientation in the multilayered cell sheet construct. d–h) Formation of vascular-like branching networks of endothelial cells within multilayered myoblast sheets. d,e) HUVECs formed branching networks only between two layered cell sheets. HUVECs were aligned on a single anisotropic myoblast sheet (f), and formed oriented networks within the anisotropic environment of multilayered myoblast sheets (g,h). Endothelial cells and actin fibers were stained with green and red, respectively. i,j) Fluorescence images of myosin heavy chain-positive myotubes. Myoblast sheets were transferred to normal culture dishes. After Day 5 of culturing in differentiation medium (2% horse serum), i) randomly oriented myoblasts, and j) aligned myoblasts differentiated into myotubes. (k) The comparison of the length of myotubes forming in cell sheets of randomly oriented (Random) and aligned myotubes (Aligned). The length was increased remarkably by regulating myoblast orientation. Scale bar: 100 µm. Reproduced with permission.[88,96] Copyright 2013, Elsevier. Copyright 2014, Wiley-VCH.

cell source due to their important role in the development of mature skeletal muscle. Early in the development stage, myoblasts align and fuse to form multinucleated myotubes. The newly formed myotubes elongate unidirectionally and consequently mature muscle fibers form a bundle structure in native skeletal muscle. This anisotropic architecture is a crucial component to produce the mechanical function of skeletal muscle. From this viewpoint, regulation of myoblast alignment has been the focus in studies of muscle tissue engineering. In some previous studies, various kinds of microfabricated substrates were

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used to control the orientation of myoblasts.[17,32,33,60,87] In fact, myoblasts and myotubes are well aligned on these substrates; however, to produce 3D muscle tissue mimicking native tissue structure, the 2D oriented muscle cells need to be expanded three-dimensionally. As described above, multiple cell sheets can be layered to produce 3D thick tissue constructs. Based on this technique, 3D anisotropy in engineered muscle tissue can be obtained.[63] On the Polymer-patterned substrate, human skeletal muscle myoblasts are aligned and reach confluence simply by one-pot cell seeding (Figure 5a,b). Finally, the aligned




4.2.2. Formation of Oriented Cellular Network for Vascularization in Muscle Tissue Large-scale tissue construction is a major challenge in the field of tissue engineering research. However, large-scale engineered tissues require a sufficient supply of oxygen and nutrients to survive long-term. To date, therefore, many research groups have investigated methods to promote vascularization into engineered tissues.[82,89,90] Although the cell sheet layering technique is useful for the production of 3D tissues, vascularization is also necessary for multilayered cell sheet constructs to prevent necrosis in the construct. For example, whereas threelayered cardiomyocyte sheets can survive without necrosis inside the construct, constructs greater than four-layered cell sheets are limited in thickness due to hypoxia. Vascularization overcomes the diffusion limit of oxygen and nutrients to engineered tissues. After implantation, microvessel network formation begins within the transplanted multilayered cell sheets from the host's vessels. Consequently, a well-defined vascular network forms within a few days inside the transplanted tissue. Therefore, step-by-step transplantations of three-layered cardiomyocyte sheets allows for the creation of thicker tissues in vivo.[91] However, it should be noted that this strategy is applicable only for in vivo transplantation. For the in vitro creation of thick tissues, an advanced culture method using a bioreactor system has been developed to create vascularized cell sheet constructs.[92,93] Briefly, in multilayered cell sheets composed of co-cultured cardiomyocyte and endothelial cells, the endothelial cells form vascular-like branching networks. When the construct is cultured on a vascular bed (e.g., collagen gel), the network-forming endothelial cells connect to the microchannels fabricated in the vascular bed.[92] This vasculature matures in the bioreactor culture system, and eventually provides the supply of oxygen and nutrients into the multilayered cell sheet construct. This newly developed technique avoids necrotic complications and allows for the production of thicker cell sheet constructs in vitro. Vascularization can be promoted by incorporating endothelial cells within tissue constructs.[90] Typically they are co-cultured by mixing with other types of cells to form tissue constructs. In a cell sheet engineering strategy, on the other hand, endothelial


