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/INTRODUCTION: Adipose-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 METHODS: Adipose-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. RESULTS: The 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. CONCLUSIONS: Transplantation 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.

Rat Mesenchymal Stromal Cell Sheets Suppress Renal Fibrosis via Microvascular Protection.

Renal fibrosis is one of the largest global health care problems, and microvascular (MV) injury is important in the development of progressive fibrosis. Although conventional cell therapy suppresses kidney injury via the role of vasoprotective cytokines, the effects are limited due to low retention of administered cells. We recently described that transplantation of hepatocyte growth factor (HGF)-transgenic mesothelial cell sheets showed a remarkable cell survival and strong therapeutic effects in a rat renal fibrosis model. Due to the translational hurdles of transgenic cells, we here applied this technique for allogeneic transplantation using rat bone marrow mesenchymal stromal cells (MSCs). MSC sheets were transplanted onto the kidney surface of a rat renal ischemia-reperfusion-injury model and the effects were compared between those in untreated rats and those receiving intravenous (IV) administration of the cells. We found that donor-cell survival was superior in the cell sheet group relative to the IV group, and that the cell sheets secreted HGF and vascular endothelial growth factor (VEGF) up to day 14. Transplantation of cell sheets increased the expression of activated HGF/VEGF receptors in the kidney. There was no evidence of migration of transplanted cells into the kidney parenchyma. Additionally, the cell sheets significantly suppressed renal dysfunction, MV injury, and fibrosis as compared with that observed in the untreated and IV groups. Furthermore, we demonstrated that the MSC sheet protected MV density in the whole kidney according to three-dimensional microcomputed tomography. In conclusion, MSC sheets strongly prevented renal fibrosis via MV protection, suggesting that this strategy represents a potential novel therapy for various kidney diseases. Stem Cells Translational Medicine 2019;8:1330&1341.

Renal subcapsular transplantation of hepatocyte growth factor-producing mesothelial cell sheets improves ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a clinically important cause of acute kidney injury leading to chronic kidney disease. Furthermore, IRI in renal transplantation still remains a risk factor for delayed graft function. Previous studies on IRI have had some limitations, and few of the studied therapies have been clinically applicable. Therefore, a new method for treating renal IRI is needed. We examined the effects of human mesothelial cell (MC) sheets and hepatocyte growth factor (HGF)-transgenic MC (tg MC) sheets transplanted under the renal capsule in an IRI rat model and compared these two treatments with the intravenous administration of HGF protein and no treatment through serum, histological, and mRNA analyses over 28 days. MC sheets and HGF-tg MC sheets produced HGF protein and significantly improved acute renal dysfunction, acute tubular necrosis, and survival rate. The improvement in necrosis was likely due to the cell sheets promoting the migration and proliferation of renal tubular cells, as observed in vitro. Expression of α-smooth muscle actin at day 14 and renal fibrosis at day 28 after IRI were significantly suppressed in MC sheet and HGF-tg MC sheet treatment groups compared with the other groups, and these effects tended to be reinforced by the HGF-tg MC sheets. These results suggest that the cell sheets locally and continuously affect renal paracrine factors, such as HGF, and support recovery from acute tubular necrosis and improvement of renal fibrosis in chronic disease.

Hepatocyte Growth Factor-Secreting Mesothelial Cell Sheets Suppress Progressive Fibrosis in a Rat Model of CKD.

BACKGROUND: Although hepatocyte growth factor (HGF) has antifibrotic effects and is involved in angiogenesis and vasodilation, systemic administration of HGF to prevent kidney fibrosis is not a feasible strategy for suppressing interstitial fibrosis in patients with CKD. METHODS: We investigated a novel therapy involving HGF transgenic cell sheets grown in culture from human mesothelial cells and administered to rats with unilateral ureteral obstruction (UUO). We compared progression of fibrosis in rats with UUO that received one of five interventions: HGF-transgenic mesothelial cell sheets transplanted to the kidney surface, HGF-transgenic mesothelial cell sheets transplanted to thigh, mesotherial cell sheets transplanted to kidney, no sheets, or HGF injections. RESULTS: HGF transgenic cell sheets transplanted to the kidney strongly suppressed the induction of myofibroblasts and collagen in the kidney for 28 days; other interventions did not. Additionally, the HGF-secreting cell sheets ameliorated loss of peritubular capillaries and maintained renal blood flow. CONCLUSIONS: These findings suggest that cell sheet therapy is a novel and promising strategy for inhibiting progressive fibrosis in CKD.

