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During pulmonary resections, removal of visceral pleura is frequently required, resulting in lung air leakage (LAL) and bleeding. Especially persistent LAL after pulmonary surgery has negative consequences. Current surgical procedures are ineffective in closing these visceral pleural injuries. Previously, the authors' laboratory has developed a novel and effective LAL sealant using tissue-engineered cell sheets harvested from temperature-responsive culture dishes. The clinical application of fresh fibroblast sheets (FSs) is limited by several problems related to the cell culture period, mass production, preservation, and transportation. Therefore, cryopreservation of FSs and feasibility of off-the-shelf FSs for repairing visceral pleural defects were investigated. Over 3 to 6 months, harvested skin-derived FSs in Dulbecco's modified Eagle's medium supplemented with 10% dimethyl sulfoxide were stored in an atmosphere of liquid nitrogen. The amounts of cytokines (basic fibroblast growth factor [bFGF] and vascular endothelial growth factor) released from frozen–thawed FSs were determined. bFGF levels were significantly elevated in frozen–thawed FSs compared with fresh FSs. After a visceral pleural injury model was created, a frozen–thawed skin-derived FS was transplanted directly to the defect. One month after transplantation, the frozen–thawed FS remained on the pleural surface, resulting in permanent closure, suggesting that cells in the off-the-shelf FS had the ability to proliferate and release various cytokines. Frozen–thawed FSs were useful for closing LALs during pulmonary surgery as an off-the-shelf technique and would be used as a pleural substitute. [ABSTRACT FROM AUTHOR]

Thermoresponsive surfaces, prepared by grafting of poly( N -isopropylacrylamide) (PIPAAm) or its copolymers, have been investigated for biomedical applications. Thermoresponsive cell culture dishes that show controlled cell adhesion and detachment following external temperature changes, represent a promising application of thermoresponsive surfaces. These dishes can be used to fabricate cell sheets, which are currently used as effective therapies for patients. Thermoresponsive microcarriers for large-scale cell cultivation have also been developed by taking advantage of the thermally modulated cell adhesion and detachment properties of thermoresponsive surfaces. Furthermore, thermoresponsive bioseparation systems using thermoresponsive surfaces for separating and purifying pharmaceutical proteins and therapeutic cells have been developed, with the separation systems able to maintain their activity and biological potency throughout the procedure. These applications of thermoresponsive surfaces have been improved with progress in preparation techniques of thermoresponsive surfaces, such as polymerization methods, and surface modification techniques. In the present review, the various types of PIPAAm-based thermoresponsive surfaces are summarized by describing their preparation methods, properties, and successful biomedical applications. [ABSTRACT FROM AUTHOR]

Bisphosphonates (BPs) inhibit bone resorption and are frequently used to treat osteoporosis, bone metastasis, and other conditions that result in bone fragility. However, numerous studies have reported that BPs are closely related to the development of osteonecrosis of the jaw (BRONJ), which is an intractable disease. Recent studies have demonstrated that intravenous infusion of multipotent mesenchymal stromal cells (MSCs) is effective for the treatment of BRONJ-like disease models. However, the stability of injected MSCs is relatively low. In this study, the protein level of vascular endothelial growth factor in BP-treated MSCs was significantly lower than untreated-MSCs. The mRNA expression levels of receptor activator of nuclear factor κ-B ligand and osteoprotegerin were significantly decreased in BP-treated MSCs. We developed a tissue-engineered cell sheet of allogeneic enhanced green fluorescent protein (EGFP)-labeled MSCs and investigated the effect of MSC sheet transplantation in a BRONJ-like rat model. The MSC sheet group showed wound healing in most cases compared with the control group and MSC intravenous injection group (occurrence of bone exposure: 12.5% compared with 80% and 100%, respectively). Immunofluorescence staining revealed that EGFP-positive cells were localized around newly formed blood vessels in the transplanted sub-mucosa at 2 weeks after transplantation. Blood vessels were significantly observed in the MSC sheet group compared to in the control group and MSC intravenous injection group (106 ± 9.6 compared with 40 ± 5.3 and 62 ± 10.2 vessels/mm 2 , respectively). These results suggest that allogeneic MSC sheet transplantation is a promising alternative approach for treating BRONJ. Statement of Significance Bisphosphonates are frequently used to treat osteoporosis, bone metastasis of various cancers, and other diseases. However, bisphosphonate related-osteonecrosis of the jaw (BRONJ) is an intractable disease because it often recurs after surgery or is exacerbated following conservative treatment. Therefore, an alternative approach for treating BRONJ is needed. In this study, we developed a bone marrow-derived multipotent mesenchymal stromal cell (MSC) sheet to treat BRONJ and investigated the effect of MSC sheet transplantation in a rat model of BRONJ-like disease. The MSC sheet transplantation group showed wound healing in most cases, while only minimal healing was observed in the control group and MSC intravenous injection group. Our results suggest that the MSC sheet is a promising alternative approach for the treatment of BRONJ. [ABSTRACT FROM AUTHOR]

