Temperature-responsive Culture Dishes Research Articles

Assessment of human bioengineered cardiac tissue function in hypoxic and re-oxygenized environments to understand functional recovery in heart failure.

IntroductionMyocardial recovery is one of the targets for heart failure treatment. A non-negligible number of heart failure with reduced ejection fraction (EF) patients experience myocardial recovery through treatment. Although myocardial hypoxia has been reported to contribute to the progression of heart failure even in non-ischemic cardiomyopathy, the relationship between contractile recovery and re-oxygenation and its underlying mechanisms remain unclear. The present study investigated the effects of hypoxia/re-oxygenation on bioengineered cardiac cell sheets-tissue function and the underlying mechanisms.MethodsBioengineered cardiac cell sheets-tissue was fabricated with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) using temperature-responsive culture dishes. Cardiac tissue functions in the following conditions were evaluated with a contractile force measurement system: continuous normoxia (20% O2) for 12 days; hypoxia (1% O2) for 4 days followed by normoxia (20% O2) for 8 days; or continuous hypoxia (1% O2) for 8 days. Cell number, sarcomere structure, ATP levels, mRNA expressions and Ca2+ transients of hiPSC-CM in those conditions were also assessed.ResultsHypoxia (4 days) elicited progressive decreases in contractile force, maximum contraction velocity, maximum relaxation velocity, Ca2+ transient amplitude and ATP level, but sarcomere structure and cell number were not affected. Re-oxygenation (8 days) after hypoxia (4 days) was associated with progressive increases in contractile force, maximum contraction velocity and relaxation time to the similar extent levels of continuous normoxia group, while maximum relaxation velocity was still significantly low even after re-oxygenation. Ca2+ transient magnitude, cell number, sarcomere structure and ATP level after re-oxygenation were similar to those in the continuous normoxia group. Hypoxia/re-oxygenation up-regulated mRNA expression of PLN.ConclusionsHypoxia and re-oxygenation condition directly affected human bioengineered cardiac tissue function. Further understanding the molecular mechanisms of functional recovery of cardiac tissue after re-oxygenation might provide us the new insight on heart failure with recovered ejection fraction and preserved ejection fraction.

Functional changes of cocultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: Differences in transplantation sites.

AimThe formation of a secondary liver is expected in ectopic transplants in liver therapy. It is reported that the transplantation of hepatocyte sheets constitutes one of the techniques used to form a secondary liver. Accordingly, we established a subcutaneous transplant for hepatocyte/fibroblast sheets in previous studies. In this development study with hepatocyte/fibroblast sheets, we evaluated the differences in transplantation sites to promote the maturation of transplanted tissue in a liver injury model.MethodsA cocultured hepatocyte sheet of fibroblasts (TIG-118 cells) and human hepatocytes (PXB cells) was prepared on a temperature-responsive culture dish. The prepared cocultured hepatocyte sheet was either transplanted subcutaneously or on the liver surface of a persistent liver injury model (cDNA-uPA/SCID mouse: uPA mouse), and was evaluated by the human albumin concentration in mouse blood. As a control group, hepatocyte cell sheets were used that were transplanted to both areas and compared.ResultsAlthough the cocultured hepatocyte sheet led to functional improvements in the early stages of culture in subcutaneous transplantation, these did not last in the long-term after transplantation. Although coculture effects were not observed in the liver surface transplantation case, long-term functional expressions in mono- and cocultured sheets in the case of liver surface transplantation were exhibited compared with subcutaneous administration.ConclusionThese results suggest that sustained stimulation of liver regenerationvaries depending on the transplant site and is largely involved in the maturation of hepatocyte tissue.

Improvement of the therapeutic capacity of insulin-producing cells trans-differentiated from human liver cells using engineered cell sheet

