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Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration. [ABSTRACT FROM AUTHOR]

Subcutaneous tumor‐bearing mice are commonly used to evaluate antitumor activity in preclinical studies of anticancer drugs. However, these models often exhibit excessive antitumor responses to anticancer drug candidates. In this study, intrahepatic tumor‐bearing mice as orthotopic tumor models were fabricated by transplanting hepatocarcinoma cell monolayers (sheets) to investigate differences in ectopic versus orthotopic antitumor response. Cell sheets, harvested from temperature‐responsive cell culture dishes using thin gelatin gel supporters, were transferred onto mouse liver surfaces. Cell sheet transplantation drastically improved intrahepatic tumor formation compared with direct intrahepatic injection of dispersed cells. In particular, all cell sheet‐transplanted mice formed well‐developed tumors inside the liver following removal of the mesothelial membrane at the liver surface. Notably, these mice exhibited comparable life spans, indicating similar intrahepatic tumor development rates. Antitumor activity of doxorubicin (DOX) was examined using both subcutaneous and intrahepatic tumor‐bearing mice. Although DOX administration yielded decreased subcutaneous tumor volumes, intrahepatic tumors exhibited no significant antitumor response. The results were considered to represent pharmacokinetic and histological structure differences between ectopic and orthotopic tumors, and partially supported the clinical uses of DOX. Therefore, cancer cell sheet transplantation constitutes a promising method to fabricate intrahepatic tumor‐bearing mice for drug screening test in preclinical studies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1071–1079, 2019. [ABSTRACT FROM AUTHOR]

Cell sheet tissue engineering is a concept for creating transplantable two‐dimensional (2D) and three‐dimensional (3D) tissues and organs. This review describes three elements of cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting/manipulation and transplantation of cell sheets. An essential technology for the preparation of cell sheets is the use of a temperature‐responsive cell culture surface, where the surface of tissue culture polystyrene (TCPS) dish is modified with thin layer of temperature‐responsive polymer, poly(N‐isopropylacrylamide) (PIPAAm). PIPAAm‐immobilized TCPS allows cultured cells to be harvested as a contiguous cell sheet with extracellular matrices (ECMs) by reducing the temperature, while chemical and physical disruption impair ECMs, cell–cell junction, and membrane proteins. Ligand‐immobilized and porous hydrophilic PIPAAm‐grafted surfaces are able to accelerate cell sheet preparation and harvesting, respectively. In addition, the manipulation of harvested cell sheets with the aid of cell sheet manipulator facilitates the formation of 3D tissues. Cell sheet‐based tissues and their transplantation are in seven clinical settings such as heart, cornea, esophagus, periodontal, middle chamber of ear, knee cartilage, and lung. In order to create thick and large 3D tissues and organs, large production of differentiated parenchymal cells from induced pluripotent stem (iPS) cells and vascularization within 3D tissues are key issues. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 955–967, 2019. [ABSTRACT FROM AUTHOR]

For the first time, we have fabricated insect-derived cell sheets by using temperature-responsive culture surfaces having a phase-transition temperature below 25°C. We prepared the temperature-responsive cell culture surfaces (tissue culture polystyrene, TCPS) by grafting a copolymeric gel consisting of hydrophobic N-tert-butylacrylamide (tBAAm) and N-isopropylacrylamide (IPAAm) units. First, to characterize the hydrophilic and hydrophobic properties of the copolymeric gel-grafted surfaces, static water contact angles of each surface were measured at various temperatures. By increasing the amount of tBAAm in the grafted copolymeric gel, the transition temperature of the gel was shifted to lower temperatures. At 25°C, the grafted copolymeric gel was dehydrated, and the insect-derived cells (AeAl2 cells) adhered on all the copolymeric gel-grafted surfaces. At 20°C, AeAl2 cells cannot adhere on the P(IPAAm-1.62tBAAm)-TCPS surface (the initial molar ratio of IPAAm and tBAAm (tBAAm = 1.62 mol%)) better than on other surfaces (TCPS and tBAAm = 4.88, 8.17 mol%). These two findings implied that the lower critical solution temperature of the copolymeric gel-grafted-TCPS existed from 20°C to 25°C. The laminin-coated P(IPAAm-1.62tBAAm)-TCPS surface showed temperature-dependent cell attachment and detachment properties, while AeAl2 cells were not detached from the extracellular matrix uncoated P(IPAAm-1.62tBAAm)-TCPS surface. AeAl2 cells and insect muscle cells were harvested as the respective sheets. [ABSTRACT FROM AUTHOR]