Mesothelial cells are an integral part of the peritoneum and play an important role in maintaining its structural and functional properties. In recent years a number of studies on mesothelial cells have been performed to evaluate the localization, secretional properties and the ability of regeneration and transdifferentiation of these cells. They are also involved in the repair of the peritoneum damage following surgery or peritonitis. Mesothelial cells produce several cytokines, growth factors and extracellular matrix components, possessing anti-inflammatory and immunomodulatory properties. Based on previous research, cell sheet engineering has made it possible to transplant cells that retain the cells' function, and stacking different cells in layers has also become possible. Arranging blood vessels between the cell layers is a problem when stacking cells in layers. Whether adequate blood flow can be obtained for the cell layers to survive long-term is the difference between success and failure. Mesothelial cell transplantation for peritoneal regeneration needs to be performed under conditions in which the surface area of the visceral peritoneum is large and the mesothelial cell damage area is small. In this article we explain cell sheet engineering as one of the technologies for transplanting cells with a variety of intercellular adhesion and cell membrane molecules maintained intact, and its application to peritoneal regeneration. [ABSTRACT FROM AUTHOR]

Cell sheets harvested from temperature-responsive cell culture dishes (TRDs) has attracted considerable attention as effective tools for reconstructing the lost functions of tissues and organs in the regenerative medicine field. However, because of their thinness, handling problems sometimes arise when transferring cell sheets from a TRD to a target surface. In this study, we developed a facile cell transfer method referred to as in situ gelation by using both gelatin hydrogel and a support membrane. Gelation and low-temperature processes were simultaneously performed on TRD. Confluent cultured cells were efficiently harvested from TRD in less than 5 min by decreasing the incubation temperature to 20°C. Harvested cells were found to maintain their cell viability, extracellular matrix, and original shape, thus allowing transfer of the cells to another surface with a short incubation time at 37°C. This method is applicable for various cell types regardless of the formation of tight cell-cell junctions. In addition, because of the high flexibility of the gelatin-coated membrane, cells were efficiently transferred to the surface of a mouse subcutis and liver. When compared with conventional cell sheet manipulation methods, the interaction between the cell surface and membrane was reinforced by the uniformly formed gelatin gel layer without using a special device. Therefore, the in situ gelation method is a promising technique for cell sheet-based tissue engineering and regenerative medicine. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1659-1668, 2014. [ABSTRACT FROM AUTHOR]

Antibody-immobilized thermoresponsive poly( N-isopropylacrylamide- co-2-carboxyisopropylacrylamide) [poly(IPAAm- co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3883-3893, 2014. [ABSTRACT FROM AUTHOR]