BackgroundAlthough pancreatic islet transplantation therapy is ideal for diabetes patients, several hurdles have prevented it from becoming a standard treatment, including donor shortage and low engraftment efficacy. In this study, we prepared insulin-producing cells trans-differentiated from adult human liver cells as a new islet source. Also, cell sheet formation could improve differentiation efficiency and graft survival.MethodsLiver cells were expanded in vitro and trans-differentiated to IPCs using adenovirus vectors carrying human genes for PDX1, NEUROD1, and MAFA. IPCs were seeded on temperature-responsive culture dishes to form cell sheets. Differentiation efficiency was confirmed by ß cell-specific gene expression, insulin production, and immunohistochemistry. IPC suspension was injected by portal vein (PV), and IPC sheet was transplanted on the liver surface of the diabetic nude mouse. The therapeutic effect of IPC sheet was evaluated by comparing blood glucose control, weight gain, histological evaluation, and hepatotoxicity with IPC injection group. Also, cell biodistribution was assessed by in vivo/ex vivo fluorescence image tagging.ResultsInsulin gene expression and protein production were significantly increased on IPC sheets compared with those in IPCs cultured on conventional culture dishes. Transplanted IPC sheets displayed significantly higher engraftment efficiency and fewer transplanted cells in other organs than injected IPCs, and also lower liver toxicity, improved blood glucose levels, and weight gain. Immunohistochemical analyses of liver tissue revealed positive staining for PDX1 and insulin at 1, 2, and 4 weeks after IPC transplantation.ConclusionsIn conclusion, cell sheet formation enhanced the differentiation function and maturation of IPCs in vitro. Additionally, parameters for clinical application such as distribution, therapeutic efficacy, and toxicity were favorable. The cell sheet technique may be used with IPCs derived from various cell sources in clinical applications.

Bio-artificial pleura using autologous dermal fibroblast sheets to mitigate air leaks during thoracoscopic lung resection

Lung air leaks (LALs) due to visceral pleura injury during surgery are a difficult-to-avoid complication in thoracic surgery (TS). Reliable LAL closure is an important patient management issue after TS. We demonstrated both safeties of transplantation of a cultured human autologous dermal fibroblast sheet (DFS) to LALs. From May 2016 to March 2018, five patients who underwent thoracoscopic lung resection met all the inclusion criteria. Skin biopsies were acquired from each patient to source autologous dermal cells for DFS fabrication. During the primary culture, fibroblasts migrated from the dermal tissue pieces and proliferated to form cell monolayers. These fibroblasts were subcultured to confluence. Transplantable DFSs were fabricated from these subcultured fibroblasts that were trypsinized and seeded onto temperature-responsive culture dishes. After 10 days of fabrication culture, intact patient-specific DFS were harvested. DFSs were analyzed for fibroblast cell content and tissue contaminants prior to application. For closing intraoperative LAL, mean number of transplanted autologous DFS per patient was 6 ± 2 sheets. Mean chest drainage duration was 5.0 ± 4.8 days. The two patients with major LAL had a drainage duration of more than 7 days. All patients currently have no LAL recurrence after discharge. DFSs effectively maintain LAL closure via remodeling of the deposited extracellular matrix. The use of autologous DFSs to permanently close air leaks using a patient-derived source is expected to reduce surgical complications during high-risk lung resections.

Development of a nasal mucosa-removal model for evaluating cell therapy.

IntroductionEndoscopic sinus surgery is an effective surgical procedure for treating chronic sinusitis; however, extensive exposure of the bone in the nasal cavity can result in permanent disability postoperatively. Particularly, closure of the sinus drainage pathway due to bone hyperplasia associated with bone exposure can trigger the recurrence of sinusitis. It is essential to regenerate the nasal mucosa after surgery to avoid bone hyperplasia. Regenerative medicine, including cell therapy, could be one of the leading options for nasal mucosa regeneration. To date, there is a lack of effective models for evaluating treatments for prevention of bone hyperplasia that occurs after sinus surgery. The purpose of this study was to develop a model of nasal mucosal removal to evaluate cellular therapies.MethodsThe model was created in rabbits, a species with a wide nasal structure, and was generated by approaching the maxillary sinus from the nasal bone side and solely removing the maxillary sinus mucosa without destroying the structures in the nasal cavity. Adipose-derived mesenchymal stromal cell sheets prepared in temperature-responsive cell culture dishes were examined for the effect of transplantation in the animal model. Intranasal evaluation was assessed by micro-computed tomography and tissue staining.ResultsSignificant bone hyperplasia in the maxillary sinus occurred on the side of mucosal removal, and no bone hyperplasia occurred in the control sham side in the same rabbits on postoperative day 28. Bone hyperplasia was observed over a short time period, with the presence of bone hyperplasia in the maxillary sinus on day 14 and calcification of the bone on day 28. The adipose-derived mesenchymal stromal cell (ADSC) sheet was transplantable in a nasal mucosa-removal model. No significant differences in bone hyperplasia were found between the transplantation side and the sham side in terms of the effect of transplantation of the ADSC sheet; however, bone hyperplasia tended to be suppressed on the transplantation side.ConclusionsThis animal model is simple, highly reproducible, and does not require special equipment or drugs. In addition, this model can be used for various therapeutic interventions, including cell therapy. The presence or absence of the nasal mucosa affects bone remodeling, which highlights the importance of regeneration of the nasal mucosa. In the nasal mucosal regeneration therapy, the ADSC sheet had an inhibitory effect on bone hyperplasia. The nasal mucosa-removal model allows observation of conditions associated with nasal mucosa removal and evaluation of the effectiveness of cell therapy.

