Tissue-engineered Cell Sheets Research Articles

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.

Maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using oral mucosal epithelial cell sheets.

We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets.

Tissue-Engineered Myoblast Cell Sheets or the Treatment of Enteral Defects in a Rat Model

Tissue-Engineered Myoblast Cell Sheets or the Treatment of Enteral Defects in a Rat Model

When is pre-emptive treatment necessary after endoscopic mucosal resection of early esophageal neoplasm?

When is pre-emptive treatment necessary after endoscopic mucosal resection of early esophageal neoplasm?

Anti‐Angiogenic Factors of Tissue‐Engineered Oral Mucosal Epithelial Cell Sheets for Corneal Regeneration

Corneal regeneration by autologous oral mucosal epithelial cell sheet transplantation has been successfully performed in limbal stem cell deficiency (LSCD) patients. However, the molecular mechanism by which cell sheet grafting efficiently suppresses corneal vascularization, a major symptom of LSCD, is not fully understood. Here, we report the differential expression of pro‐ and anti‐angiogenic factors in cell sheets engineered from isolated rabbit oral mucosal epithelial cells, harvested on thermo‐responsive culture dish (UpCell®, CellSeed, Inc., Japan). A gene expression profiling‐directed study, followed by protein and histological analyses of cell sheets, showed a down‐regulation of pro‐angiogenic MMP‐3, and an up‐regulation of wound healing‐related proteins, MMP‐13 and collagen VIII. MMPs inhibitors, TIMP‐1 and TIMP‐3, were found to be expressed in cell sheets, thus reflecting a fine tuned balance between the pro‐ and anti‐angiogenic factors. These results indicated that tissue‐engineered cell sheets acquired a suppressive capability against corneal vascularization.

Muscle-derived Stem Cell Sheets Support Pump Function and Prevent Cardiac Arrhythmias in a Model of Chronic Myocardial Infarction

Direct intracardiac cell injection for heart repair is hindered by numerous limitations including: cell death, poor spreading of the injected cells, arrhythmia, needle injury, etc. Tissue-engineered cell sheet implantation has the potential to overcome some of these limitations. We evaluated whether the transplantation of a muscle-derived stem cell (MDSC) sheet could improve the regenerative capacity of MDSCs in a chronic model of myocardial infarction. MDSC sheet-implanted mice displayed a reduction in left ventricle (LV) dilation and sustained LV contraction compared with the other groups. The MDSC sheet formed aligned myotubes and produced a significant increase in capillary density and a reduction of myocardial fibrosis compared with the other groups. Hearts transplanted with the MDSC sheets did not display any significant arrhythmias and the donor MDSC survival rate was higher than the direct myocardial MDSC injection group. MDSC sheet implantation yielded better functional recovery of chronic infarcted myocardium without any significant arrhythmic events compared with direct MDSC injection, suggesting this cell sheet delivery system could significantly improve the myocardial regenerative potential of the MDSCs.

Quantitative evaluation system for tissue-engineered corneal epithelial cell sheets using image-based technology

Abstract Background: Tissue-engineered corneal epithelial cell sheets have been applied for treating ocular surface diseases. It is essential to establish a standardized validation system for fabricated cell sheets

Thermoresponsive Cell Culture Substrates Based on PNIPAM Brushes Functionalized with Adhesion Peptides: Theoretical Considerations of Mechanism and Design

Thermoresponsive tissue culture substrates based on PNIPAM brushes are used to harvest confluent cell sheets for tissue engineering. The prospect of clinical use imposes the utilization of culture medium free of bovine serum, thus suggesting conjugation with adhesion peptides containing the RGD minimal recognition sequence. The optimum position of the RGD along the chain should ensure both cell adhesion at 37 °C and cell detachment at T(L) below the lower critical solution temperature of PNIPAM. Design guidelines are formulated from considerations of brush confinement by the cells: (i) Cell adhesion at 37 °C is controlled by the RGDs accessible without brush compression. (ii) Cell detachment at T(L) is driven by a disjoining force due to confinement of the swollen brush by cells retaining integrin-RGD bonds formed at 37 °C. These suggest placing the RGDs at the grafting surface or its vicinity. Randomly placed RGDs do not enable efficient detachment because a large fraction of the integrin-RGD bonds are not sufficiently tensioned at T(L), in line with experimental observations (Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Immobilization of celladhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Eng. 2004, 10, 1125-1135). The theory framework enables analysis of culture media based on polymer brushes conjugated with adhesion peptides in general.

Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix - based scaffold

Stem cell mediated tissue engineering has been acknowledged as a prospective strategy for repairing and replacing damaged and lost tissues. However, the low survival rate of implanted stem cells proves to be a major challenge in the management of transplantation failures. While previous studies have indicated the effectiveness of tissue engineered cell sheets in improving the survival rate of implanted cells, we have recently demonstrated the use of treated dentin matrix (TDM) as a biological scaffold and dental follicle cells (DFCs) as the seeding cells for dentinogenesis and tooth root construction. This study proposes a strategy utilizing TDM with human dental follicle cell sheets (DFCSs) for root regeneration. The biological characteristics and changes of human DFCSs under the effect of TDM were studied with scanning electron microscopy, transmission electron microscopy, immunofluorescence microscopy, immunohistochemistry and quantitative real-time PCR. DFCSs combined with TDM were implanted subcutaneously into the dorsum of mice. Histological examination of the harvested grafts revealed a whirlpool-like alignment of the DFCs in multiple layers that were positive for COLI, integrinβ1, fibronectin and alkaline phosphatase (ALP), suggestive of the formation of a rich extracellular matrix. DFCSs, under the effect of TDM, highly expressed DMP-1 and bone sialoprotein (BSP), indicating their potential for odontogenesis and osteogenesis. Importantly, in vivo, TDM could induce and support DFCSs to develop new dentin-pulp like tissues and cementum-periodontal complexes that were positive for markers such as DSP, nestin and VIII factors, COLI and cementum attachment protein (CAP), implying successful root regeneration. Therefore, DFCSs combined with TDM may prove to be a better strategy for the construction of tooth root, and is a prospective approach that could be utilized for the treatment of root or tooth defect or loss in future.

Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Human Immature Dental Pulp Stem Cells

To determine the outcome of the use of a tissue-engineered cell sheet composed of human undifferentiated immature dental pulp stem cells (hIDPSC) for ocular surface reconstruction in an animal model of total limbal stem cell deficiency (LSCD). LSCD was induced by the application of 0.5 M NaOH to the right eye of rabbits for 25 seconds (mild chemical burn [MCB]) and for 45 seconds (severe chemical burn [SCB]). After 1 month, a superficial keratectomy was performed to remove the fibrovascular pannus that covered the animals' burned corneas. A tissue-engineered hIDPSC sheet was transplanted onto the corneal bed and then covered with deepithelialized human amniotic membrane (AM). In the respective control groups, the denuded cornea was covered with AM only. After 3 months, a detailed analysis of the rabbit eyes was performed with regard to clinical aspect, histology, electron microscopy, and immunohistochemistry. Corneal transparency of the rabbit eyes that underwent hIDPSC transplantation was improved throughout the follow-up, while the control corneas developed total conjunctivalization and opacification. Rabbits from the MCB group showed clearer corneas with less neovascularization. The clinical data were confirmed by histologic analysis that showed healthy uniform corneal epithelium, especially in the MCB group. The presence of hIDPSC was detected using an anti-hIDPSC antibody. The corneal tissue also showed positive immunostaining with anti-human antibodies. In the control corneas, none of these antigens were detected. Overall, these data showed that transplantation of a tissue-engineered hIDPSC sheet was successful for the reconstruction of corneal epithelium in an animal model of LSCD.

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

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

A Fully Automated Approach to Quantitatively Determine Thickness of Tissue-Engineered Cell Sheets

Sheet-based tissue engineering is an innovative field that has provided the scientific community with new tissue-engineered products such as skin, cornea, heart valves, and vascular grafts. As this area of tissue engineering progresses toward clinical implementation, quality control becomes more and more important. Imaging methods advertise themselves because of their high resolution and good tissue-fluid contrast. We present and compare two methods, one based on a custom-designed automatized large-area confocal scanner that uses backscattered light for image formation, and one based on optical coherence tomography (OCT). In both modalities, additional image processing is used to extract sheet thickness and density information and to create a quantitative tissue thickness map in a fully automated fashion. In test objects (glass of known thickness and scattering samples) and engineered tissue sheets with artificially introduced defects we found high agreement between the two methods in the measurement of thickness and the visual representation of the defects. Both the OCT and the confocal scanner were able to provide high-detail images visually consistent to those obtained with brightfield microscopy. Both OCT and large-area confocal scanning in combination with specialized image processing algorithms promise to provide information on tissue homogeneity, density, and the presence of potential defects in tissue sheets in an unsupervised fashion and thus help establish new quality control methods in sheet-based tissue engineering.