Thready stripe-polyacrylamide (PAAm) pattern was fabricated on a thermoresponsive poly( N-isopropylacrylamide) (PIPAAm) surface, and their surface properties were characterized. A PIPAAm surface spin-coated with positive photoresist was irradiated through a 5 µm/5 µm or a 10 µm/10-µm black and white striped photomask, resulting in the radical polymerization of AAm on the photoirradiated area. After staining with Alexa488 bovine serum albumin, the stripe-patterned surface was clearly observed and the patterned surface was also observed by a phase contrast image of an atomic force microscope. NIH-3T3 (3T3) single cells were able to be cultured at 37°C on the patterned surfaces as well as on a PIPAAm surface without pattern, and the detachment of adhered cells was more rapidly from the patterned surface after reducing temperature. Furthermore, the rate of detachment of 3T3 confluent cell sheet on the patterned surface was accelerated, compared with on a conventional PIPAAm surface under the static condition. The rate control of cell sheet recovery should contribute the preservations of cell phenotype and biological functions of cell sheet for applying to clinical trials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2849-2856, 2014. [ABSTRACT FROM AUTHOR]

There has been an upsurge in regenerative medicine in recent years. In particular, because oral mucosal epithelial cells can be obtained noninvasively, cultured epithelial cell sheets have been used in a number of ectopic transplantations. Additionally, the verification of the properties of experimental animals' cultured cells has accelerated the application of regenerative medicine. In the present study, the properties of oral mucosal epithelial cells were compared between six animal species. The human and pig epithelia were relatively thicker than the epithelia of the other species. The colony-forming efficiency of the rat was the highest, followed by those of the dog, human, rabbit, and pig, whereas the colonies of the mouse cells were all paraclone and uncountable in the colony-forming assay. We also found that the rabbit and pig cells proliferated poorly and were unable to form cell sheets without feeder layers. In contrast, even in the absence of feeder layers and cholera toxin, cultured dog and mouse cells formed contiguous sheets, when the cell seeding density was high. These results indicate that interspecies variation is considerable in oral mucosal epithelial cells and that specific experimental animal or human cells must be chosen according to the intended use. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1829-1837, 2014. [ABSTRACT FROM AUTHOR]

The oral mucosa is an easily accessible source of cells. Oral mucosal collection will be an essential surgical procedure for regenerative medicine and cell biological research. However, there is no current report that describes the details of the surgical procedure used for oral mucosal collection. Moreover, the number of cells that can be obtained has not been determined. Two different procedures, the punch biopsy and the spindle-shaped biopsy, were performed for the fabrication of transplantable autologous epithelial cell sheets. The mean values of the cells collected per square centimeter of tissue using the punch biopsy and the spindle-shaped biopsy were 76.8 ± 45 × 104 cells/cm2 and 195.7 ± 120 × 104 cells/cm2, respectively. There was no significant difference between the punch biopsy and the spindle-shaped biopsy. The coefficient of variation of the punch biopsy and the spindle-shaped biopsy was 58.9% and 69.8%, respectively. This result indicated that both procedures showed variations in the number of collected cells. Although the punch biopsy may be easier and simpler than the spindle-shaped biopsy, multiple punch biopsies may result in a more complicated procedure, and the spindle-shaped biopsy may be preferable when a large number of cells is necessary. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2849-2854, 2012. [ABSTRACT FROM AUTHOR]

Purpose: The objective of this study was to establish a method for accurate cell counting from matrix-rich cell sheets in the clinical setting. Materials and Methods: Human periodontal ligament (HPDL) cells were obtained from healthy donors to prepare PDL cell sheets. To obtain single cell suspensions, the cell sheets were treated with three different enzymatic formulations: collagenase alone, trypsin-ethylenediaminetetraacetic acid (EDTA) alone, and a combination of collagenase and trypsin-EDTA. After cell dispersion, cell numbers and cell survival rates were measured. To evaluate damage to the cell surfaces from the enzymes, the dispersed cells were analyzed by a low cytometer with an anti-alkaline phosphatase antibody. Results: Treatment with collagenase alone or trypsin-EDTA alone dispersed few cells from HPDL cell sheets. In contrast, combined treatment with collagenase and trypsin-EDTA successfully produced a large amount of single cells from cell sheets. Flow cytometry analysis showed that single cells obtained by combined use of collagenase and trypsin-EDTA preserved alkaline phosphatase epitopes on the cell surfaces. Conclusions: Cell sheets rich with extracellular matrix were dispersed via combined treatment with collagenase and trypsin-EDTA without destroying the expression of cell surface markers. The results suggest that this method would be useful for determining the accurate cell number of cell sheets for cell therapies and should also be applicable for other kinds of matrix-rich cell sheets. [ABSTRACT FROM AUTHOR]

