Temperature-responsive Culture Dishes Research Articles

Abstract 368: Ex vivo Fabrication of Heart Tissue Model With Perfusable Blood Vessels Using Cell Sheet

Introduction: Creation of 3-D tissue model similar to a biological tissue using tissue engineering method are expected as an alternative approach to 2-D culture or animal test in a research for developing a new medicine. We have already developed functional 3-D heart tissue with perfusable blood vessels in vitro using cell sheet based tissue engineering approach. Present study assessed the possibility as 3-D tissue model by performing drug evaluation in the engineered heart tissues. Methods: Firefly luciferase positive cardiac cells co-cultured with endothelial cells were harvested as cell sheets from temperature-responsive culture dishes. Triple-layer cell sheets were overlaid on the vascular bed with an artery and a vein in vitro. Layered cardiac cell sheets were maintained in custom made bioreactor system with perfusion medium. After neovascularization within the first triple-layer tissue, the second triple-layer cardiac cells sheets were overlaid on the first graft after cultivation for 3 days and then the whole construct was further perfused for an additional 3 days. After 6-day perfusion culture, isoproterenol or doxorubicin were administrated to the engineered heart tissues from artery side. The sympathetic and toxic effects were measured by in vitro bioluminescence imaging, electrical potential, and blood pressure. Results: In case of isoproterenol administration, bioluminescence imaging (BLI) demonstrated that adenosine triphosphate (ATP) of the heart tissue improved. In addition, electrograms demonstrated increasing beating rate and amplitude. Moreover, sphygmomanometer showed decreasing arterial pressure. In case of doxorubicin administration, BLI showed that the ATP of the heart tissue worsened after 24 hours. It is now possible to analyze the pharmacological effects such as metabolism and the blood pressure as well as action potential by using our vascularized heart tissue model. Conclusions: We believe that our 3-D heart tissue model offers a new possibilities for evaluation of drug efficacy.

The effect of tendon stem/progenitor cell (TSC) sheet on the early tendon healing in a rat Achilles tendon injury model

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 3mm of Achilles tendon defect. At 2 and 4weeks 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 4weeks 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 SignificanceTendon 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.

Harvesting epithelial keratinocyte sheets from temperature-responsive dishes preserves basement membrane proteins and improves cell survival in a skin defect model.

Cultured epithelial autograft (CEA) therapy has been used in clinical applications since the 1980s. However, there are some issues related to this treatment that still remain unsolved. Enzymatic treatment is typically used in the collection of epithelial keratinocyte sheets, but it tends to break the adhesion and basement membrane proteins. It is thought that the loss of proteins after enzymatic treatment is responsible for the poor survival of transplanted cell sheets. Our laboratory has developed a temperature-responsive culture dish that does not require enzymatic treatment to harvest the cells. In this study, we compare morphological and survival results from rat epithelial keratinocyte cell sheets harvested by temperature-reducing treatment (TT sheets) against cell sheets harvested by enzymatic (dispase) treatment (DT sheets). TT sheets preserve keratin structure in better conditions and express higher levels of collagen IV and laminin 5 than DT sheets. In order to evaluate cell sheet survival after transplantation, we created an in vivo transplant model. Keratinocyte sheets obtained from GFP-positive animals were transplanted into athymic rats. The survival rate 7days after transplantation of TT sheet was higher than that of DT sheets. Collagen IV and Laminin 5 expression was observed in the TT sheet transplantation group. These results indicate that the remaining basement membrane proteins are important for initial attachment and cell survival. We believe that the cell sheet harvesting method using temperature-responsive culture dishes provides superior cell survival and can solve one of the roadblocks in CEA therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Efficacy of Multilayered Hepatocyte Sheet Transplantation for Radiation-Induced Liver Damage and Partial Hepatectomy in a Rat Model.