cells can be sandwiched between multiple cell sheets.[94–96] For example, human umbilical vein endothelial cells (HUVECs) were sandwiched between two normal myoblast sheets via the cell sheet layering process. Whereas HUVECs simply adhere as single cells onto a myoblast sheet (Figure 5d), the cell sheet layering triggers the formation of a branching structure (Figure 5e).[96] It is expected that this network formation will promote connections with the host vasculature and supply oxygen and nutrients to the tissue. Interestingly, endothelial cells form no branching networks when culturing on a single cell sheet, indicating the importance of the 3D cell sheet environment for the network formation. This cell sheet effect on endothelial cells can be uniquely revealed by the layer-by-layer construction technique. Since this construction method enables communication between myoblasts and endothelial cells, endothelial cells recognize the 3D cell environment, resulting in the formation of a self-organized network. The scaffold-free tissue architecture also allows endothelial cells to recognize anisotropy of the 3D environment.[96] When HUVECs are cultured on a single anisotropic myoblast sheet, they align in the same direction as the myoblasts, but do not form branching networks (Figure 5f). The cell sheet layering triggers the network formation (Figure 5g) and consequently the endothelial cells form anisotropic branching structures (Figure 5h). The directionality of the networks indicates that endothelial cells recognize the anisotropy of the 3D microenvironment composed of aligned myoblasts. The native vascular structure is highly organized in skeletal muscle for efficient blood circulation.[16] Therefore, this structural organization must be important to produce biomimetic muscle tissues. In addition, it should be noted that the recognition of the anisotropic environment by endothelial cells and the subsequent self-organization of the anisotropic network is due to the scaffold-free cell-dense tissue architecture made only of cells and ECM proteins. As described above, it is possible that endothelial cells can communicate physically and/or biologically with aligned myoblasts in the cell sheet construct. In future work, this structurally organized tissue needs to be functionalized. Recently, functional blood vessels can be produced within a multilayered cell sheets construct in a newly developed bioreactor system.[92,93] Therefore, the combination of this tissue organization method and maturation system will achieve the functionalization of complex tissues, required for the development of a sophisticated in vitro tissue model.

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myoblasts are harvested as an anisotropic myoblast sheet by lowering the culture temperature.[88] Although anisotropic myoblast sheets shrink after cell sheet detachment, the manipulation technique using a gelatin gel-coated plunger allows for the transfer of the cell sheet while maintaining cell orientation. The anisotropy of the transferred cell sheet is maintained, even onto a normal cell culture dish for at least 3 weeks. Because of the transferable anisotropy, multiple myoblast sheets can be layered successfully to arrange a single cell orientation in a thick tissue construct. For example, three-layered myoblast sheets showed unidirectional actin fibers after 7 days of layering (Figure 5c). Based on this cell sheet layering process, an oriented architecture in 3D tissues is possible without the use of scaffolds. The uniqueness of this technique is suitable for closely mimicking native tissue such as skeletal muscle that is composed of densely packed myofibers.

4.2.3. Differentiation of Aligned Myoblasts in Anisotropic Cell Sheets In the development of native muscle, myoblasts fuse and differentiate into myotubes. Therefore, a number of studies have reported that differentiation behavior is a key factor in muscle tissue engineering.[4,58] A myoblast sheet can be transferred onto a normal cell culture dish using a gelatin gel-coated plunger while maintaining the cell directionality. Using culturing differentiation medium (e.g., 2% horse serum containing medium), a transferred myoblast sheet shows differentiation into aligned myotubes, even on normal (non-patterned) cell



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Figure 6. 3D arrangement of cell orientation within tissue constructs through the cell sheet layering process. a) Illustration of the cell sheet layering process using a gelatin gel-coated plunger to design 3D cell orientation. b) Fluorescence image of perpendicularly layered fibroblast sheets. Two dermal fibroblast sheets were layered perpendicularly using a gelatin gel manipulator. The 3D orientation was maintained until Day 7 after layering. c) Fluorescence image of herringbone patterned MSCs and their orientation distribution. Three anisotropic MSC sheets were layered at different angles to mimic the herringbone-like formation of SMCs in an artery. The orientation distribution showed that each cell layer had different directionality of cell alignment as designed. Reproduced with permission.[73,101] Copyright 2011, Elsevier.

culture substrates.[88] Their morphologies are clearly changed microscopically, and immunostaining reveals that myosin heavy chain-positive myotubes are aligned on the surface (Figure 5i,j). Importantly, the orientation of myoblasts is maintained during the differentiation. In conventional studies, micropatterns provide a cue for cells to align by compulsion. However, aligned myoblasts prefer keeping the orientation even on a non-patterned surface. Also, in the differentiation process, they form multinucleated myotubes while maintaining the original anisotropic formation on normal culture substrates. Taken together, while the micropatterned thermoresponsive substrate provides a cue to form preferable orientation for myoblasts, it is not necessary to control myoblast orientation in further behaviors. Once the anisotropic structure is formed, they mimic the bundle structure of native muscle tissue by themselves. In addition, while the diameter of the myotubes is not significantly different between cell sheets composed of aligned (Aligned sheet) and randomly oriented myoblasts (Random sheet), the length of the myotubes increases remarkably by forming the aligned structure (Figure 5k). This morphologic advantage also suggests that this structural organization is a key component in skeletal muscle tissue engineering.