Abstract Cell transplantation is a promising approach for promoting tissue regeneration in the treatment of damaged tissues or organs. Although cells have conventionally been delivered by direct injection to damaged tissues, cell injection has limited efficiency to deliver therapeutic cells to the target sites. Progress in tissue engineering has moved scaffold-based cell/tissue delivery into the mainstream of tissue regeneration. A variety of scaffolds can be fabricated from natural or synthetic polymers to provide the appropriate culture conditions for cell growth and achieve in-vitro tissue formation. Tissue engineering has now become the primary approach for cell-based therapies. However, there are still serious limitations, particularly for engineering of cell-dense tissues. "Cell sheet engineering" is a scaffold-free tissue technology that holds even greater promise in the field of tissue engineering and regenerative medicine. Thermoresponsive poly(N -isopropylacrylamide)-grafted surfaces allow the fabrication of a tissue-like cell monolayer, a "cell sheet", and efficiently delivers this cell-dense tissue to damaged sites without the use of scaffolds. At present, this unique approach has been applied to human clinical studies in regenerative medicine. Furthermore, this thermally triggered cell manipulation system allows us to produce various types of 3D tissue models not only for regenerative medicine but also for tissue modeling, which can be used for drug discovery. Here, new cell sheet-based technologies are described including vascularization for scaled-up 3D tissue constructs, induced pluripotent stem (iPS) cell technology for human cell sheet fabrication and microfabrication for arranging tissue microstructures, all of which are expected to produce more complex tissues based on cell sheet tissue engineering. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Background: This study investigated the effects of mesenchymal stem cell (MSC) sheet transplantation on healing of chemically induced oral ulceration in a rabbit animal model.Methods: Oral mucosal ulcers were induced by topical application of filter paper soaked with 70% acetic acid to the anterior gingiva and buccal mucosa of 12 New Zealand white rabbits. The animals were randomly assigned to two groups: with (treatment group, n = 6) or without (control group, n = 6) cell sheets applied to ulcers. Gross findings were sequentially evaluated, and histologic examination was performed on day 7.Results: Based on gross inspection, ulceration resolved before day 5 in the treatment group; however, in the control group, healing was incomplete on day 7. In the treatment group, the total area of the ulcer decreased significantly from day 2 to day 5 (P < 0.001) and from day 5 to day 7 (P = 0.020), whereas the area decreased significantly from day 5 to day 7 in the control group (P < 0.001). Histologic and immunofluorescence examination revealed full-thickness mucosa healing and complete basal cell coverage in the treatment group; in contrast, only partial healing was observed on day 7 in the control group.Conclusions: Cell sheet technology using MSC can be an alternative treatment for oral ulcerations in that it can decrease healing time without invasive properties. [ABSTRACT FROM AUTHOR]

Tissue-engineering approaches have a great potential to improve the treatment of tendon injuries that affect millions of people. The present study tested the hypothesis that introduction of a tendon derived stem/progenitor cell (TSC) sheet accelerates tendon healing and tendon regeneration in a rat model. TSC sheets were produced on temperature-responsive culture dishes. Then, they were grafted on unwounded Achilles tendons and at sites of a 3 mm of Achilles tendon defect. At 2 and 4 weeks after implantation tendons were examined by histology, immunohistochemistry, transmission electron microscopy (TEM) and mechanical testing. The results showed that the implanted TSC sheet remained stably attached on the tendon surface at 4 weeks after implantation. Moreover, in the tendon defect model, tendon defect area where TSC sheet was implanted was well regenerated and had better organized collagen fibers with elongated spindle shaped cells, compared to relatively disorganized collagen fibers and round shaped cells in the control group. TEM observations revealed longitudinally aligned collagen fibers and thick collagen fibrils in the TSC sheet implanted group. Finally, at 4 weeks mechanical property of the TSC sheet implanted tendon had better ultimate load than the control. In conclusion, this study demonstrates the feasibility of implanting TSC sheets on tendons in vivo . Introduction of the cell sheets into a tendon defect significantly improved histological properties and collagen content at both 2 and 4 weeks after implantation, indicating that TSC sheets may effectively promote tendon remodeling in the early stages of tendon healing. Statement of Significance Tendon injury is a highly prevalent clinical problem that debilitates millions of people worldwide in both occupational and athletic settings. It also costs billions of healthcare dollars in treatment every year. In this study, we showed the feasibility of using tendon derived stem cell sheet to deliver biologically active tenogenic-constructs and promote tendon regeneration. This work has the potential to impact the orthopaedic surgery and sports medicine fields in the treatment of tendon injury. [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]