Although cell sheet technology has recently been developed for use in both animal experiments and in the clinical setting, it remains unclear whether transplanted hepatocyte sheets improve the liver function in vivo. Radiation-induced liver damage (RILD) combined with partial hepatectomy (PH) has been reported to suppress the proliferation of host hepatocytes and induce critical liver failure. The aim of this study was to improve the liver function in the above-mentioned diseased rat model (RILD + PH) using multilayered hepatocyte sheet transplantation. In this study, we used Fischer rats as a donor for primary hepatocytes and dermal fibroblast isolation. Cocultured multilayered hepatocyte sheets were generated by disseminating hepatocytes onto fibroblasts cultured beforehand on temperature-responsive culture dishes. Four cell sheets were transplanted into the recipient rats subcutaneously. Prior to transplantation, RILD (50 Gy) with 2/3PH was induced in the recipients. The same model was applied in the control group without transplantation. The serum was collected each week. The rats in both groups were sacrificed at 2 months after transplantation for the histological analysis. Consequently, the serum albumin concentrations were significantly higher in the transplant group than in the control group (54.3 ± 9.6 vs. 32.7 ± 5.7 mg/ml; p < 0.01) after 2 months and comparable to the serum albumin levels in the normal rats (58.1 ± 6.4 mg/ml). In addition, treatment with the transplanted sheets significantly improved the survival rate (57% vs. 22%, p < 0.05), and the hepatocyte sheets showed the storage of albumin, glycogen, and bile canaliculus structures. Some hepatocytes and fibroblasts were positive for Ki-67, and vascularization was observed around the cell sheets. Transplanted multilayered hepatocyte sheets can survive with additional proliferative activity, thereby maintaining the liver function in vivo for at least 2 months, providing metabolic support for rats with RILD.

Pericyte-based collagen scaffold for vascular tissue engineering

Event Abstract Back to Event Pericyte-based collagen scaffold for vascular tissue engineering Betül Çelebi-Saltik1, 2 and Beyza Gökçinar-Yagci1, 2 1 Hacettepe University, Department of Stem Cell Sciences, Graduate School of Health Sciences, Türkiye 2 Hacettepe University, Center for Stem Cell Research and Development, Türkiye Pericytes are a perivascular cell population embedded on the capillary wall in a shared basement membrane with the endothelium. During vasculogenesis and angiogenesis, endothelial cells secrete and/or present cell bound molecular cues that stimulate pericytes to proliferate, migrate and attach to nascent capillary tubes. It has been previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches. In this study our aim is to isolate human umbilical cord vein pericytes then differentiate into smooth muscle cells and fibroblasts then co-culture these cells on 3D collagen scaffold that will be used for generating tissue engineering vascular grafts. Pericytes were enzymatically isolated from human umbilical cord vein and were purified by using MACs cell separation with CD146 microbeads. They were cultured in fibroblast growth medium and smooth muscle growth medium for 21 days on poly(N-isopropylacrylamide) to obtain cell sheets. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibroblast markers; Tenascin-C and Collagen type I, smooth muscle cell markers; Caldesmon, Calponin and Alpha smooth muscle actin). Collagen gel was prepared with human collagen type I (3mg/mL) + 10X DMEM (adjustment pH of mixture to 7.2–7.6 using sterile 0.1 M NaOH). Collagen gel was covered by cell sheets (sandwich system) and vascular graft was incubated in 37oC for 7 days (Figure 1). The results indicated that perivascular cells could differentiate into fibroblast and smooth muscle and formed vascular graft with collagen gel. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support. The Scientific and Technological Research Council of Turkey (Project Number: 113S815) Keywords: Tissue Engineering, cell, 3D scaffold, tissue niche Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in mesenchymal and hematopoietic stem cell biology Citation: Çelebi-Saltik B and Gökçinar-Yagci B (2016). Pericyte-based collagen scaffold for vascular tissue engineering. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02836 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Scholar Betül Çelebi-Saltik Beyza Gökçinar-Yagci PubMed Betül Çelebi-Saltik Beyza Gökçinar-Yagci Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 15038: Transplantation of Genomic Integration-free Human iPS Cell-derived Cardiac Tissue Sheets Into Porcine Infarct Hearts Produced No Lethal Arrhythmia: A Preclinical Study for a Safer Human iPS Cell Therapy