Perfusion culture maintained with an air-liquid interface to stimulate epithelial cell organization in renal organoids in vitro.

BACKGROUND: Organoids derived from induced pluripotent stem (iPS) or embryonic stem (ES) cells have been evaluated as in vitro models of development and disease. However, maintaining these cells under long-term static culture conditions is difficult because of nutrition shortages and waste accumulation. To overcome these issues, perfusion culture systems are required for organoid technology. A system with a stable microenvironment, nutrient availability, and waste removal will accelerate organoid generation. The aim of this study was to develop a novel perfusion system for renal organoids by maintaining the air-liquid interface with a device fabricated using a 3D printer. RESULTS: Our results revealed slow flow at the organoid cultivation area based on microbead movement on the membrane, which depended on the perfusion rate under the membrane. Moreover, the perfused culture medium below the organoids via a porous membrane diffused throughout the organoids, maintaining the air-liquid interface. The diffusion rates within organoids were increased according to the flow rate of the culture medium under the membrane. The perfused culture medium also stimulated cytoskeletal and basement membrane re-organization associated with promotion tubular formation under 2.5 µL/min flow culture. In contrast, tubules in organoids were diminished at a flow rate of 10 µL/min. CONCLUSIONS: Our liquid-air interface perfusion system accelerated organization of the renal organoids. These results suggest that suitable perfusion conditions can accelerate organization of epithelial cells and tissues in renal organoids in vitro.

Pathological Process of Prompt Connection between Host and Donor Tissue Vasculature Causing Rapid Perfusion of the Engineered Donor Tissue after Transplantation.

The shortage of donors for transplantation therapy is a serious issue worldwide. Tissue engineering is considered a potential solution to this problem. Connection and perfusion in engineered tissues after transplantation is vital for the survival of the transplanted tissue, especially for tissues requiring blood perfusion to receive nutrients, such as the heart. A myocardial cell sheet containing an endothelial cell network structure was fabricated in vitro using cell sheet technology. Transplantation of the three-dimensional (3D) tissue by layering myocardial sheets could ameliorate ischemic heart disease in a rat model. The endothelial cell network in the 3D tissue was able to rapidly connect to host vasculature and begin perfusion within 24 h after transplantation. In this review, we compare and discuss the engineered tissue⁻host vasculature connection process between tissue engineered constructs with hydrogels and cell sheets by histological analysis. This review provides information that may be useful for further improvements of in vivo engineered tissue vascularization techniques.

Introduction of vasculature in engineered three-dimensional tissue.

BACKGROUND: With recent developments in tissue engineering technology, various three-dimensional tissues can be generated now. However, as the tissue thickness increases due to three-dimensionalization, it is difficult to increase the tissue scale without introduction of blood vessels. MAIN TEXT: Many methods for vasculature induction have been reported recently. In this review, we introduced several methods which are adjustable vascularization in three-dimensional tissues according to three steps. First, "selection" provides potents for engineered tissues with vascularization ability. Second, "assembly technology" is used to fabricate tissues as three-dimensional structures and simultaneously inner neo-vasculature. Third, a "perfusion" technique is used for maturation of blood vessels in three-dimensional tissues. In "selection", selection of cells and materials gives the ability to promote angiogenesis in three-dimensional tissues. During the cell assembly step, cell sheet engineering, nanofilm coating technology, and three-dimensional printing technology could be used to produce vascularized three-dimensional tissues. Perfusion techniques to perfuse blood or cell culture medium throughout three-dimensional tissues with a unified inlet and outlet could induce functional blood vessels within retransplantable three-dimensional tissues. Combination of each step technology allows simulation of perivascular microenvironments in target tissues and drive vascularization in three-dimensional tissues. CONCLUSION: The biomimetic microenvironment of target tissues will induce adequate cell-cell interaction, distance, cell morphology, and function within tissues. It could be accelerated for vascularization within three-dimensional tissues and give us the functional tissues. Since vascularized three-dimensional tissues are highly functional, they are expected to contribute to the development of regenerative medicine and drug safety tests for drug discovery in the future.