Evaluation of human keratinocyte sheets transplanted onto porcine excised esophagus after submucosal dissection in an exvivo model.

BackgroundThe utility of endoscopic transplantation of epithelial cell sheets to ulcer sites after endoscopic submucosal dissection (ESD) has been shown to prevent scar stenosis after ESD of early esophageal cancer. Previously, our group reported use of an endoscopic transplantation device fabricated with a 3-dimensional printer. Cell sheets are transplanted to the esophageal wound site with the following procedure: first, a cell sheet harvested from temperature-responsive culture dishes is placed on the device's deflated balloon surface and transported to the wound site with endoscopic forceps; second, by applying pressure from inflating the balloon locally at the wound site, the cell sheet is successfully transferred and adhered to the wound tissue; third, the balloon is deflated, and the device is removed. By repeating the procedure, several cell sheets can be safely transplanted to a wider ESD area. Nonetheless, possible damage to cell sheets using this procedure has not yet been assessed.ObjectiveEffects of endoscopic transplantation balloon inflation on cell viability and damage of normal human epidermal keratinocyte sheets resident on the device's balloon surface were evaluated by histology after sheet placement onto lumenal surfaces in the ex vivo porcine submucosal dissection esophagus model. Endoscopic transplantation of these same cell sheets with conventional methods using a polyvinylidene fluoride (PVDF) cell sheet support membrane, balloon device transfer, and also using a novel modified balloon transfer procedure was also examined. Cell sheet transfer results obtained with these three procedures were compared.MethodNormal human epidermal keratinocyte sheets were fabricated on temperature-responsive culture inserts. By temperature reduction to 20 °C, all cells were harvested as a single contiguous cell sheet. Freshly excised porcine esophagi purchased in a slaughter house were turned inside-out, and the exposed lumenal mucosa and submucosal layers were removed by Cooper scissors. This luminal surface was then utilized as a transplantation bed in ex vivo cell sheet experiments. Cell sheets were adhered to the endoscopic transfer device balloon, expanded by balloon inflation and resulting cell viability was evaluated by trypan blue exclusion test after cell sheet trypsinization and dispersion. Cell sheets were transferred onto the esophagus lumen ex vivo using forceps and the balloon device, and also using a modified balloon transfer method. The obtained results were compared with those without balloon expansion, and evaluated for sheet thickness and lumenal histology. Finally, TUNEL staining was performed to examine cell apoptosis.ResultCell sheets thinned after cell sheet balloon expansion, but no apoptosis was observed after these procedures.ConclusionExpanding keratinocyte cell sheets on a balloon endoscopic transfer device did not damage the cell sheets. This sheet transplantation method using the endoscopic balloon transfer device may be considered as a future standard cell sheet endoscopic transplantation procedure for repairing the esophagus.

Abstract 14377: Therapeutic Potential of Clinical Grade Human Induced Pluripotent Stem Cell-derived Cardiac Tissues for a Rat Myocardial Infarction Model: A Pre-clinical Study for a Cell Transplantation Therapy