Background: Any preclinical study of a human iPS cell(hiPSC)-based cardiac regenerative therapy must confirm that the therapy in question does not produce lethal arrhythmias. In a recent study, we succeeded in long-term (&lt;9 months) tumor-free transplantation of genomic integration-free hiPSC-derived cardiac tissue sheets (CTS) into a rat myocardial infarction (MI) model. In the present study, we attempted to prove that the integration-free CTS do not produce life-threatening arrhythmias after transplantation into a porcine MI model. We chose porcine hearts because they have a heart rate similar to that of the human heart. METHODS &amp; RESULTS: We induced a mixture of cardiomyocytes (CMs) and vascular cells (endothelial cells [ECs] and vascular mural cells [MCs]) from integration-free hiPSCs and generated CTSs using temperature-responsive culture dishes (UpCell, CellSeed, Tokyo, Japan). Self-pulsating CTSs were approximately 3.5cm diameter with approximately 7x106 of cells containing cTnT+ CMs (41±11.9%), VE-cadherin+ ECs (7.0±2.6%) and PDGFRβ+ MCs (32.7±25.0%). In the transplantation group (Tx group: n=4), we induced MI in microminipigs (13-21 months old) by two-stage ligation of the left anterior descending coronary artery, and transplanted CTSs at 2 weeks after MI induction (4 sheets per recipient heart) under immunosuppression. Neither arrhythmia-related sudden death nor tumor formation was observed in any transplanted animals during the observation period (8 weeks). In the Tx group, echocardiogram showed a significant improvement in systolic function of the left ventricle (fractional shortening: 36.7%±2.3 vs 25.2%±2.2, p&lt;0.05, sham-operated animals [Sham]: n=3 ). Holter electrocardiogram showed no episode of ventricular tachycardia in the Tx group during the observation period, whereas 67% (2/3) of Sham exhibited ventricular tachycardia. In the Tx group, beat to beat variability of ventricular repolarization, a surrogate marker of ventricular tachycardia, was lower than that in the Sham (1.95±0.6 ms vs 3.65±0.47 ms). CONCLUSIONS: This preclinical study using genomic integration-free CTS indicated a promising feature of hiPSC-based cardiac regenerative therapy for clinical use.

Rapid fabrication system for three-dimensional tissues using cell sheet engineering and centrifugation.

Three-dimensional (3D) tissues can be reconstructed by cell sheet technology, and various clinical researches using these constructed tissues have already been initiated to regenerate damaged tissues. While 3D tissues can be easily fabricated by layering cell sheets, the attachment period for cell adhesion between a cell sheet and a culture dish, or double-layered cell sheets normally takes 20-30 min. This study proposed a more rapid fabrication system for bioengineered tissue using cell sheet technology and centrifugation. A C2C12 mouse myoblast sheet harvested from a temperature-responsive culture dish will attach tightly to a culture dish or another cell sheet at 37°C after a 20 min-incubation. However, the same cell sheet centrifuged (12-34 × g) for 3 min also attached tightly to a dish or another cell sheet at 37°C after only a 3 min-incubation. The manipulation time was reduced by approximately two-thirds by centrifugation. The rapid attachments were also cross-sectionally confirmed by optical coherence tomography. These rapidly constructed cell sheet-tissues using centrifugation showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor, like those prepared by the conventional method; indicating complete cell sheet-attachment without any cell damage. This new system will be a powerful tool in the fields of cell sheet-based tissue engineering and regenerative medicine, and accelerate the use of cell sheets in clinical applications.

Rapid production of engineered human primary hepatocyte/fibroblast sheets.