Abstract: The retinal pigment epithelium (RPE) plays an important role in maintaining a healthy neural retina. With changes due to age, morbidity or removal of choroidal neovascularis developed as a means ofation, damage or defects of the RPE occur. Accordingly, RPE transplantation techniques have been repairing the damaged RPE. We conducted a study to transplant tissue-engineered RPE cell sheets in a rabbit model. RPE cells were isolated from pigmented rabbit eyes and seeded on temperature-responsive culture surfaces. Cultured RPE cells were arranged as a monolayer with a cobblestone cell shape that is characteristic of native RPE. The pigmented RPE cell sheets were non-invasively harvested without enzymatic treatment simply by reducing the culture temperature. Using 3-port vitrectomy, RPE cell sheets were transplanted into the subretinal space of albino rabbits. Seven days after surgery, the rabbits were sacrificed, and the eyes were enucleated and examined under both light and electron microscopy. After transplantation, our results show that the RPE cell sheets attached to the host tissues in the subretinal space more effectively than with the injection of isolated cell suspensions. Although the cell sheets maintained a monolayer structure in most areas, they were slightly folded or wrinkled in some regions. We conclude that tissue-engineered RPE cell sheets harvested from temperature-responsive culture dishes can be effectively transplanted beneath the neural retina. [Copyright &y& Elsevier]

Aim: The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue. Material and Methods: To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing. Results: Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed. Conclusions: Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration. [ABSTRACT FROM AUTHOR]

Aim: Hepatocyte transplantation to host livers by single cell suspension injection from the portal vein has been clinically successful in cases where the host liver architecture is intact. However, further investigation is still needed to achieve regeneration of the liver architecture when the host liver is destroyed, since single cell suspension injection often results in the formation of small hepatocyte colonies or islands. We show a hepatocyte transplantation strategy to ectopic sites. Methods: Primary hepatocytes isolated from green fluorescent protein (GFP)-transgenic rats were cultured on temperature-responsive culture dishes. After harvest as intact cell sheets, triple-layered cell sheets were transplanted over the superficial caudal epigastric artery and vein of athymic rats which had operation of 70% partial hepatectomy. Results: The transplanted hepatocytes were integrated to host tissue with a laminar cell arrangement at transplanted sites within one week after surgery. But the transplanted hepatocytes were hardly detected four weeks after transplantation, when the partially hepatectomized host liver was completely regenerated. GFP-positive bile duct-like tubes and functional blood vessels were observed. Conclusion: These results imply the usefulness of hepatocyte sheets in ectopic transplantation, as well as the need of trophic factors for hepatocyte survival. [ABSTRACT FROM AUTHOR]

Abstract: The field of tissue engineering has yielded several successes in early clinical trials of regenerative medicine using living cells seeded into biodegradable scaffolds. In contrast to methods that combine biomaterials with living cells, we have developed an approach that uses culture surfaces grafted with the temperature-responsive polymer poly(N-isoproplyacrylamide) that allows for controlled attachment and detachment of living cells via simple temperature changes. Using cultured cell sheets harvested from temperature-responsive surfaces, we have established cell sheet engineering to create functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, tracheal resection, and cardiac failure. Additionally, by exploiting the unique ability of cell sheets to generate three-dimensional tissues composed of only cultured cells and their deposited extracellular matrix, we have also developed methods to create thick vascularized tissues as well as, organ-like systems for the heart and liver. Cell sheet engineering therefore provides a novel alternative for regenerative medicine approaches that require the re-creation of functional tissue structures. [Copyright &y& Elsevier]