4.3. Layer-by-layer Orientation in 3D Tissue Construct using Anisotropic Cell Sheets In some native tissues, cells and ECM are organized threedimensionally and the 3D arrangement plays an important role in mechanical and biological functions.[97,98] For example, native myocardial tissue is arranged three-dimensionally with a stack of sheet-like tissues comprised of aligned cardiomyocytes

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oriented to various directions throughout the whole tissue transmurally, from the epicardium to the endocardium.[21,99] The complex tissue anisotropy is essential for producing its unique electrical propagation.[22,25,100] The anisotropic structure of an artery is crucial to provide the contractile and mechanical properties of the vessel. Mainly circumferentially aligned SMCs and the associated ECM are thought to produce the anisotropic property.[19] Furthermore, each SMC layer has a different helical pitch, resulting in a complex “herringbone” structure. Although some recent studies have produced the appropriate structure of circumferentially oriented SMCs for mechanical tissue function, the precise arrangement of 3D anisotropy remains elusive. To mimic the unique tissue anisotropy such as that found in myocardium and vessels, the layer-by-layer construction technique may be an effective approach. The manipulating technique for cell sheets using a gelatin gel-coated plunger can be used for stacking multiple sheets, resulting in the layer-by-layer cell organization. Through this layering technique, cell alignment can be controlled simply by rotating the manipulator through a desired angle relative to the next sheet (Figure 6a).[73,101] Since cell sheets can be harvested with the associated ECM, they connect to each other simply by incubating the layered cell sheets for 30 min at 28 °C. For example, two anisotropic fibroblast sheets attach significantly and form the desired perpendicularly orientated 3D tissue construct (Figure 6b).[73] The actin fibers of the top and bottom layers are oriented individually in the single tissue construct. This 3D arrangement should be useful to mimic complex structures found in corneal tissues and myocardial tissues that have three-dimensionally organized cell/ECM orientations. Although some previous studies have also created 3D oriented tissue constructs using well-designed scaffolds, this cell sheet-based





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technology requires no scaffolds to arrange complex 3D anisotropy in engineered tissues. In vascular tissue engineering, an anisotropic cell sheet composed of human mesenchymal stem cells (MSCs) may be able to produce a tissue structure mimicking the circumferential and herringbone orientations of the native artery.[67] In the same manner as fibroblast sheets, MSC sheets can also be layered at different angles relative to each other. For mimicking the herringbone structure of the media layer in an artery, the cell alignment changes from 25° in sheet 1 to 60° in sheet 2 to 5° in sheet 3 with respect to the x-axis of the images (Figure 6c).[101] Theoretically, based on this technique, any number of anisotropic cell sheets can be stacked at any alignment. Recently with the development of the bioreactor culture system, twelvelayered cell sheets were produced without the induction of necrosis inside the construct.[92] Therefore, in the near future, thick tissues can be produced with complex, but well-organized cell orientation. To mimic complex structures in regular connective tissue like tendons and ligaments, not only cells, but also the ECM is required to be organized with the preferential orientation.[98,102,103] The ECM has important roles in providing 3D architectures and producing mechanical forces for myocardium and vessels.[104] From this viewpoint, the anisotropy of ECM proteins is also a key factor for mimicking native tissues. Collagen is the major component of the ECM in a variety of connective tissues including bone, tendons, and cornea.[74,103,105] Importantly, in these tissues, collagen fibers show a highly oriented structure depending on the tissue and its function. For example in tendon, collagen fibers are all oriented parallel to the long axis of the tissue, while cornea shows a layer-by-layer arrangement of aligned collagen fibrils. These specific oriented structures provide the appropriate mechanical properties for the native tissues.[106] In the native myocardium, cardiac fibroblasts that produce collagen are known to be well-organized three-dimensionally with myocytes.[15,21,97,107] In mature bone tissues, to arrange the directionality of collagen fibrils and apatite deposition, the orientation of osteoblasts is known as a key factor.[74,76,108] That is to say, collagen production is directly influenced by organizing the cell alignment in these tissues. In fact, the ECM proteins associated with an anisotropic cell sheet also show the directionality. Whereas collagen deposited with a normal cell sheet has no any specific orientation, the collagen deposition with an anisotropic fibroblast sheet shows directionality in a direction parallel to the cell orientation.[73,109] When cultured in a medium without L-ascorbic acid, aligned fibroblasts produce intracellularly collagen on a micropatterned surface (Figure 7a). After transferring onto a non-patterned surface, on the other hand, extracellular collagen deposition is promoted by the addition of L-ascorbic acid to the culture medium,[110] and the anisotropic fibroblast sheet produces extracellularly aligned collagen fibers on a normal cell culture dish (Figure 7b).[73] Therefore, anisotropic cell sheets could be used to mimic ECM orientation in native tendons and ligaments. More importantly, collagen production is not influenced by the surface patterning. This indicates that the orientation of deposited ECM can be organized three-dimensionally via the layer-by-layer construction technique. After layering multiple cell sheets, they may produce an oriented ECM with