Functional closure of visceral pleural defects by autologous tissue engineered cell sheets☆

The occurrence of intraoperative air leaks is an unavoidable complication during pulmonary surgeries. However, current surgical methods are generally ineffective in closing these visceral pleural defects, resulting in a decreased quality of life for patients. Here, we examined novel tissue engineered cell sheets for the closure of pleural defects in a porcine model. Skin biopsies were harvested from juvenile swine and tissue sheets composed of dermal fibroblasts were created using ex vivo culture on temperature-responsive dishes. After creating a visceral pleural injury model, the tissue engineered autologous dermal fibroblast sheets were transplanted directly to the defects without the use of sutures or additional adhesive agents, such as fibrin glue. The tissue engineered autologous dermal fibroblast sheets attached directly to the lung surface providing an immediate seal against up to 25 cm H2O of airway pressure. Four weeks after transplantation, the dermal fibroblast sheets remained present on the pleural surface, providing permanent closure. The dermal fibroblast sheets were also responsive to changes in lung volume due to mechanical ventilation. No recurrences of air leaks were observed throughout the follow-up period. This study presents the development of an effective sealant for visceral pleural defects using autologous cells that have the flexibility to respond to expansion and contraction during respiration.

Therapeutic Angiogenesis Using Tissue Engineered Human Smooth Muscle Cell Sheets

Peripheral arterial disease (PAD) can have severe consequences on patient mortality and morbidity. In contrast to approaches using growth factor administration or isolated cell transplantation, we attempted to develop an alternative method for ischemic therapy using the transplantation of tissue engineered cell sheets with angiogenic potential. Human smooth muscle cell (SMC) and fibroblast cell (FbC) sheets were harvested from temperature-responsive culture dishes and transplanted into ischemic hind limbs of athymic rats. ELISA showed significantly increased in vitro secretion of angiogenic factors by SMCs in comparison to FbCs. Twenty-one days after transplantation, laser doppler analysis demonstrated significantly increased blood perfusion in the SMC group. Perfusion with Indian ink and immunohistochemistry also revealed significantly greater numbers of functional capillaries in the SMC group. Finally, cell tracing experiments revealed that some SMCs from the transplanted cell sheets migrated into the ischemic tissues, contributing to newly formed vessels. SMC sheet transplantation allows for controlled and localized delivery of cells that possess angiogenic potential directly to ischemic tissues. Through the secretion of angiogenic factors, as well as cell migration and integration with newly formed vessels, SMC sheet transplantation provides an effective method for the revascularization of ischemic tissues.

Dynamic sealing of lung air leaks by the transplantation of tissue engineered cell sheets

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

A Novel Inflatable Surgical Device Used in Endoscopic Transplantation of Tissue Engineered Cells Sheets for Regenerative Treatments of Esophageal Ulcerations: A Preliminary Report