Abstract: Current methods including the use of various biological and synthetic sealants are ineffective in the closure of intraoperative air leaks that often occur during cardiothoracic surgeries, resulting in a decreased quality of life for patients. We present the development of a novel lung air leak sealant using tissue engineered cell sheets. In contrast to previous materials such as fibrin glue, these bioengineered cell sheets immediately and permanently seal air leaks in a dynamic fashion that allows for the extensive tissue contraction and expansion involved in respiration, without any postoperative recurrences. Additionally, we demonstrate that mesothelial cells migrate to cover the transplanted cells sheets, thereby confirming excellent biocompatibility and integration with the host tissues. Finally, we present the use of skin fibroblasts as an effective and readily available autologous cell source that can be easily applied. This study shows for the first time, the development of an immediate and permanent lung air leak sealant, suitable for future clinical applications. [Copyright &y& Elsevier]

Abstract: While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field''s progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds. [Copyright &y& Elsevier]

Akizuki T, Oda S, Komaki M, Tsuchioka H, Kawakatsu N, Kikuchi A, Yamato M, Okano T, Ishikawa I. Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs. J Periodont Res 2005; 40: 245–251.© Blackwell Munksgaard 2005The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs.Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically.No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group.The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy. [ABSTRACT FROM AUTHOR]

Myocardial tissue engineering has now emerged as one of the most promising treatments for the patients suffering from severe heart failure. Tissue engineering has currently been based on the technology using three-dimensional (3-D) biodegradable scaffolds as alternatives for extracellular matrix. According to this most popular technique, several types of 3-D myocardial tissues have been successfully engineered by seeding cardiomyocytes into poly(glycolic acid), gelatin, alginate or collagen scaffolds. However, insufficient cell migration into the scaffolds and inflammatory reaction due to scaffold biodegradation remain problems to be solved. In contrast to these technologies, we now propose novel tissue engineering methodology layering cell sheets to construct 3-D functional tissues without any artificial scaffolds. Confluent cells on temperature-responsive culture surfaces can be harvested as a viable contiguous cell sheet only by lowering temperature without any enzymatic digestions. Electrical communications are established between layered cardiomyocyte sheets, resulting in simultaneous beating 3-D myocardial tissues. Layered cardiomyocyte sheets in vivo present long survival, macroscopic pulsation and characteristic structures of native heart tissue. Cell sheet engineering should have enormous potential for fabricating clinically applicable myocardial tissues and should promote tissue engineering research fields. [Copyright &y& Elsevier]

Thermoresponsive cell-culture polystyrene (PS) surfaces that are grafted with poly(N-isopropylacrylamide) (PIPAAm) facilitate the cultivation of cells at 37 °C and the detachment of cultured cells as a sheet with an underlying extracellular matrix (ECM) by reducing the temperature. However, the ECM and cell detachment mechanisms are still unclear because the detachment of cells from thermoresponsive surfaces is governed by complex interactions among the cells/ECM/surface. To explore the dynamic behavior of serum protein adsorption/desorption, thermoresponsive surfaces that correspond to thermoresponsive tissue-culture PS dishes were formed on sensor chips for quartz crystal microbalance with dissipation (QCM-D) measurements. X-ray photoelectron spectroscopy (XPS) measurements and temperature-dependent frequency and dissipation shifts, Δf and ΔD, using QCM-D revealed that the thermoresponsive polymers were successfully grafted onto oxidized, thin PS films on the surfaces of the sensor chips. Increased amounts of adsorbed bovine serum albumin (BSA) and fibronectin (FN) were observed on the thermoresponsive polymer-grafted surfaces at 37 °C when compared with those at 20 °C because of enhanced hydrophobic interactions with the hydrophobic, thermoresponsive surface. While the calculated masses of adsorbed BSA and FN using QCM-D were 3–5 times more than those that were obtained from radiolabeling, the values were utilized for relative comparisons among the same substrate. More importantly, the thermoresponsive, dynamic behavior of serum protein adsorption/desorption was monitored using the QCM-D technique. Observations of this dynamic behavior revealed that the BSA and FN that were adsorbed at 37 °C remained on both surfaces after decreasing the temperature to 20 °C. [ABSTRACT FROM AUTHOR]