Comparison of angiogenic potential between prevascular and non-prevascular layered adipose-derived stem cell-sheets in early post-transplanted period.

Layered adipose-derived stem cell (ADSC) sheet transplantation is attracting attention as a new stem cell therapeutic strategy for damaged hearts. To prolong the function of tissue-engineered constructs after transplantation, a rapid and sufficient vascularization of engrafted tissue is essential. The in vitro formation of network structures derived from endothelial cells (ECs) in grafts before transplantation contributes to the induction of functional anastomosis in vivo. This study compared the angiogenic potential of ADSC sheets containing dissociated ECs (non-prevascular cell-sheets) and networked ECs (prevascular cell-sheets) after transplantation. For preparing the two different types of ECs-containing layered cell-sheets, human umbilical vein endothelial cells (HUVECs) were sandwiched between two human ADSC sheets. Non-prevascular cell-sheets were obtained immediately after sandwiching without further cultivation. Prevascular cell-sheets were harvested form temperature-responsive culture dishes following re-cultivation for allowing them to form an EC network structure. In transplant experiments in the subcutaneous tissues of immune-deficient rat for 4 days, prevascular cell-sheets were observed to promote neovascularization with HUVEC-lined microvessels. In contrast, neovessels were hardly observed in non-prevascular cell-sheets. These results suggested that prefabricated EC network in layered cell-sheet was effective for making a rapid connection to the host vasculature in the early post-transplanted period.

Vascularization in 3D tissue using cell sheet technology.

Tissue engineering is a field of study unto itself, but in reality, it is a fusion of medicine, pharmacology, chemistry, cell biology, molecular biology and engineering. The field has been developing at an ever-increasing pace and already provides benefits to regenerative medicine in areas such as the skin and cornea. However, the problem facing all of these technologies is the diffusion limitation, which has impeded the fabrication of thicker 3D tissue. Overcoming this problem requires vascularization of 3D tissue, which is critical to any future advances. Here, we introduce our own cell sheet technology and compare it with other technologies for the fabrication of vascularized 3D tissue.

Hormone supplying renal cell sheet in vivo produced by tissue engineering technology.

Regenerative medicine is a new medical field and is expected to have a profoundly positive effect in curing difficult-to-treat diseases. Cell sheet fabrication is an important tissue engineering technology used in regenerative medicine. This study investigated the creation of a hormone-releasing tissue using cell sheet technology, which could be utilized in future therapy for chronic renal disease. Renal cell sheets were fabricated on a temperature-responsive cell culture surface with primary renal cells from adult porcine kidney. These sheets contained various kinds of renal cells that showed cyst-like formation. An important renal function is the synthesis of 1,25-dihydroxyvitamin D3, and this was confirmed in the cell sheets in vitro. Erythropoietin (EPO) production is another important renal function. This ability was also observed in the renal cell sheets in vitro, and then again after transplantation in a nude rat. In particular, the relative expression of EPO mRNA increased more under cell sheet culture conditions compared with exponential cell growth conditions. Histological analysis of the implanted renal cell sheets showed them to be Dolichos biflorus agglutinin-positive and to have regenerated renal tubular-like morphology. These results indicated that both functional and morphological regenerative renal tissues were fabricated by cell sheet technology. This study introduces a hormone-supplying treatment for renal dysfunctional diseases using engineered renal tissues. Moreover, since our renal cell sheets developed renal tubular-like structures in vivo, it holds promise for fabricating artificially engineered true renal tissue in the future.

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro.

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

"Deep-media culture condition" promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro.

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a "deep-media culture conditions." Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.

Three-dimensional cell-dense constructs containing endothelial cell-networks are an effective tool for in vivo and in vitro vascular biology research.