Figure 7. Collagen secretion by human dermal fibroblasts within anisotropic cell sheets on micropatterned and non-patterned surfaces. a) An anisotropic fibroblast sheet was cultured on the Polymer-patterned surface for 5 days. Since collagen was produced intracellularly in medium without L-ascorbic acid, a fluorescence image showed aligned fibroblasts. b) The cell sheet was transferred onto a normal cell culture surface, and further incubated for 7 days. To promote producing extracellularly collagen fibers, the transferred cell sheet was incubated in medium containing L-ascorbic acid. After 7days of culturing, collagen fibers deposited with a cell sheet showed anisotropy on the non-patterned surface. Scale bar: 100 µm. Reproduced with permission.[73] Copyright 2011, Elsevier.

individual directions independent of neighboring cells. For example, corneal stroma consists of alternating lamellae of collagen fibrils, and the cells are oriented in parallel with aligned collagen fibers in the fibrous tissues.[13,16,55,111] Since collagen is organized uniquely into the sheets of parallel fibrils, which are arranged perpendicular to its neighbor sheet in the transparent cornea, the layer-by-layer construction technique may be useful to organize stroma having native mechanical and optical properties.

5. Unique Cell Behaviors Found through a Cell Sheet Layering Approach 5.1. Self-Organization of Myoblasts in Multilayered Cell Sheets Unlike in conventional methods, aligned myoblasts can be manipulated as a single continuous myoblast sheet using thermoresponsive substrates. The transferable anisotropy allows the production of three-dimensionally oriented myotube constructs through the layer-by-layer construction technique, since myoblasts prefer forming an aligned structure even after being transferred onto non-patterned surfaces. After differentiation into myotubes, two anisotropic myotube sheets layered perpendicularly show different vertical directionality in the single tissue construct. Myotube architecture can be flexibly produced using the cell sheet manipulation technique. Interestingly, on the other hand, the cell-dense 3D environment of multilayered cell sheets induces the self-organization of myoblasts.[88] Since cell sheets are layered together with only their associated ECM, the aligned myoblasts are in contact with each other both physically and biologically. When two anisotropic myoblast sheets are layered perpendicularly, before the differentiation into myotubes, they rearrange their orientation by themselves to form a unidirectional structure within the multilayered cell sheets



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Figure 8. The self-organization of myoblast orientation in multilayered cell sheet constructs. a) Two anisotropic myoblast sheets were layered perpendicularly via the cell sheet layering process. Actin fibers were stained just after cell sheet layering or 7 days after layering. The layered myoblast sheets showed a single orientation after 7 days. b) Fluorescence images of bottom cell sheets at 0, 6, 12, and 18 h post-layering. An unstained anisotropic myoblast sheet was layered perpendicularly onto a fluorescently stained cell sheet (CellTracker Green). The aligned bottom cells changed their orientation to match the same direction as the orientation of the top cells within 24 h. c) Orientation changes of randomly oriented myoblasts in two- and six-layered cell sheets. A single anisotropic myoblast sheet (Aligned) was placed onto a single or five-layered cell sheets composed of randomly oriented myoblasts (Random). Only the bottom cells were stained fluorescently (green). In the two-layered cell sheets, random cells self-organized their orientation to a single alignment by layering a single Aligned sheet. In the six-layered sheet construct, most of the bottom cells showed random orientation even after 3 days. Only a few cells (indicated by white arrows) changed their orientation to match the same direction as the alignment of top aligned cells. This indicates that the rearrangement of myoblasts influences through several cell layers, but is limited. d) Self-organized formation of aligned myotubes in multilayered cell sheet constructs. A top Aligned sheet triggered the self-organization of two-layered Random sheets into a single orientation in the cell sheet construct. After the rearrangement, the three-layered myoblast sheets were cultured in differentiation medium. After 5 days of culturing, the self-organized myotube construct showed a single cell orientation. Scale bar: 100 µm. Reproduced with permission.[88] Copyright 2013, Elsevier.