Introduction: Transplantation of three-dimensional bioengineered cardiac tissues composed of pluripotent stem cell-derived cardiovascular cell lineages is reported to hold potential for functional recovery on pre-clinical studies. We aimed to evaluate therapeutic and myocardial regenerative potential of clinical grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) on a rat myocardial infarction (MI) model. Methods: Clinical grade hiPSC lines established from a healthy volunteer were simultaneously differentiated into cardiovascular cell lineages. We seeded the cells on temperature responsive culture dishes to form cell sheets. HiCTs were generated by stacking 5 cell sheets with insertion of gelatin hydrogel microspheres (GHMs) to promote oxygen and nutrition supply and transplanted onto an athymic rat MI model (n=6). Echocardiography and histological analysis at 12 weeks after surgery were conducted and compared to those in animals with sham surgery (n=9) and with cell sheet stacks without GHMs [GHM(-), n=6]. Some of HiCT-transplanted rats were subjected to tissue clearing and two-photon excitation microscopy (TPEM) to assess graft vascularization. Results: Flow cytometry revealed cellular components after differentiation as follows: 52.0±1.4% of cardiomyocytes (cardiac isoform of troponin-T + :cTnT), 9.9±0.7% of vascular endothelial cells (VE-cadherin + ) and 14.1±1.8% of vascular mural cells (PDGFRβ + ). Echocardiography revealed significantly lower left ventricular end diastolic volume (LVEDV) and higher left ventricular ejection fraction (LVEF) in HiCT group [sham vs GHM(-) vs HiCT: LVEDV; 1.5±0.1 vs 1.3±0.05 vs 0.9±0.03 mL, p<0.0001 / LVEF; 59.7±2.2 vs 67.4±0.6 vs 82.7±0.9 %, p<0.0001]. HiCT group showed significantly larger engraftment [GHM(-) vs HiCT; 12 week; 0.1 ± 0.1 vs 1.8 ± 0.6 mm 2 ; p<0.05]. Engrafted graft tissues were composed of cTnT / α-Actinin-positive cardiomyocytes which exhibited obvious striated structure. TPEM revealed host to graft vascular connection at 2 weeks after HiCT transplantation. Conclusions: HiCTs derived from clinical grade hiPSC potentially serve as a stem cell-derived cellular product in cardiac regenerative therapy for foreseeable clinical applications.

Transplantation of clinical-grade human induced pluripotent stem cell derived cardiac tissues contributes to functional recovery in a rat myocardial infarction model

Abstract Background Clinical-grade human induced pluripotent stem cells (iPSCs) established from a healthy volunteer are currently being considered as a quality controlled cell source for regenerative therapy. Transplantation of three-dimensional bioengineered cardiac tissues composed of human iPSC-derived cardiovascular cell lineages is reported to hold potential for cardiac functional recovery. Purpose The aims of this study were to evaluate tissue conformation and cellular viability of human iPSC-derived cardiac tissues (HiCTs) generated from clinical-grade cells and to validate functional efficacy of HiCT transplantation. Methods Clinical-grade human iPSC lines were simultaneously differentiated into cardiovascular cell lineages by a high-density monolayer culture. The differentiation efficacy was analyzed by flow cytometry. We seeded the cells on temperature responsive culture dishes to form cell sheets. HiCTs are generated by stacking 5 cell sheets with insertion of gelatin hydrogel microspheres (GHMs) between each sheet to promote oxygen and nutrition supply. Characteristics of the HiCTs are histologically and immunohistochemically evaluated. The HiCTs were transplanted onto an athymic nude rat myocardial infarction (MI) model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging (MRI) until 4 weeks after surgery, and compared to those in animals with sham operation and with cell sheet stacks without GHMs [GHM(−)]. Results Flow cytometry at differentiation day15 revealed cellular components as follows: 52.5±1.4% of cardiomyocytes (cardiac isoform of troponin-T+), 9.8±0.7% of vascular endothelial cells (VE-cadherin+), 14.8±1.8% of vascular mural cells (PDGFRβ+) and 0.2±0.1% of undifferentiated cells (TRA-1-60+). HiCTs were significantly thicker [GHM(−) vs HiCT: 357.3±81.5 vs 723.0±84.0μm, p<0.05], composed of higher area of cardiomyocytes (27.7±7.9 vs 71.9±15.5mm2, p<0.05) and endothelial cells (CD31+) (1.6±0.7 vs 9.2±1.5mm2, p<0.05), free from hypoxia (HIF-1α+) (3.1±0.1 vs 0.8±0.2%, p<0.05) and cell death (TUNEL+) (3.2±0.1 vs 1.4±0.3%, p<0.05) after 7 days of in vitro culture. Echocardiography revealed significantly lower left ventricular end diastolic volume (LVEDV) and higher left ventricular ejection fraction (LVEF) in HiCT group [sham (n=27) vs GHM(−) (n=12) vs HiCT (n=12): LVEDV; 1.4±0.1 vs 1.3±0.1 vs 0.9±0.1mL, p<0.0001/LVEF; 55.3±1.1 vs 58.2±2.3 vs 78.2±1.5%, p<0.0001]. Cardiac MRI showed lower LVEDV and higher LVEF as well [sham (n=8) vs GHM(−) (n=6) vs HiCT (n=6): LVEDV; 0.7±0.03 vs 0.7±0.03 vs 0.6±0.02mL, p<0.01 / LVEF; 39.2±2.1 vs 43.8±1.4 vs 54.0±2.8%, p<0.001]. Conclusions We conclude that HiCTs generated from clinical-grade cells hold sufficient viability and tissue conformation suitable for functional recovery validated by a rat MI model. Clinical-grade human iPSCs potentially serve as a reasonable cell source for stem cell-derived product transplantation therapy with foreseeable clinical applications. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Japan Agency for Medical Research and Development (AMED), Invited Research Project of Institute for Advancement of Clinical Transnational Science, Kyoto University Hospital

Evaluation of Multi-Layered Pancreatic Islets and Adipose-Derived Stem Cell Sheets Transplanted on Various Sites for Diabetes Treatment.