Angiogenesis is a complicated natural process, and understanding the mechanism by which it occurs is important for medical, pharmaceutical, and cell biological sciences. Many techniques for investigating angiogenesis have been reported. In this study, we introduced a novel application of a cell culture technique that can be used in in vitro and in vivo vascular biology research. Cultivated endothelial cells (ECs) were harvested from temperature responsive culture dishes by reducing the temperature, without the need for a proteinase treatment. For this technique, the direct contact of ECs with fibroblasts was important for the formation of a capillary-like network in vitro. Moreover, layered cell sheets containing EC-networks produced lumen and vascular structures in the three-dimensional constructs, as well as in the construct transplanted into a living body. Thus, our culture technique was able to create cell sheets and three-dimensional constructs containing EC-networks, because they preserved normal and intrinsic cell-cell direct contact and various cell adhesive factors. Moreover, the thickness of these three-dimensional (3-D) constructs could be controlled by the number of layered cell sheets. These observations indicated that our novel technology contributed to the progress of vascular biology and lead to a new tool that can be used in in vivo and in vitro vascular biology research.

Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.

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

Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology.

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

Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts.

BACKGROUND: Regenerative therapies, including myocardial tissue engineering, have been pursued as a new possibility to repair the damaged myocardium, and previously the transplantation of layered cardiomyocyte sheets has been shown to be able to improve cardiac function after myocardial infarction. We examined the effects of promoting neovascularization by controlling the densities of cocultured endothelial cells (ECs) within engineered myocardial tissues created using our cell sheet-based tissue engineering approach. METHODS AND RESULTS: Neonatal rat cardiomyocytes were cocultured with GFP-positive rat-derived ECs on temperature-responsive culture dishes. Cocultured ECs formed cell networks within the cardiomyocyte sheets, which were preserved during cell harvest from the dishes using simple temperature reduction. We also observed significantly increased in vitro production of vessel-forming cytokines by the EC-positive cardiac cell sheets. After layering of 3 cardiac cell sheets to create 3-dimensional myocardial tissues, these patch-like tissue grafts were transplanted onto infarcted rat hearts. Four weeks after transplantation, recovery of cardiac function could be significantly improved by increasing the EC densities within the engineered myocardial tissues. Additionally, when the EC-positive cardiac tissues were transplanted to myocardial infarction models, we observed significantly greater numbers of capillaries in the grafts as compared with the EC-negative cell sheets. Finally, blood vessels originating from the engineered EC-positive cardiac tissues bridged into the infarcted myocardium to connect with capillaries of the host heart. CONCLUSIONS: In vitro engineering of 3-dimensional cardiac tissues with preformed EC networks that can be easily connected to host vessels can contribute to the reconstruction of myocardial tissue grafts with a high potential for cardiac function repair. These results indicate that neovascularization can contribute to improved cardiac function after the transplantation of engineered cardiac tissues.

Cellular control of tissue architectures using a three-dimensional tissue fabrication technique.

Tissue engineering seeks to provide regenerated tissue architectures in vitro but has not yet successfully created thick, highly vascularized, multi-functional tissues replicating native structure. We describe a novel method to fabricate pre-vascularized tissue equivalents using multi-layered cultures combining micro-patterned endothelial cells as vascular pre-cursors with fibroblast monolayer sheets as tissue matrix. Stratified tissue equivalents are constructed by alternately layering fibroblast monolayer sheets with patterned endothelial cell sheets harvested from newly developed thermo-responsive micro-patterned surfaces alternating 20 microm-wide cell-adhesive lanes with 60 microm non-adhesive zones. Cell culture substrates covalently grafted with different thermo-responsive polymers permit spatial switching of cell adhesion and detachment using applied small temperature changes. Endothelial cell patterning fidelity was maintained within the multi-layer tissue constructs after assembly, leading to self-organization into microvascular-like networks after 5-day tissue culture. This novel technique holds promise for the study of cell-cell communications and angiogenesis in reconstructed, three-dimensional environments as well as for the fabrication of tissues with complex, multicellular architecture.

Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential.

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