(Figure 8). Just after stacking, the two myoblast sheets have different angles relative to each other. However, the 3D orientation changes to a single direction through the self-organization of myoblasts (Figure 8a). In every case, the most interesting point is that the top anisotropic cell sheet determines the orientation of the bottom cells. The bottom myoblasts change

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their orientation to align with the same direction as the anisotropy of top cell sheet. This behavior actually occurs rapidly (within 1 day) (Figure 8b). This self-organization behavior is also observed in three-layered myoblast sheets. For example, when a single anisotropic myoblast sheet is placed on two-layered myoblast sheets composed of randomly oriented cells, the




rearrangement occurs step-by-step from the top to the bottom. In the case of four-layered random sheets, even though three cell sheets are between the top anisotropic sheet and the bottom sheet, most of the cells change their orientation to align with the top sheet. On the other hand, when five myoblast sheets are layered underneath one anisotropic myoblast sheet, the bottom cells show no change in orientation, even 3 days after layering (Figure 8c). Taken together, the direction of the top cell sheet influences multiple cell sheets; however, this rearrangement is limited to no more than four layered cell sheets. Skeletal muscle myoblasts are originally capable of organizing into an aligned structure in native muscle tissues. Although the mechanism of this rearrangement behavior has not yet been elucidated, the native myoblast characteristic is probably a key factor in this unique behavior to organize the 3D orientation in a cell sheet construct. In fact, anisotropic fibroblast sheets layered perpendicularly show individual orientations even 7 days after layering.[73] Some factors such as N-cadherin binding and fibronectin-integrin interaction contribute to regulate myoblast alignment in the development of native muscle tissues.[112] These factors may play important roles in this self-organization process. Since muscle fibers are natively oriented through the self-organization of myoblasts, the orientation change of myoblasts found in this cell sheet study probably occurred though a biological process mimicking the native myoblast behavior. It should be noted that this cell behavior is probably impossible to be found in scaffold-based tissue constructs due to the limitations of cell–cell communication and flexibility of cell migration. By adjusting the directionality of multiple cell sheets, a thick tissue can be created with a single orientation. However, to adjust precisely the alignment, simply rotating the manipulator is insignificant. Fortunately in cell sheet-based muscle construction, myoblasts self-organize their orientation threedimensionally which is simply induced by layering of an anisotropic myoblast sheet. Therefore, the adjustment of cell alignment is not required in this tissue construction approach, and just one anisotropic myoblast sheet is necessary to create unidirectional thick muscle tissues. For example, when a single anisotropic myoblast sheet is placed on two layered normal myoblast sheets, the randomly oriented myoblasts change their


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Figure 9. Formation of separate assemblies of neurons and endothelial cells on an anisotropic myoblast sheet. a) Human iPS cell-derived neurons adhered on an anisotropic myoblast sheet and then formed oriented networks along the myoblast orientation. Neurons were stained with anti-beta III tubulin antibody (red). b) Neurons (red) and green fluorescent protein (GFP)-expressing HUVECs (green) were mixed and seeded onto a single myoblast sheet. When the seeding density of HUVECs was 5 × 104 cells/cm2, neurons were unable to spread and formed small aggregates. c) When the seeding density was decreased to 2 × 104 cells/cm2, neurons formed oriented networks only in regions where no endothelial cells adhered. HUVEC also formed assemblies of only endothelial cells, even though a mixture of both types of cells was seeded onto the cell sheet. Scale bar: 50 µm. Reproduced with permission.[96] Copyright 2014, Wiley-VCH.

orientation to align with the same direction as the top cells. Consequently, they self-organize into a single orientation in three-layered myoblast cell sheets.[88] After the rearrangement, differentiated myotubes show unidirectionally oriented myotube architecture (Figure 8d). This feature may represent the self-organization process in natively oriented muscle tissues.

5.2. Intermingling Cellular Network Formation Triggered by Cell Sheet Layering In the regenerative medicine approach and development of in vitro tissue models, more complex tissues composed of multiple cell types are required to be produced biomimetically.[12,31] In mature native skeletal muscle, not only the highly oriented muscle fibers, but also vasculatures and neurons are essential components to perform mature contractile functions of skeletal muscle. As described above, cell sheet layering triggered the formation of endothelial cell networks.[94,96] Although the network-forming endothelial cells need to be functionalized to supply oxygen and nutrients to engineered tissues, it was interesting that the orientation of the branching network was regulated by anisotropic myoblast sheets. On the other hand, since the muscle fibers are innervated by the nervous system in skeletal muscle, structurally and biologically organized neurons must also be considered to produce sophisticated tissue functions mimicking native skeletal muscles. In a previous study, an oriented neural and vascular network was produced through a simple cell sheet layering process.[96] For incorporation of endothelial cells and neurons, these two kinds of cells were sandwiched between two anisotropic myoblast sheets. In the study, human iPS cell-derived neurons formed an oriented network on a single anisotropic myoblast sheet (Figure 9a). Importantly, neurite outgrowth was guided by the myoblast orientation, indicating that neurons recognized the anisotropic environment. In addition, unlike endothelial cells, they required no cell sheet layering to form the branching structure. The coordination between nerves and vessels has been thoroughly investigated in the field of cell biology, specifically in developmental biology.[113] In an approach to incorporation of neurons and endothelial cells within a cell sheet construct,