Postoperative regeneration of the middle ear mucosa and pneumatization of the middle ear cavity are of great importance after middle ear surgery. This study developed a new method to transplant autologous nasal mucosal epithelial cell sheets into the damaged middle ear cavity. The aim of this study was to evaluate postoperative healing after the transplantation of the cell sheets. Rabbit nasal mucosal epithelial cell sheets were fabricated on a temperature-responsive culture dish, and transplanted into the damaged middle ear of rabbit, which was surgically created. The healing of middle ears was evaluated by histology and X-ray computed tomography after transplantation. Functional evaluation was performed by measuring the maximum middle ear total pressure reflecting a trans-mucosal gas exchange function. Two control groups were used: the normal control group and the mucosa-eliminated control group. Transplantation of cell sheets suppressed the bone hyperplasia and the narrowing of pneumatic space in the middle ear cavity compared with the mucosa-eliminated control group. The mucosal gas exchange function was also better in the cell sheet-transplanted group. Nasal mucosal epithelial cell sheet was confirmed to be useful as an effective graft material after middle ear surgery and hopefully become a novel therapy in the future. [ABSTRACT FROM AUTHOR]

Abstract: Heparin-functionalized poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [P(IPAAm-co-CIPAAm)] grafted surface was designed for the switching of cell growth/detachment, achieved by the regulation of affinity binding between basic fibroblast growth factor (bFGF) and immobilized heparin through the temperature-dependent conformational change of grafted P(IPAAm-co-CIPAAm) chains. At 37 °C, bFGF-bound heparin-thermoresponsive surfaces were able to hold the two- to three-fold number of mouse fibroblast (NIH/3T3) cells than both bFGF-physisorbed surface and PIPAAm surface with soluble bFGF after a 3-day cultivation. Bound bFGF via heparin on shrunken grafted P(IPAAm-co-CIPAAm) chains at 37 °C was able to reinforce the formation and stabilization of bFGF-FGF receptor complex, although the activity of physisorbed bFGF on PIPAAm-grafted surfaces was decreased by non-specific and randomly oriented adsorption. At 20 °C, the cultured NIH/3T3 cell sheet with bFGF detached from heparin-functionalized thermoresponsive surface. The release of bFGF from the surfaces was induced by reducing the affinity binding between bFGF and immobilized-heparin due to increasing the mobility of the swollen grafted P(IPAAm-co-CIPAAm) chains. Therefore, heparin-functionalized thermoresponsive surface was able to enhance cell proliferation, and confluent cells detached themselves as a contiguous cell sheet due to switching cell growth by changing temperature. A cell culture system using this surface is useful for rapid cell sheet fabrication and manipulation. [Copyright &y& Elsevier]

Abstract: A comb-type grafted poly(N-isopropylacrylamide) (PIPAAm) gel modified surface was newly developed for providing a rapid cell sheet recovery for tissue engineering. PIPAAm macromonomer was prepared by the etherification reaction of the hydroxyl terminal moieties of PIPAAm with acryloyl chloride, followed by the radical telomerization reaction of N-isopropylacrylamide (IPAAm) monomer using 2-mercaptoethanol as a chain transfer agent. Solution containing IPAAm monomer and PIPAAm macromonomer was spread on the surface of tissue culture polystyrene (TCPS), and then the surface was subjected to electron beam irradiation for grafting the monomer and macromonomer on the surfaces, resulting in comb-type grafted PIPAAm gel modified TCPS (GG-TCPS). Besides the difference of the amount of the modified PIPAAm, no distinct difference was found between the properties of GG-TCPSs and normal-type PIPAAm gel modified TCPS (NG-TCPS) through XPS, AFM and a contact angle measurement. At 37 °C, bovine aortic endothelial cells (BAECs) were well adhered and spread on GG-TCPS as well as NG-TCPS regardless of the macromonomer concentration. By lowering temperature to 20 °C, BAECs detached themselves more rapidly from GG-TCPS compared with NG-TCPS. Upon lowering temperature, the grafted polymer was speculated to accelerate the hydration of modified PIPAAm gel, resulting in a rapid cell sheet detachment. [ABSTRACT FROM AUTHOR]