This data article contains data related to the research article entitled “Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice,” published in Biomaterials[1]. Engineered hepatocyte/fibroblast sheets (EHFSs) are used for construction of vascularized subcutaneous liver tissue without a pre-transplant vascularization procedures. Here, we described a rapid production technique of EHFSs by controlling fibroblast density and coating fetal bovine serum (FBS) onto temperature-responsive culture dishes (TRCDs). The human fibroblast monolayer formed on FBS-coated TRCDs within 1 h when seeded at a high density (at least 1.56×105 cells/cm2). The most rapid EHFS production was achieved soon after the adhesion of human primary hepatocytes onto the fibroblast layer.

Time Course of Cell Sheet Adhesion to Porcine Heart Tissue after Transplantation.

Multilayered cell sheets have been produced from bone marrow-derived mesenchymal stem cells (MSCs) for investigating their adhesion properties onto native porcine heart tissue. Once MSCs reached confluence after a 7-day culture on a temperature-responsive culture dish, a MSCs monolayer spontaneously detached itself from the dish, when the culture temperature was reduced from 37 to 20°C. The basal extracellular matrix (ECM) proteins of the single cell sheet are preserved, because this technique requires no proteolytic enzymes for harvesting cell sheet, which become a basic building block for assembling a multilayer cell sheet. The thickness of multilayered cell sheets made from three MSC sheets was found to be approximately 60 μm. For investigating the adhesion properties of the basal and apical sides, the multilayered cell sheets were transplanted onto the surface of the heart’s left ventricle. Multilayered cell sheets were histological investigated at 15, 30, 45 and 60 minutes after transplantation by hematoxylin eosin (HE) and azan dyes to determine required time for the adhesion of the multilayered sheets following cell-sheet transplantation. The results showed that only the basal side of multilayered cell sheets significantly enhanced the sheets adhesion onto the surface of heart 30 minutes after transplantation. This study concluded that (1) cell sheets had to be transplanted with its basal side onto the surface of heart tissue and (2) at least 30 minutes were necessary for obtaining the histological adhesion of the sheets to the heart tissue. This study provided clinical evidence and parameters for the successful application of MSC sheets to the myocardium and allowed cell sheet technology to be adapted clinical cell-therapy for myocardial diseases.

A Tissue-Engineered Chondrocyte Cell Sheet Induces Extracellular Matrix Modification to Enhance Ventricular Biomechanics and Attenuate Myocardial Stiffness in Ischemic Cardiomyopathy.

There exists a substantial body of work describing cardiac support devices to mechanically support the left ventricle (LV); however, these devices lack biological effects. To remedy this, we implemented a cell sheet engineering approach utilizing chondrocytes, which in their natural environment produce a relatively elastic extracellular matrix (ECM) for a cushioning effect. Therefore, we hypothesized that a chondrocyte cell sheet applied to infarcted and borderzone myocardium will biologically enhance the ventricular ECM and increase elasticity to augment cardiac function in a model of ischemic cardiomyopathy (ICM). Primary articular cartilage chondrocytes of Wistar rats were isolated and cultured on temperature-responsive culture dishes to generate cell sheets. A rodent ICM model was created by ligating the left anterior descending coronary artery. Rats were divided into two groups: cell sheet transplantation (1.0 × 10(7) cells/dish) and no treatment. The cell sheet was placed onto the surface of the heart covering the infarct and borderzone areas. At 4 weeks following treatment, the decreased fibrotic extension and increased elastic microfiber networks in the infarct and borderzone areas correlated with this technology's potential to stimulate ECM formation. The enhanced ventricular elasticity was further confirmed by the axial stretch test, which revealed that the cell sheet tended to attenuate tensile modulus, a parameter of stiffness. This translated to increased wall thickness in the infarct area, decreased LV volume, wall stress, mass, and improvement of LV function. Thus, the chondrocyte cell sheet strengthens the ventricular biomechanical properties by inducing the formation of elastic microfiber networks in ICM, resulting in attenuated myocardial stiffness and improved myocardial function.