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the two kinds of cells show a unique behavior in the construct. Unlike endothelial cells, neurons form anisotropic networks whether they are on a single cell sheet or sandwiched between two layered cell sheets (Figure 9a). Nevertheless, neurons are unable to spread significantly with the surrounding endothelial cells. When neurons are seeded with endothelial cells (a seeding density of HUVEC: 5 × 104 cells/cm2) onto a single cell sheet without layering, neurons are surrounded by the adhering endothelial cells, and form sphere-shaped neuron aggregates (Figure 9b). When the seeding density of HUVEC is decreased (2 × 104 cells/cm2) to provide room for neuron spreading, neurons form a branching network by avoiding intermingling with endothelia cells (Figure 9c). Consequently, both types of cells form separate assemblies on a myoblast sheet. HUVECs also form cell assemblies composed only of endothelial cells. The polarity of endothelial cells is well regulated and the apical surface of the endothelium is normally non-adhesive for cells, resulting in the non-thrombogenic property in vascular endothelium.[114] This characteristic may be one reason that prevents the spread of neurons on the HUVEC assemblies. However, even when HUVECs are seeded onto a neuron-spreading surface, they avoid adhering onto the areas where neurons have already spread. Taken together, irrespective of rank or standing, neurons and endothelial cells prefer forming individual cell assemblies. Interestingly, this behavior is only seen on a single cell sheet, but not in multilayered cell sheets. When sandwiched between two cell sheets, they show no separate assemblies of neurons or endothelial cells.[96] As described above, the cell sheet layering probably alters some biological cellular signaling in the endothelial cells (Figure 5d–h). This change in the endothelial cells may permit neurons to elongate in the same space of the tissue construct. Consequently, they form uniform intermingling networks composed of both neurons and endothelial cells throughout the whole area within the layered cell sheet construct. That is to say, this 3D cell microenvironment allows all three types of cells (myoblasts, neurons and endothelial cells) to self-organize native-like microstructures. Although the relationship between neurons and endothelial cells on a cell sheet has not been fully revealed, their coordinated cell behavior emphasizes the importance of cell sheet layering for not only the formation of vascular-like branching structures, but also to create a complex intermingling of the two kinds of networks in the tissue construct.

5.3. Reorientation of Mesenchymal Stem Cells in an Anisotropic Cell Sheet The alignment of human bone-marrow MSCs has been regulated by using the Protein-patterned and Polymer-patterned substrates. Interestingly, in the cell sheet preparation, the aligned MSCs changed their orientation on the Protein-patterned substrate.[101] When MSCs are seeded onto the surface in serumfree medium, cells form stripe-shaped micropatterns and orient parallel to the FN-patterns (Figure 10a). By adding serum to the medium, the aligned MSCs proliferate outside the FN-patterns and finally reach confluence within 5–6 days. Although the resultant cell sheet shows directionality, as do the other types