Islet cell transplantation is considered an ideal treatment for insulin-deficient diabetes, but implantation sites are limited and show low graft survival. Cell sheet technology and adipose-derived stem cells (ADSCs) can be useful tools for improving islet cell transplantation outcomes since both can increase implantation efficacy and graft survival. Herein, the optimal transplantation site in diabetic mice was investigated using islets and stem cell sheets. We constructed multi-layered cell sheets using rat/human islets and human ADSCs. Cell sheets were fabricated using temperature-responsive culture dishes. Islet/ADSC sheet (AI sheet) group showed higher viability and glucose-stimulated insulin secretion than islet-only group. Compared to islet transplantation alone, subcutaneous AI sheet transplantation showed better blood glucose control and CD31+ vascular traits. Because of the adhesive properties of cell sheets, AI sheets were easily applied on liver and peritoneal surfaces. Liver or peritoneal surface grafts showed better glucose control, weight gain, and intraperitoneal glucose tolerance test (IPGTT) profiles than subcutaneous site grafts using both rat and human islets. Stem cell sheets increased the therapeutic efficacy of islets in vivo because mesenchymal stem cells enhance islet function and induce neovascularization around transplanted islets. The liver and peritoneal surface can be used more effectively than the subcutaneous site in future clinical applications.

Measuring the Contractile Force of Multilayered Human Cardiac Cell Sheets.

Three-dimensional (3D) cardiac tissue reconstruction using tissue engineering technology is a rapidly growing area of regenerative medicine and drug screening development. However, there remains an urgent need for the development of a method capable of accurately measuring the contractile force of physiologically relevant 3D myocardial tissues to facilitate the prediction of human heart tissue drug sensitivity. To this end, our laboratory has developed a novel drug screening model that measures the contractile force of cardiac cell sheets prepared using temperature-responsive culture dishes. To circumvent the difficulties that commonly arise during the stacking of cardiomyocyte sheets, we established a stacking method using centrifugal force, making it possible to measure 3D myocardial tissue. Human induced pluripotent stem cell-derived cardiomyocytes were seeded in a temperature-responsive culture dish and processed into a sheet. The cardiac cell sheets were multilayered to construct 3D cardiac tissue. Measurement of the contractile force and cross-sectional area of the multilayered 3D cardiac tissue were then obtained and used to determine the relationship between the cross-sectional area of the cardiac tissue and its contractile force. The contractile force of the 1-, 3-, and 5-layer tissues increased linearly in proportion to the cross-sectional area. A result of 6.4 mN/mm2, accounting for one-seventh of the contractile force found in adult tissue, was obtained. However, with 7-layer tissues, there was a sudden drop in the contractile force, possibly because of limited oxygen and nutrient supply. In conclusion, we established a method wherein the thickness of the cell sheets was controlled through layering, thus enabling accurate evaluation of the cardiac contractile function. This method may enable comparisons with living heart tissue while providing information applicable to regenerative medicine and drug screening models.

A novel method to align cells in a cardiac tissue-like construct fabricated by cell sheet-based tissue engineering.

Fabrication of cardiac tissue from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has received great interest, but a major challenge facing researchers is the alignment of cardiomyocytes in the same direction to optimize force generation. We have developed a novel method of fabricating a cardiac tissue-like construct with aligned cells based on the unidirectional stretching of an hiPS-CM sheet. A square cell sheet was harvested from a temperature-responsive culture dish and placed on a silicone surface, and an extending force was imposed on the silicone to stretch the cell sheet along one direction. To enable evaluation of cardiomyocyte morphology in vitro, a cell sheet was constructed by coculture of hiPS-CMs and human adipose-derived stem cells. In separate experiments, a stretched double-layered cell sheet constructed from hiPS-CMs alone was transplanted onto the muscle of an athymic rat, and its features were compared with those of a nonstretched (control) cell sheet. Immediately after stretching, the stretched cell sheet was significantly longer than the control cell sheet. Immunohistological analysis revealed that the cardiomyocytes showed unidirectional alignment in the stretched cell sheet but random directionality in the control cell sheet. Two weeks after transplantation, immunohistology demonstrated that the stretched cell sheet had retained the unidirectionality of its myocardial fibers and had an orientation intensity that was higher than that of the control cell sheet after transplantation or the stretched cell sheet before transplantation. Our technique provides a simple method of aligning an hiPS-CM-derived cardiac tissue-like construct without the use of a scaffold.