A Method for Performing Islet Transplantation Using Tissue-Engineered Sheets of Islets and Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are known to have a protective effect on islet cells. Cell sheets developed using tissue engineering help maintain the function of the cells themselves. This study describes a tissue engineering approach using islets with MSC sheets to improve the therapeutic effect of islet transplantation. MSCs were obtained from Fischer 344 rats and engineered into cell sheets using temperature-responsive culture dishes. The islets obtained from Fischer 344 rats were seeded onto MSC sheets, and the islets with MSC sheets were harvested by low-temperature treatment after coculture. The functional activity of the islets with MSC sheets was confirmed by a histological examination, insulin secretion assay, and quantification of the levels of cytokines. The therapeutic effects of the islets with MSC sheets were investigated by transplanting the sheets at subcutaneous sites in severe combined immunodeficiency (SCID) mice with streptozotocin-induced diabetes. Improvement of islet function and viability was shown in situ on the MSC sheet, and the histological examination showed that the MSC sheet maintained adhesion factor on the surface. In the recipient mice, normoglycemia was maintained for at least 84 days after transplantation, and neovascularization was observed. These results demonstrated that islet transplantation in a subcutaneous site would be possible by using the MSC sheet as a scaffold for islets.

Xenotransplantation of Bone Marrow-Derived Human Mesenchymal Stem Cell Sheets Attenuates Left Ventricular Remodeling in a Porcine Ischemic Cardiomyopathy Model.

Bone marrow-derived autologous human mesenchymal stem cells (MSCs) are one of the most promising cell sources for cell therapy to treat heart failure. The cell sheet technique has allowed transplantation of a large number of cells and enhanced the efficacy of cell therapy. We hypothesized that the transplantation of MSC sheets may be a feasible, safe, and effective treatment for ischemic cardiomyopathy (ICM). Human MSCs acquired from bone marrow were positive for CD73, CD90, and CD105 and negative for CD11b and CD45 by flow cytometry. Ten MSC sheets were created from a total cell number of 1×10(8) MSCs using temperature-responsive culture dishes. These were successfully transplanted over the infarct myocardium of porcine ICM models induced by placing an ameroid constrictor on the left anterior descending coronary artery without any procedural-related complications (MSC group=6: sheet transplantation; sham group=6, oral intake of tacrolimus in both groups). Premature ventricular contractions were rarely detected by Holter electrocardiogram (ECG) in the MSC group in the first week after transplantation. On echocardiography, the cardiac performance of the MSC group was significantly better than that of the sham group at 8 weeks after transplantation. On histological examination 8 weeks after transplantation, left ventricular (LV) remodeling was significantly attenuated compared with the sham group (cardiomyocyte size and interstitial fibrosis were measured). Immunohistochemistry of the von Willebrand factor showed that the vascular density in the infarct border area was significantly greater in the MSC group than the sham group. Expression of angiogenesis-related factors in the infarct border area of the MSC group was significantly greater than that of the sham group, as measured by real-time polymerase chain reaction. Bone marrow-derived MSC sheets improved cardiac function and attenuated LV remodeling in ICM without major complications, indicating that this strategy would be applicable in clinical settings.