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of anisotropic cell sheets, the aligned MSCs collectively reorient approximately 16° off the pattern direction during proliferation (Figure 10b). Interestingly, the MSCs still form an aligned orientation even although they change their orientation. The reorientation behavior is related to cell seeding density. Low seeding density gives cells more space to reorient on the patterned surface, while higher seeding density restricts the cell reorientation. Although the influence of cell seeding density on the reorientation is recognized, the specific mechanism is still not clear. This cell behavior may be guided biochemically or physically via cell–cell contacts, and will be of interest in future studies of cell-material interactions. Human MSCs can be also harvested as an anisotropic cell sheet using the Polymer-patterned substrate (unpublished data). Cells are aligned parallel to the direction of the stripeshaped polymer patterns, and then harvested as a single continuous cell sheet by lowering the culture temperature (e.g., 20 °C). Even after being transferred to a normal culture dish using a gelatin gel manipulator, the cell alignment can be maintained for at least 3 weeks in MSC growth medium (Figure 10c). Bone marrow MSCs can differentiate into a range of cell types including osteoblasts, chondrocytes and adipocytes.[115] Due to this capability, anisotropic MSC sheets are expected to be a platform to produce a variety of anisotropic cell sheets.[6,67,80,116] To confirm that the transferred MSCs can differentiate into multiple cell types, the transferred cell sheet was cultured in adipogenic or osteogenic differentiation medium on a normal culture dish. Unfortunately, in adipogenic differentiation, even though they showed the capability of differentiation into adipocytes, the spindle-shaped morphology changed to a more round-shape due to the original morphology of adipocytes (Figure 10d). As a result, the differentiated cells lost the directionality of the cell sheet. On the other hand, the aligned MSCs showed an interesting behavior when being cultured in osteogenic differentiation medium. As shown in Figure 10e, the aligned MSCs reoriented during 2 weeks of culturing. While the angle shifted from being parallel to the direction of the MSC alignment, the cells still showed a specific orientation. Some areas of cells showed no reorientation and kept the original orientation. As a result, a single MSC sheet had two different orientations. Confocal 3D imaging revealed that the two alignments were observed only on the bottom side of the cell sheet, and the top side showed only a single alignment of the reoriented cells (Supporting Information Video 1). This suggested that, although only a few cells kept the original orientation, most cells reoriented to a different direction relative to the original alignment. Since the alignment was maintained in MSC growth medium for 3 weeks, this reorientation was induced by culturing in osteogenic differentiation medium. On the other hand, the relationship between the reorientation and osteogenic differentiation is still unknown. This behavior is similar to the reorientation behavior of aligned MSCs on the Protein-patterned substrate as described above. However, one was observed for growing on the Protein-patterned surface, another occurred when they differentiated on a normal TCPS surface (after being transferred from the Polymer-patterned surface). Therefore, it is not certain whether these two kinds of reorientation behavior originate from the MSC-specific property. Although these




PROGRESS REPORT Figure 10. Reorientation of aligned MSCs found in cell sheet engineering. a,b) Microscopic images of aligned MSCs on a thermoresponsive surface prepared using a micro-contact printing method (the Protein-patterned surface). Scale bar: 100 µm. a) Human MSCs were aligned along FN-patterns on the thermoresponsive surface in serum-free culture. b) They proliferated outside the patterns by the addition of serum. The aligned cells reoriented their alignment while they reached confluence. The MSCs were aligned even after the reorientation (the angles shifted to 16° relative to the FN-patterns). c–e) Phase contrast and fluorescence microscopic images of anisotropic MSC sheets after transfer onto a normal culture dish. Scale bar: 100 µm. c) Human MSCs were aligned on a micropatterned thermoresponsive substrate (the Polymer-patterned substrate), and then harvested as an anisotropic cell sheet. Using a gelatin gel-coated plunger, the MSC sheet was transferred onto a normal cell culture dish. The transferred MSC sheet showed the original orientation even at 3 weeks after the transfer. d) The transferred cell sheet was cultured in adipogenic differentiation medium for 1 week. The cell morphology was changed by the differentiation into adipocyte. Neutral lipid in adipocytes was stained with LipidTOX Green. e) The transferred cell sheet was cultured in osteogenic differentiation medium for 2 weeks. Most cells altered their alignment and reoriented at a different angle, while some aligned cells kept the original orientation (the original direction of cell alignment is indicated by the white arrow). Consequently, one anisotropic MSC sheet exhibited two different orientations. A 3D image of actin fibers in this cell sheet was observed through z-stack imaging, and separated into top and bottom sides. Both cell alignments were observed in the bottom side, while the image of the top side showed only a single orientation shifted from the original alignment. These images suggest that a few cells kept the original orientation, but most cells reoriented to the different direction within 2 weeks. Reproduced with permission.[101] Copyright 2011, Elsevier.

phenomena would not directly influence cell sheet-based tissue construction, the reorientation behaviors are of interest to understand the importance of cell orientation in the field of cell biology. If the mechanism of these unique behaviors of myoblasts, endothelial cells, and MSCs could be revealed, it may provide a clue to propose a new strategy for future tissue engineering.