Fabrication of blood capillaries in tissue-engineered tissue is necessary for creating thick three-dimensional (3D) tissue with a high cellular density. For inducing blood capillaries in the tissue in vitro, a molded hyaluronic acid (HA) capillary-shaped gel was made as a template for blood capillaries by photolithogra-phy and power free pumping techniques. The fabricated HA capillary-shaped gel was sandwiched between two cell sheets consisting of neonatal normal human dermal fibroblasts (NHDFs), human umbilical vein endothelial cells (HUVECs), or co-cultured NHDFs and HUVECs, and eventually covered with the cells. Although a slight degradation of the HA gel was observed in the sandwiched tissue with HUVEC or NHDF cell sheets, significant degradation of the HA gel was observed in the sandwiched tissue with co-cultured cell sheets. Moreover, by continuing to culture the co-cultured tissue with HA gel, a tube formation was observed at the HA gel site. A sandwiched HA capillary-shaped gel with two cell sheets has a potential for creating blood capillaries in vitro and fabricating vascularized artificial organs. [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]

Because esophageal ulceration is extensive after endoscopic submucosal dissection, we have developed a new treatment that combines endoscopic dissection with the endoscopic transplantation of oral mucosal epithelial cell sheets to prevent esophageal stenosis. Cell sheets created from the patient''s own oral mucosa were attached directly to the bed of the esophageal ulcer using endoscopic forceps immediately after endoscopic resection. The first application of regenerative medicine to endoscopic treatment was performed in 2008 and more patients are being accumulated. We are working on several advanced research methods using regenerative medicine for endoscopic treatment. [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]

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]

We developed a novel simple method for making functional myocardial cell sheets that may be used as transplants. Polymerized human fibrin-coated dishes were prepared with fibrinogen monomers mixed with thrombin. Neonatal rat cardiomyocytes cultured on these dishes formed myocardial cell sheets within 4 days. These cell sheets were easily dissociated intact from the polymerized fibrin layer, because the fibrin had been digested by intrinsic protease. Two overlaid myocardial cell sheets exhibited synchronized spontaneous beating and captured artificial pacing. Optical mapping confirmed that the conduction of the action potential between two partially overlaid myocardial cell sheets was established, and the action potential propagated across the junction without any delay. Transplanted three-layered myocardial cell sheets exhibited strong spontaneous beating and showed well-differentiated striations and an increase in cell size. This simple method of cell sheet engineering may also be applicable for various other cell types. [ABSTRACT FROM AUTHOR]

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation. [ABSTRACT FROM AUTHOR]

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]