Abstract: Regenerative medicine involving injection of isolated cells and transplantation of tissue-engineered myocardial patches, has received significant attention as an alternative method to repair damaged heart muscle. In the present study, as the next generation of myocardial tissue engineering we demonstrate the in vitro fabrication of pulsatile myocardial tubes using cell sheet engineering technologies. Three neonatal rat cardiomyocyte sheets, which were harvested from temperature-responsive culture dishes, were wrapped around fibrin tubes using a novel cell sheet-wrapping device. The tubular constructs demonstrated spontaneous, synchronized pulsation within 3h after cell sheet wrapping. Contractile force measurements showed that the contractile force increased in accordance with both increasing rest length (Starling mechanism) and increasing extracellular Ca2+ concentration. Furthermore, the tissue-engineered myocardial tubes presented measurable inner pressure changes evoked by tube contraction (0.11±0.01mmHg, max 0.15mmHg, n=5). Histological analyses revealed both well-differentiated sarcomeres and diffuse gap junctions within the myocardial tissues that resembled native cardiac muscle. These data indicate that tissue-engineered myocardial tubes have native heart-like structure and function. These new myocardial tissue constructs should be useful for future applications in physiological studies and pharmacological screening, and present a possible core technology for the creation of engineered tissues capable of independent cardiac assistance. [Copyright &y& Elsevier]

Abstract: Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive culture dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro. [Copyright &y& Elsevier]

Abstract: Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature. [Copyright &y& Elsevier]

Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27°C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm–BMA) co-grafted domains (1-mm∅ area), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37°C, achieving patterned co-cultures. Reducing culture temperature to 20°C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets. [Copyright &y& Elsevier]

Highlights: [•] Endometrial cell sheets reconstructed endometrium-like tissues in vitro and in vivo. [•] Endometrium-like tissues retained a function of hormonal receptors. [•] Endometrium-like tissues in vivo formed uterus-specific glandular structures. [•] Endometrium-like tissues in vivo formed functional secretory glands. [ABSTRACT FROM AUTHOR]

Abstract: In our previous studies, we have demonstrated effective regeneration of cartilage through the creation and application of layered cell sheets that combine both chondrocytes and synovial cells. In this study, we were able to demonstrate that cells derived from cell sheets can survive for long periods after transplantation into rat knee joints having osteochondral defects. We established a method for generating cell sheets from firefly luciferase-expressing chondrocytes obtained from transgenic Lewis rats, and carried out allogenic transplantation of these cell sheets into wild-type Lewis rats. We then administered luciferin and monitored the survival of the transplanted cells by using bioluminescence imaging (BLI). Our data showed that the transplanted cells survived and could be detected for more than 21 months, which was longer than expected. Furthermore, the BLI data showed that the transplanted cells remained in the knee joint and did not migrate to other parts of the body, thus confirming the safety of the cell sheets. In this study, we monitored the duration of survival of cell sheets composed of only chondrocytes, only synovial cells, or both chondrocytes and synovial cells, and found that all three types of cell sheets survived for an extended period of time. [Copyright &y& Elsevier]

Abstract: In some parts of native tissues, the orientation of cells and/or extracellular matrixes is well organized. We know that because anisotropy produces important tissue functions, an appropriate anisotropy needs to be designed to biomimetically construct complex tissue. Here, we show the unique features of anisotropic myoblast sheets for organizing the three-dimensional (3D) orientation of myoblasts and myotubes. Utilizing a micropatterned thermoresponsive surface, human skeletal muscle myoblasts were aligned on the surface, and manipulated as a single anisotropic cell sheet by reducing the culture temperature. Consequently, layering of anisotropic myoblast sheets using gelatin gel allowed 3D myotube constructs to be built up with the desired anisotropy. We also discovered a surprising myoblast behavior. An anisotropic cell sheet placed on top of other cell sheets in fabricating thick tissue was able to change the cell orientation in several layered cell sheets underneath. This self-organization is believed to provide the uniqueness required in designing 3D cell orientation architecture for reconstructed muscle tissue. [Copyright &y& Elsevier]

Abstract: Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14days produced around 8×107 cells/100ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. [Copyright &y& Elsevier]