633. Reconstruction of Vascularized Subcutaneous Liver Tissue for Minimally Invasive Cell Therapy

Subcutaneous liver tissue construction is an attractive and minimally invasive approach used to overcome the chronic shortage of donor livers for the curative treatment of hepatic failure. However, graft failure occurs frequently due to lack of blood vessels. In this study, we describe a subcutaneous human liver reconstruction approach allowing for rapidly vascularized grafts by transplanting engineered hepatocyte/fibroblast sheets (EHFSs).Human primary hepatocytes were isolated and purified from surgically resected human liver tissues by the collagenase perfusion method. EHFSs were fabricated using temperature-responsive culture dishes (TRCDs) (UpCell; CellSeed, Tokyo). The hepatocytes were plated onto a confluent layer of normal human dermal fibroblasts (TIG-118 cells). After 4 days in culture, EHFSs were harvested and transplanted subcutaneously into immunodeficient mice. The expression levels of angiogenic factors in vitro, liver-specific functions and structures in vivo, and therapeutic potential of serious liver diseases were evaluated.EHFSs exhibited significantly higher synthesis rates of VEGF, TGF-b1, and HGF than the hepatocyte-only sheets (HSs) from 1 to 3 days in culture. This result indicate the possibility of using a one-step subcutaneous hepatocyte transplantation procedure without a pre-transplant vascularization step such as implanting a FGF-releasing device. The transplanted EHFSs were laminated and formed a vascularized subcutaneous human liver tissues (VSLTs). The VSLTs had liver-specific features such as glycogen stores and albumin, A1AT, and coagulation factor IX syntheses. The aggregated hepatocytes formed several linear structures. Furthermore, subacute hepatic failure model mice (TK-NOG mice) with VSLT were alive at least 7 weeks after liver damage.The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel minimally invasive cell therapies for liver diseases.

175. Double-Layered Cell Sheets Transplantation That Achieves Durable Factor VIII Delivery in the Mouse Model of Hemophilia A

Hemophilia A is an inherited bleeding disorder caused by a deficiency of the procoagulant cofactor VIII (FVIII). Currently, the patients with hemophilia A are being treated through repeated intravenous injection of FVIII concentrates. Since FVIII is a secreted protein and the tight regulation of its expression is not necessary, ex vivo gene therapy strategies for hemophilia A might be good for the therapeutic alternative. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et.al. PLoS One 2013 8(12):e83280). To enhance the viability of the implanted BOECs which boost the FVIII expression, double-layered cell sheets transplantation was performed by placing the recovered BOECs sheet onto the fibroblast sheet directly. FVIII transduced BOECs and non-transduced fibroblasts were cultured on temperature-responsive culture dish independently. Both cells were recovered as a contiguous cell sheet. Subcutaneous co-transplantation studies of BOEC and fibroblast sheet indicated that all hemophilia A mice treated with cyclophosphamide expressed between 40-50 mU/mL levels of FVIII without developing anti-FVIII inhibitory antibodies over 200 days. In contrast, the hemophilia A mice without any prior immuno-suppressive treatment developed neutralizing anti-FVIII antibodies between 5-10 Bethesda units. These therapeutic FVIII levels were two times higher than the group of hemophilia A mice that were transplanted BOECs sheet alone. Histological examination in the part of the mice revealed that the combination of BOEC and fibroblast sheet was structured as flat clusters without any cells infiltration. In conclusion, tissue engineering approach using double-layered cell sheets are viable for persistent tissue survival and providing therapeutic values. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A and merits further assessment.

Engineered bone marrow-derived cell sheets restore structure and function of radiation-injured rat urinary bladders.

Previously, we reported that implantation of isolated single bone marrow-derived cells into radiation-injured urinary bladders could restore structure and function. However, injections of isolated single cells had some limitations. Thus, in this study, we produced bone marrow-derived cell sheets in temperature-responsive culture dishes that release the monolayer sheets intact. We then determined whether the produced cell sheets could restore function to irradiated urinary bladders. Twenty female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2 gray once a week for 5 weeks. Bone marrow cells harvested from two male 17-week-old green fluorescence protein-transfected SD rats were placed in primary culture for 7 days. Bone marrow cell-derived outgrowths were harvested by enzymatic digestion and transferred into the atelocollagen-coated temperature-responsive culture dishes for 2 days. To harvest the secondarily cultured cells as monolayer sheets, a support membrane was put in each culture dish, and then the temperature was reduced to 20°C. Each released cell sheet was then patched onto the irradiated anterior bladder wall (n=10). As controls, cell-free sheets were similarly patched (n=10). After 4 weeks, transplanted cells were detected on the bladder walls. The cell sheet-transplanted bladders had smooth muscle layers and acetylcholinesterase-positive nerve fibers in proportions that were significantly larger than those of the control bladders. In addition, the cell sheet-transplanted bladders had reduced prolyl 4-hydroxylase beta (P4HB)-positive regions of collagen synthesis and apoptosis within the smooth muscle layers. In cystometric investigations, threshold pressures, voiding interval, micturition volume, and bladder capacity in the cell sheet-transplantation group were significantly higher than those in the control group. Residual volume of the cell sheet-transplantation group was significantly lower compared with the control. There were 24 growth factor mRNAs in the cell sheet-transplanted urinary bladders that were expressed greater than or equal to two-fold over the controls. In conclusion, cell sheet engineering has great potential to restore damaged urinary bladders.