Construction of three-dimensional (3D) tissues with pre-isolated cells is a promising achievement for novel medicine and drug-discovery research. Our laboratory constructs 3D tissues with an innovative and unique method for layering multiple cell sheets. Cell sheets maintain a high-efficiently regenerating function, because of the higher cell density and higher transplantation efficiency, compared to other cell-delivery methods. Cell sheets have already been applied in clinical applications for regenerative medicine in treating patients with various diseases. Therefore, in our search to develop a more efficient treatment with cell sheets, we are constructing 3D tissues by layering cell sheets. Native animal tissues and organs have an abundance of capillaries to supply oxygen and nutrients, and to remove waste molecules. In our investigation of vascularized cardiac cell sheets, we have found that endothelial cells within cell sheets spontaneously form blood vessel networks as in vivo capillaries. To construct even thicker 3D tissues by layering multiple cell sheets, it is critical to have a medium or blood flow within the vascular networks of the cell sheets. Therefore, to perfuse medium or blood in the cell sheet vascular network to maintain the viability of all cells, we developed two types of vascular beds; (1) a femoral muscle-based vascular bed, and (2) a synthetic collagen gel-based vascular bed. Both vascular beds successfully provide the critical flow of culture medium, which allows 12-layer cell sheets to survive. Such bioreactor systems, when combined with cell sheet engineering techniques, have produced functional vascularized 3D tissues. Here we explain and discuss the various processes to obtain vascular networks by properly connecting cell sheets and the engineering of 3D tissues. [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]

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]

After the biotech medicine era, regenerative medicine is expected to be an advanced medicine that is capable of curing patients with difficult-to-treat diseases and physically impaired function. Our original scaffold-free cell sheet-based tissue engineering technology enables transplanted cells to be engrafted for a long time, while fully maintaining their viability. This technology has already been applied to various diseases in the clinical setting, including the cornea, esophagus, heart, periodontal ligament, and cartilage using autologous cells. Transplanted cell sheets not only replace the injured tissue and compensate for impaired function, but also deliver growth factors and cytokines in a spatiotemporal manner over a prolonged period, which leads to promotion of tissue repair. Moreover, the integration of stem cell biology and cell sheet technology with sufficient vascularization opens possibilities for fabrication of human three-dimensional vascularized dense and intact tissue grafts for regenerative medicine to parenchymal organs. [ABSTRACT FROM AUTHOR]

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: Cell transplantation is attracting considerable attention as the next-generation therapy for treatment of cardiovascular diseases. We have developed cell sheet engineering as a type of scaffold-less tissue engineering for application in myocardial tissue engineering and the repair of injured heart tissue by cell transplantation. Various types of cell sheet transplantation have improved cardiac function in animal models and clinical settings. Furthermore, cell-based tissue engineering with human induced pluripotent stem cell technology is about to create thick vascularized cardiac tissue for cardiac grafts and heart tissue models. In this review, we summarize the current cardiac cell therapies for treating heart failure with cell sheet technology and cell sheet-based tissue engineering. [Copyright &y& Elsevier]

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: Construction of bio-mimetic well-organized three-dimensional (3D) tissue with various cells in vitro is one of the ultimate goals of tissue engineering. In particular, fabrication of vasculature in 3D tissue is one of the most important tasks in tissue engineering, because a vascular network is indispensable for almost every tissue in our body. Here, we sandwiched stripe patterned endothelial cells by randomly cultured fibroblast sheets to control the formation of vasculature in the tissue. The endothelial cells left the original pattern and formed a random network between the two sheets, but, where fibroblasts were focally oriented, some endothelial cells changed their orientation to the same direction as the surrounding fibroblasts. Based on this phenomenon, we sandwiched stripe-patterned endothelial cells between parallel-oriented fibroblast sheets to construct a continuous pre-vascular structure. In the tissue, endothelial cells maintained the shape of their original pattern. On the other hand, stripe-patterned endothelial cells that were vertically sandwiched between oriented fibroblast sheets diverged from the original pattern at right angles, so that they were aligned with the surrounding fibroblasts. These data indicates that, 3D design with consideration of cell-to-cell interaction is critical to fabricate a specific 3D tissue structure. The 3D-designed tissue will become a powerful tool for the study of pharmacology and biology, the substitution of animal models and the fabrication of vascularized tissue grafts. [Copyright &y& Elsevier]

Abstract: Autologous transplantation of myoblast sheet has attracted attention as a new technique for curing myocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function via the facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheet after transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaid on human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVEC behavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape and migrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted in network formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued to migrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Image processing was performed to determine the variation in the extent of network formation at different HUVEC densities. It was found that the extent of formed network depended on the frequency of encounters among HUVECs in the middle of the sheet. The present system, which can evaluate network formation, is considered to be a promising in vitro angiogenesis model. [Copyright &y& Elsevier]