Three-dimensional periodontal tissue regeneration using a bone-ligament complex cell sheet

Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.

Ameliorated healing of biliary anastomosis by autologous adipose-derived stem cell sheets.

IntroductionCell sheets consisting of adipose-derived stem cells (ADSCs) have been reported to be effective for wound healing. We conducted this study to clarify the efficacy of ADSC sheets in wound healing at the duct-to-duct biliary anastomotic site in pigs.MethodsEleven female pigs (20–25 kg) were divided into two groups: biliary anastomosis with an ADSC sheet (n = 6) or without an ADSC sheet (n = 5). To follow the transplanted ADSCs, PKH26GL-labeled sheets were used in one of the ADSC pigs. Two weeks prior to laparotomy, ADSCs were isolated from the lower abdominal subcutaneous adipose tissue. After three passages, ADSCs were seeded on temperature-responsive culture dishes and collected as cell sheets. ADSC sheets were gently transplanted on the anastomotic site. We evaluated specimens by PKH26GL labeling, macroscopic changes, infiltration of inflammatory cells, and collagen content.ResultsLabeled ADSCs remained around the bile duct wall. In the no-ADSC group, more adhesion developed at the hepatic hilum as observed during relaparotomy. Histopathological examination showed that the diameter and cross-sectional area of the bile duct wall were decreased in the ADSC group. In the no-ADSC group, a large number of inflammatory cells and more collagen fibers were identified in the bile duct wall.ConclusionsThe present study demonstrated that autologous ADSC sheet transplantation reduced hypertrophic changes in the bile duct wall at the anastomotic site. A long-term follow-up is required to evaluate the efficacy of this mechanism in prevention of biliary anastomotic strictures.

The Efficacy of Autologous Myoblast Sheet Transplantation to Prevent Perforation After Duodenal Endoscopic Submucosal Dissection in Porcine Model

The recent advent of endoscopy has enabled the endoscopic submucosal dissection (ESD) of superficial nonampullary duodenal epithelial tumors. However, the substantially thin wall and presence of bile and pancreatic juice make it technically difficult to perform duodenal ESD without perforation, which leads to lethal complications. The present study evaluated the efficacy of autologous myoblast sheet transplantation for the prevention of late perforation after duodenal ESD in a porcine model. Two weeks before ESD, skeletal muscle was surgically excised from the femur of pigs, and myoblasts were isolated and seeded in temperature-responsive culture dishes to prepare sheets. Immediately after ESD, the autologous myoblast sheets were attached to the serosal surface at the ESD site with omentopexy. The pigs were divided into two groups: the autologous myoblast sheet group (n = 5), where the myoblast cell sheet was attached to the ESD ulcer part from the duodenal serous side, and the Omentum group (n = 5), where only the omentum was used. The pigs were sacrificed and analyzed macroscopically and histologically on postoperative day 3. The macroscopic examination of the abdominal cavity revealed perforation in the ESD ulcer area and leakage of bile in the Omentum group but no perforation in the Sheet group. A histopathological examination revealed that continuity of the duodenal wall at the ESD site was maintained with dense connective tissue in the Sheet group. In conclusion, autologous myoblast sheets were useful for preventing perforation after duodenal ESD.

Transplantation of adipose-derived mesenchymal stem cell sheets directly into the kidney suppresses the progression of renal injury in a diabetic nephropathy rat model.