Osteogenic cell sheets reinforced with photofunctionalized micro-thin titanium.

Cell sheet technology has been used to deliver cells in single-sheet form with an intact extracellular matrix for soft tissue repair and regeneration. Here, we hypothesized that titanium-reinforced cell sheets could be constructed for bone tissue engineering and regeneration. Fifty-µm-thick titanium plates containing apertures were prepared and roughened by acid etching, some of which were photofunctionalized with 12 min of UV light treatment. Cell sheets were prepared by culturing rat calvarial periosteum-derived cells on temperature-responsive culture dishes and attached to titanium plates. Titanium-reinforced osteogenic cell sheet construction was conditional on various technical and material factors: cell sheets needed to be double-sided and sandwich the titanium plate, and the titanium plates needed to be micro thin and contain apertures to allow close apposition of the two cell sheets. Critically, titanium plates needed to be UV-photofunctionalized to ensure adherence and retention of cell sheets. Single-sided cell sheets or double-sided cell sheets on as-made titanium contracted and deformed within 4 days of incubation. Titanium-reinforced cell sheets on photofunctionalized titanium were structurally stable at least up to 14 days, developed the expected osteogenic phenotypes (ALP production and mineralization), and maintained structural integrity without functional degradation. Successful construction of titanium-reinforced osteogenic cell sheets was associated with increased cell attachment, retention, and expression of vinculin, an adhesion protein by photofunctionalization. This study identified the technical and material requirements for constructing titanium-reinforced osteogenic cell sheets. Future in vivo studies are warranted to test these titanium-reinforced cell sheets as stably transplantable, mechanically durable, and shape controllable osteogenic devices.

Adipose-derived stem cell sheet transplantation therapy in a porcine model of chronic heart failure

Adipose-derived stem cells (ASCs) are a promising resource for cell transplantation therapy for damaged heart tissue. Cell death in the graft early after transplantation represents the main cause of unsatisfactory therapeutic efficacy, but tissue-engineered cell sheets grown in temperature-responsive cell culture dishes may enable improved engraftment of transplanted cells. We investigated the therapeutic potential of this method in chronic myocardial ischemia in swine. We created a porcine model of chronic heart failure by implanting an ameroid constrictor around the main trunk of the left anterior descending artery, just distal to the circumflex branch. Simultaneously, ASCs were obtained from a piece of subcutaneous adipose tissue and expanded to form ASC sheets using temperature-responsive dishes. Four weeks after ameroid constrictor placement, triple-layered ASC sheets were transplanted onto the area of the ischemic myocardium (sheet group, n=7). Controls (n=7) received no sheet. Just before and 4weeks after transplantation, left ventriculography (LVG) and coronary angiography (CAG) were performed. LVG revealed a significant improvement in the left ventricular ejection fraction of the sheet group compared with controls (47.6±2.9% vs 41.4±2.8%, P<0.05). Furthermore, development of collateral vessels was only detected in the sheet group with right CAG. Histologic analysis demonstrated that engrafted ASC sheets grew to form a thickened layer that included newly formed vessels. ASC sheet transplantation therapy is an intriguing therapeutic method for ischemic heart failure.