Abstract: Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies. [Copyright &y& Elsevier]

Abstract: Cytotherapeutic approaches have been investigated to overcome the limitations of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences between cell sources in a canine severe defect model (one-wall intrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from each dog; a total of four dogs were used. Three-layered cell sheets of each cell source supported with woven polyglycolic acid were autologously transplanted to the denuded root surface. One-wall intrabony defects were filled with a mixture of β-tricalcium phosphate (β-TCP) and collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDLC group than in the other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLC group. The amount of alveolar bone regeneration was highest in the PDLC group, although it did not reach statistical significance among the groups. These results indicate that PDLC sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration. [Copyright &y& Elsevier]

Abstract: Reconstructing a vascular network is a common task for three-dimensional (3-D) tissue engineering. Three-dimensional stratified tissues were created by stacking cell sheets, and the co-culture with endothelial cells (ECs) in the tissues was found to lead to in vitro pre-vascular network formation and promoted in vivo neovascularization after their transplantation. In this study, to clarify the effect of tissue fabrication process on a pre-vascular network formation, human origin ECs were introduced into the stratified tissue in several different ways, and the behavior of ECs in various positions of the 3-D tissue were compared each other. Human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), and their mixture were harvested as an intact cell sheet from temperature-responsive culture dish at low-temperature (<20 °C). Single mono-culture EC sheet was stacked with two NHDF-sheets in different orders, and 3 co-cultured cell sheets were layered by a cell sheet collecting device. Morphological analyses revealed that pre-vascular networks composing of HUVECs were formed in all the triple layer constructs. Confocal microscope observation showed that the pre-vascular networks formed tube structures like a native microvasculature. These data indicate that the primary EC positioning in 3-D tissues may be critical for vascular formation. [Copyright &y& Elsevier]

Abstract: To survive three-dimensional (3-D) cell-dense thick tissues after transplantation, the improvements of hypoxia, nutrient insufficiency, and accumulation of waste products are required. This study presents a strategy for the initiation of prevascular networks in a 3-D tissue construct by sandwiching endothelial cells between the cell sheets. For obtaining a stable stacked cell sheet construct, a sophisticated 3-D cell sheet manipulation system using temperature-responsive culture dishes and a cell sheet manipulator was developed. When sparsely cultured human umbilical vein endothelial cells (HUVECs) were sandwiched between two myoblast sheets, the inserted HUVECs sprouted and formed network structures in vitro. Additionally, when myoblast sheets and HUVECs were alternately sandwiched, endothelial cell connections through the layers and capillary-like structures were found in a five-layer construct. Moreover, the endothelial networks in the five-layer myoblast sheet construct were observed to connect to the host vessels after transplantation into the subcutaneous tissues of nude rats, resulted in a neovascularization that allow the graft to survive. These results indicated that the prevascularized myoblast sheet constructs could induce functional anastomosis. Consequently, our prevascularizing method using a cell sheet stacking manipulation technology provides a substantial advance for developing various types of three-dimensional tissues and contributes to regenerative medicine. [Copyright &y& Elsevier]

Abstract: The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20°C for 20min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature-responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method. [Copyright &y& Elsevier]

Abstract: The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture. [Copyright &y& Elsevier]

Abstract: The proper function of many tissues depends critically on the structural organization of the cells and matrix of which they are comprised. Therefore, in order to engineer functional tissue equivalents that closely mimic the unique properties of native tissues it is necessary to develop strategies for reproducing the complex, highly organized structure of these tissues. To this end, we sought to develop a simple method for generating cell sheets that have defined ECM/cell organization using microtextured, thermoresponsive polystyrene substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges (50μm wide, 5μm deep). Vascular smooth muscle cells cultured on these substrates produced intact sheets consisting of cells that exhibited strong alignment in the direction of the micropattern. These sheets could be readily transferred from patterned substrates to non-patterned substrates without the loss of tissue organization. Ultimately, such sheets will be layered to form larger tissues with defined ECM/cell organization that spans multiple length scales. [Copyright &y& Elsevier]