Aims/IntroductionAdipose‐derived mesenchymal stem cell (ASC) transplantation is a promising therapy for diabetic nephropathy (DN). However, intravascular administration of ASCs is associated with low engraftment in target organs. Therefore, we considered applying the cell sheet technology to ASCs. In this study, ASC sheets were directly transplanted into the kidneys of a DN rat model, and therapeutic consequences were analyzed.Materials and MethodsAdipose‐derived mesenchymal stem cells were isolated from adipose tissues of 7‐week‐old enhanced green fluorescent protein rats, and ASC sheets were prepared using a temperature‐responsive culture dish. A DN rat model was established from 5‐week‐old Spontaneously Diabetic Torii fatty rats. Seven‐week‐old DN rats (n = 21) were assigned to one of the following groups: sham‐operated (n = 6); ASC suspension (6.0 × 106 cells/mL) administered intravenously (n = 7); six ASC sheets transplanted directly into the kidney (n = 8). The therapeutic effect of the cell sheets was determined based on urinary biomarker expression and histological analyses.ResultsThe ASC sheets survived under the kidney capsule of the DN rat model for 14 days after transplantation. Furthermore, albuminuria and urinary tumor necrosis factor‐α levels were significantly lower in the ASC sheets transplanted directly into the kidney group than in the sham‐operated and ASC suspension administered intravenously groups (P < 0.05). Histologically, the ASC sheets transplanted directly into the kidney group presented mild atrophy of the proximal tubule and maintained the renal tubular structure.ConclusionsTransplantation of ASC sheets directly into the kidney improved transplantation efficiency and suppressed renal injury progression. Therefore, the ASC sheet technology might be a promising novel treatment for DN.

Pulsatile tubular cardiac tissues fabricated by wrapping human iPS cells-derived cardiomyocyte sheets.

The purpose of this study was to fabricate pulsatile tubular cardiac tissue using cell sheet based-tissue engineering. First, we fabricated human induced pluripotent stem cell (hiPSc)-derived cardiomyocyte sheets and normal human dermal fibroblast (NHDF) sheets which are harvested from temperature responsive culture dishes only by lowering the temperature. Then tubular cardiac tissues are formed by wrapping one hiPSc-derived cardiomyocyte sheet and three NHDF sheets around an octagonal column, and both ends of the tubular tissue were covered with fibrin and collagen gel. The octagonal column with the tubular tissue was connected to an in vitro circulation system in a culture box. After four-day culture, the cardiac tissue survived and pulsated spontaneously in the circulation system. Furthermore, the analysis with a Millar catheter inserted into the cardiac tubes revealed significant inner pressure changes generated by their beating. In addition, the tubular cardiac tissue pulsated in response to the electrical stimulation. Although histological analyses demonstrated that cardiac troponin T-positive cells stratified the inner surface of the tubular tissues, gene expression analyses showed an immature state of these cardiomyocytes. Thus, cell sheet-based tissue engineering realized human pulsatile tubular cardiac tissue fabrication and we believe that these tubular cardiac tissues should contribute to future drug screening and regenerative therapy for heart diseases.

Invitro periodontal ligament cell expansion by co-culture method and formation of multi-layered periodontal ligament-derived cell sheets.

ObjectivePer-implantitis is one of the implant treatment complications. Dentists have failed to restore damaged periodontium by using conventional therapies. Tissue engineering (stem cells, scaffold and growth factors) aims to reconstruct natural tissues. The paper aimed to isolate both periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) and use them in a co-culture method to create three-layered cell sheets for reconstructing natural periodontal ligament (PDL) tissue.Materials and methodsBMMSCs were isolated from rabbit tibia and femur, and PDLSC culture was established from the lower right incisor. The cells were co-cultured to induce BMMSC differentiation into PDL cells. Cell morphology, stem cells and PDL-specific markers (CD90, CD34, and periostin) were also detected using immunofluorescent assay. Co-cultured cell monolayers were detached using temperature-responsive tissue culture dishes and collagen graft to create the three-layer construct. The 3D-engineered tissue was examined histologically and by field emission scanning electron microscopy (FESEM).ResultsBMMSCs co-cultured with PDLSCs successfully induced more PDL cells. The newly induced PDL cells exhibited periostin and CD90 expression. Fluorescence green intensity was measured for the co-cultured cells that were stained with periostin, the mean fluorescence green intensity (periostin expression) was significantly higher for the newly induced PDL cells after 1, 2, and 3 weeks when compared with control (BM-MSCs), at 21 days non-significant difference was measured when compared with control (PDLSCs).The results showed the successful formation of 3D multilayer PDL tissue. Histological cross-section showed cell sheets and the stable adhesion between them. FESEM examination was conducted for the cross-section, showing three-layered cell sheets with stable adhesion between cells.ConclusionsThe results of this paper report that the three layered-cell sheets were successfully constructed by the novel use of collagen graft as a scaffold to be used in treatment of periodontitis and to envelop the dental implants to create biohybrid implant.