The effect of transplantation of nasal mucosal epithelial cell sheets after middle ear surgery in a rabbit model

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 17227: Transplantation of Cardiac Cell Sheet Including Human Ips Cell-derived Cardiomyocytes and Vascular Cells to Infarcted Porcine Heart Restores Impaired Left Ventricular Global Function and Dyssynchrony

BACKGROUNDS: Previously we reported beneficial effects of transplantation of all mouse ES cell-derived cardiac cell sheets composed of CMs and vascular cells (endothelial cells [ECs]/ vascular mural cells [MCs]) in a rat myocardial infarction (MI) model and importance of co-existence of vascular cells in the cell sheets (Stem Cells, 2012). Here we report a transplantation of human induced pluripotent stem cell (hiPSC)-derived cardiac cells to a porcine MI model. METHODS &amp; RESULTS: We induced a mixture of CMs and vascular cells from hiPSCs with a 2-D serum-free method (modified from Uosaki, PLoS One, 2011). We generated cell sheets using 10cm-sized temperature-responsive culture dishes (UpCell; CellSeed, Tokyo, Japan). Self-pulsating cardiac cell sheets were approximately 3.5cm in diameter with 6.8х106±0.8 (n=5) of cells containing cTnT+-CMs (19.4±5.9%), VE-cadherin+-ECs (3.8±4.4%) and PDGFRβ+-MCs (67.2±7.8%). We induced MI in micromini-pigs (12-45 month old) by ameroid constriction of coronary artery and transplanted cell sheets (Tx) 2 weeks after MI induction (4 sheets / recipient) under immunosuppression. In Tx group, echocardiogram showed a significant improvement of systolic function of left ventricle (fractional shortening: 22.6±5.0 vs 39.7±8.7%, p&lt;0.01, n=5). Ejection fraction evaluated by left ventriculogram improved significantly in Tx group (25.3±6.2% vs 39.8±4.2%, p&lt;0.01, n=5). Speckle tracking echocardiogram showed significant increase of attenuated circumference strain in infarcted and border regions leading to restored dyssynchrony (anterior: 10.8±4.1 vs 20.6±3.5%, p&lt;0.01, antero-lateral: 13±6.5% vs 24.8±7.6%, p&lt;0.05, n=5, 2 weeks after Tx). Capillary density in the border region significantly elevated indicating angiogenic effect of the sheet transplantation (75.9±42.6/mm2 vs 137.4±44.8/mm2, p&lt;0.001, n=5). CONCLUTION: HiPSC-derived cardiac cell sheets potentially ameliorate cardiac dysfunction of human-size infarct heart.

Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing

Cell-sheet-engineering based regenerative medicine is successfully applied to clinical studies, though cell sheets contain uniformly distributed cells. For the further application to complex tissues/organs, cell sheets with a multi-cellular pattern were highly demanded. Micro-contact printing is a quite useful technique for patterning proteins contained in extracellular matrix (ECM). Because ECM is a kind of cellular adherent molecules, ECM-patterned cell culture surface is capable of aligning cells on the pattern of ECM. However, a manual printing is difficult, because a stamp made from polydimethylsiloxane (PDMS) is easily deformed, and a printed pattern is also crushed. This study focused on the deformability of PDMS stamp and discussed an appropriate stamping force in micro-contact printing. Considering in availability in a medical or biological laboratory, a method for assessing the stamp deformability was developed by using stiffness measurement with a general microscope. An automated stamping system composed of a load cell and an automated actuator was prepared and allowed to improve the quality of stamped pattern by controlling an appropriate stamping force of 0.1 N. Using the system and the control of appropriate stamping force, the pattern of 8-mm-diameter 80-μm-stripe fibronectin was fabricated on the surface of temperature-responsive cell culture dish. After cell-seeding and cell culture, a co-culture system with the micro-pattern of both fibroblasts and endothelial cells was completed. Furthermore, by reducing temperature to 20 °C, the co-cultured cell sheet with the micro-pattern was successfully harvested. As a result, the method would not only provide a high-quality ECM pattern but also a breakthrough technique to fabricate multi-cellular-patterned cell sheets for the next generation of regenerative medicine and tissue engineering.