6. Conclusions Cell sheet-based technology has been applied to construct a variety of engineered tissues. With the advantages of scaffold-free tissue architecture, this unique tissue engineering approach had opened up new possibilities in regenerative medicine and the development of tissue models for drug discovery. On the other hand, the researchers understand that



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the present techniques are limited in the production of many kinds of tissues. Although scaling-up of engineered tissues is required, improvement of the techniques is progressing. As well as the production of large-scale tissue constructs, tissue engineering also needs to move forward producing more complex tissues with micro-scale specific structures. In some native tissues, specific mechanical and biological functions are produced by organizing 3D anisotropy. The strategy of cell sheet engineering reported here focus on mimicking the microstructures found in native tissues of the body. Using micropatterned thermoresponsive substrates, some kinds of cells that naturally prefer forming oriented structures can be used as an anisotropic tissue unit in cell sheet-based tissue construction. The layer-by-layer construction of anisotropic cell sheets allows for the arrangement of 3D anisotropy in scaffold-free cell-dense tissue constructs. Particularly in skeletal muscle and vascular tissue engineering, this unique tissue construction will provide well-organized tissue constructs having mechanically and biologically biomimetic features. In addition, this technology will also be applied to the creation of other types of biomimetic tissues, such as a myocardial tissue with 3D anisotropy. In this scaffold-free 3D tissue construct, cell sheets can be layered with only their associated ECM, and the aligned cells are uniquely in contact with each other, both physically and biologically. This architecture allows not only the mimicking of native tissues, but also finding biologically unique cell behaviors. Some types of cells, such as human skeletal muscle myoblasts and human umbilical vein endothelial cells, recognized the anisotropic cell environments, and consequently they selforganized the oriented formation in multilayered cell sheet constructs. The layer-by-layer tissue construction technique helps to identify these cell behaviors. In addition, human bone marrow mesenchymal stem cells exhibited the reorientation behavior in cell sheet engineering. It is important to understand the key factors to organize the unique response of aligned cells in the future. These may have great potentials for better understanding the rule in a 3D cell environment and applying these cell behaviors to engineer complex tissues in the field of tissue engineering. In vitro tissue-engineered models are now highly prized tools for clinical applications including regenerative medicine. Well-organized tissue models will be used to better understand the mechanism of specific diseases and develop therapeutic agents to treat these diseases. Compared with conventional 2D tissue models, the 3D complex tissue models provide a microenvironment that more closely mimics native tissues. The cell sheet-based technology reported here has the potential to create precise microstructures in complex tissues. We can now create human cell-based tissue models based on iPS cell technology. Tissue engineering researchers believe that tissue models using disease-specific human iPS cells will become a powerful tool as a platform for producing a personalized tissue model based on a patient-specific disease. Therefore, in tissue modeling, a technology to flexibly and precisely organize the attractive autologous cells is required. With cell sheet technology, sophisticated 3D tissue models are expected to be made of biomimetic microstructures of appropriate cell sources.


Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was supported by Grant-in-Aid for Young Scientists (A) (JSPS KAKENHI Grant Number 26702018) and Grant-in-Aid for Scientific Research (MEXT KAKENHI Grant Number 23106009) on Innovative Areas “Bio Assembler” (Area No. 2305) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. We thank Allan Nisbet for his useful comments and editing assistance. Received: March 18, 2015 Revised: April 22, 2015 Published online: June 1, 2015

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Strategies affording prevascularized cell-based constructs for myocardial tissue engineering.

Dual-Scale Polymeric Constructs as Scaffolds for Tissue Engineering.

Skeletal muscle tissue engineering: strategies for volumetric constructs.

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering.

Analyzing Biological Performance of 3D-Printed, Cell-Impregnated Hybrid Constructs for Cartilage Tissue Engineering.

Collagen-Based Substrates with Tunable Strength for Soft Tissue Engineering.

Biomaterial-mesenchymal stem cell constructs for immunomodulation in composite tissue engineering.

Experimental approaches to vascularisation within tissue engineering constructs.

Cell and tissue engineering for liver disease.

Cell-Based Strategies for Meniscus Tissue Engineering.

Stem Cell-Biomaterial Interactions for Tissue Engineering.

tissue microenvironment engineering and monitoring in tissue engineering, regenerative medicine, and in vitro tissue models.

Cardiac tissue engineering and regeneration using cell-based therapy.

Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.

Bone Tissue Engineering with Multilayered Scaffolds-Part I: An Approach for Vascularizing Engineered Constructs In Vivo.

Pre-clinical characterization of tissue engineering constructs for bone and cartilage regeneration.

Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen-elastin constructs.

Tissue engineered constructs for peripheral nerve surgery.

Electrospun fiber constructs for vocal fold tissue engineering: effects of alignment and elastomeric polypeptide coating.

polycaprolactone hydrogel-fiber composites for heart valve tissue engineering.

Engineering muscle constructs for the creation of functional engineered musculoskeletal tissue.

Tissue engineering in periodontal tissue.

Engineering structure and function using thermoresponsive biopolymers.

Anisotropic silk biomaterials containing cardiac extracellular matrix for cardiac tissue engineering.