Introduction: Large en-bloc resection of esophageal cancers by endoscopic submucosal dissection (ESD) often requires the use of balloon dilation after endoscopic operations to prevent post-operative esophageal constriction. We therefore have previously developed a novel method to transplant tissue engineered oral mucosal epithelial cell sheets to promote wound healing after ESD, in an effort to improve patient quality of life. (Gut, 1704-1720, 2006) Although we have demonstrated the successful transplantation of two cell sheets, it has remained difficult to transplant cell sheets to the entire surface of esophageal ulcerative wall using our previous methods using endoscopic forceps have been. We therefore sought to develop a novel device for the endoscopic delivery and transplantation of cell sheets after ESD. Methods: Oral mucosal cells harvested from a beagle were seeded on temperature-responsive dishes and cultured for 2 weeks. Oral mucosal epithelial cell sheets (24 × 24 mm2) were harvested by simply reducing the temperature. Using a novel inflatable device a carrier of the engineered tissues, four individual cell sheets were then simultaneously transplanted to an artificial circumferential esophageal ulceration of a deceased pig (n = 3). Results: A balloon is attached to the exterior of standard EEMR-tube with an outer cylindrical cover to physically protect the cell sheets and to retain moisture. For transplantation, the device is carefully moved to the ulcer site by endoscipy, the outer tube is retracted. Air is then pumped through the endoscope to distend the walls of the esophagus to allow for positioning of the cell sheets. The cell sheets are attached to the ulcer site by balloon inflation. Ten minutes after transplantation we macroscopically confirmed complete circumferential transplantation of oral mucosal cell sheets. Iodine staining indicated that the all of the cell sheets were successfully attached to the esophageal ulcer. Histological results also confirmed that the epithelial cell sheets were attached to the remnant submucosal layers. Conclusions: Our results show that this novel balloon device completely enables to the simultaneous, controlled transplantation of several tissue engineered cell sheets by endoscopy. This ability to simply cover circumferential esophageal ulcerations with a single procedure creates new a potential treatment in endoscopic treatment of Barrett's esophagus with high grade dysplasia, using ESD in conjunction with cell sheet transplantation. In the near future, we are planning in vivo experiments to the post-operative effects of cell sheet transplantation to larger ESD ulcers using the balloon device.

Transplantable retinal pigment epithelial cell sheets for tissue engineering

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF- β2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.

Transplantation of Tissue-Engineered Epithelial Cell Sheets after Excimer Laser Photoablation Reduces Postoperative Corneal Haze

To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.

Application of cell-sheet engineering for reconstitution of functional thyroid tissue from isolated thyroid cells

We have recently used a tissue-engineered cell sheet system with temperature-sensitive culture dishes to regenerate tissues such as corneal epithelial graft sheets, bioartificial tracheas, mucosal epithelial sheets for esophageal ulcers, and pulsatile cardiac tissue grafts, all of which can be used in clinical medicine. With regard to the thyroid gland, the three-dimensional follicular structure consists of polarized thyroid epithelial cells, and this structure is important for the production and secretion of thyroid hormones. In order to regenerate thyroid tissue from isolated cells, we used temperature-responsive culture dishes to harvest thyroid follicular cell sheets in the first step of reconstituting functional thyroid tissue. Initial trials demonstrated that the following conditions improved the recovery of cells sheets: 1) pretreatment of the dishes with serum, 2) use of medium free of thyroid stimulating hormone (TSH), and 3) co-culture with skin fibroblasts.

Tissue Cardiomyoplasty Using Bioengineered Contractile Cardiomyocyte Sheets to Repair Damaged Myocardium: Their Integration with Recipient Myocardium

We hypothesized that tissue-engineered contractile cardiomyocyte sheets without a scaffold would show histological and electrical integration with impaired myocardium, leading to the regeneration of infarcted myocardium. Neonatal rat cardiomyocytes were cultured on Poly(N-isopropylacrylamide)-grafted polystyrene dishes and detached as a square cell sheet at 20 degrees C. Two sheets were stacked to make thicker contractile cardiac sheets. In cross-section, the stacked sheets looked like homogeneous heart-like tissue. Two weeks after rats were subjected to left anterior descending (LAD) ligation, two treatments were conducted: 1) cardiomyocyte sheet implantation (T group, n=10), and 2) fibroblast sheet implantation (F group, n=10). The control group underwent no additional treatment (C group, n=10). Echocardiography demonstrated that cardiac performance was significantly ameliorated in the T group 2, 4, and 8 weeks after implantation. The cardiomyocyte sheets became attached to the infarcted myocardium, showed angiogenesis, expressed connexin-43, and appeared as homogeneous tissue in the myocardium Electrophysiological experiments showed a QRS complex with one peak in the treated scar area in the T group, but two peaks, indicative of branch block, in that of the other groups. Furthermore, the threshold for pacing of the recipient heart was lower in the T group than in the other groups. Cardiomyocyte sheets integrated with the impaired myocardium and improved cardiac performance in a model of ischemic myocardium. Techniques using such tissue-engineered cell sheets are introducing the promising concept of tissue cardiomyoplasty to the field of regenerative medicine.