Recent progress in induced pluripotent stem cell-derived cardiac cell sheets for tissue engineering

The past decade has witnessed remarkable development in tissue engineering technologies and stem cells. Our lab has developed a novel technology - "cell sheet technology" for tissue engineering. After the confluent cells are cultured on an innovative temperature-responsive culture dish, the cells can be harvested as an intact sheet by lowering temperature. We have successfully created multiple cell sheet-based tissues for therapies of a vast variety of diseases, in particular, myocardial diseases. On the other side, the discovery of human induced pluripotent stem cells (hiPSC) enables stable production of defined tissue-specific cell types and thus makes it possible to regenerate tissues or even organs for clinical application and in vitro drug screening/disease modeling. Recently, we have combined cell sheet technology and hiPSC-derived cardiac cells for fabrication of functional human cardiac tissues. This review summarizes ongoing challenges in this field and our progresses in solving issues, such as large scale culture of hiPSC-derived cardiac cells, elimination of undifferentiated iPSCs to decrease the risk of tumor formation as well as myocardial tissue fabrication technologies.

Keratinocyte sheets prepared with temperature-responsive dishes show enhanced survival after in vivo grafting on acellular dermal matrices in a rat model of staged bi-layered skin reconstruction

IntroductionBi-layered skin reconstruction can be achieved by staged grafting of acellular dermal matrices (ADMs) and cultured epithelial keratinocyte sheets (KSs). Both KSs and ADMs have been used for long; yet, their combined use has shown poor effectiveness. This outcome has been related to the enzymatic treatment used in the preparation of KSs, which impairs their adhesion potential to ADMs and the formation of a basement membrane (BM). Temperature-responsive (TR) culture dishes allow for enzyme-free preparation of KSs with preservation of BMs and intercellular adhesion proteins; yet, their use has not been previously applied to staged bi-layered skin reconstruction. Using an in vivo rat model, we tested the hypothesis that TR cultures enhance KSs survival and BM preservation after sequential grafting on ADMs.MethodsIn nude rats (n = 9/group), a 9-cm [2] full-thickness dorsal skin defect was repaired with a commercial ADM. At 2 weeks after surgery, we grafted the ADM with KSs (circular, 25 mm diameter), prepared from human cells either by enzymatic Dispase treatment (DT control group) or a TR culture dish (TR experimental group). KSs survival and BMs preservation was assessed one week later by digital imaging, histology (hematoxylin & eosin), immunohistochemistry (collagen IV, pancytokeratins) and immunofluorescence (cytokeratin 1-5-6, laminin).ResultsThe TR group showed a significantly higher KSs survival (120 ± 49 vs. 63 ± 42 mm2; p < 0.05) and epidermal thickness (165 ± 79 vs. 65 ± 54 μm; p < 0.01) compared with the control DT group, as well as higher epidermal maturation (cytokeratin) and a denser laminin and Collagen IV expression in the BMs in vitro and in vivo.ConclusionThese findings suggest that KSs prepared with TR culture dishes have significantly enhanced survival when grafted on ADMs; these outcomes could help improve current clinical strategies in wound care by skin reconstruction.

Fabrication of Functional Cell Sheets with Human Thyrocytes from Non-Tumorous Thyroid Tissue.

Engineered cell sheet transplantation has been considered an alternative physiological therapy for endocrine disorders. In this study, we attempted to fabricate functional human thyroid cell sheets using the engineering technology by culturing primary thyrocytes in free-feeder monolayers and assessed their proliferation and function in two different media. The non-tumorous tissues (approximately 2g) were dissected during surgery. Primary human thyroid cells were isolated by mechanical dispersion and treatment with isolation solution. The cells were cultured on tissue culture dishes or temperature-responsive culture dishes to induce the formation of detached cell sheets. Primary thyroid cells isolated from nine patients were positive for thyroid transcription factor 1, thyroglobulin (TG) and cytokeratin 7. Cell sheets with follicles were fabricated by cells incubated in both Dulbecco's Modified Eagle Medium (DMEM) and hepatocyte-defined medium (HDM) culture medium. The diameter and thickness of sheets fabricated in HDM were larger and thicker than those fabricated from DMEM. Furthermore, the cells incubated in HDM secreted higher levels of fT3 and fT4 than those incubated in DMEM. The thyroid peroxidase and TG mRNA of cells maintained in HDM were higher than those in cells maintained in DMEM. HDM appears suitable as a culture medium for maintaining primary thyrocytes and fabricating functional cell sheets. These in vitro findings may contribute to the development of appropriate culture conditions for human thyrocytes as well as engineered functional cell sheets.