Fibroblast Sheets Research Articles

Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model

BackgroundPulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection.ObjectiveIn this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface.ResultsOne month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet’s angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host.ConclusionsDFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

One-step fabrication of cell sheet-laden hydrogel for accelerated wound healing

Full-thickness skin wounds are have continued to be reconstructive challenges in dermal and skin appendage regeneration, and skin substitutes are promising tools for addressing these reconstructive procedures. Herein, the one-step fabrication of a cell sheet integrated with a biomimetic hydrogel as a tissue engineered skin for skin wound healing generated in one step is introduced. Briefly, cell sheets with rich extracellular matrix, high cell density, and good cell connections were integrated with biomimetic hydrogel to fabricate gel + human skin fibroblasts (HSFs) sheets and gel + human umbilical vein endothelial cells (HUVECs) sheets in one step for assembly as a cell sheet-laden hydrogel (CSH). The designed biomimetic hydrogel formed with UV crosslinking and ionic crosslinking exhibited unique properties due to the photo-generated aldehyde groups, which were suitable for integrating into the cell sheet, and ionic crosslinking reduced the adhesive force toward the substrate. These properties allowed the gel + cell sheet film to be easily released from the substrate. The cells in the harvested cell sheet maintained excellent viability, proliferation, and definite migration abilities inside the hydrogel. Moreover, the CSH was implanted into a full-thickness skin defects to construct a required dermal matrix and cell microenvironment. The wound closure rate reached 60.00 ± 6.26% on the 2nd day, accelerating mature granulation and dermis formation with skin appendages after 14 days. This project can provide distinct guidance and strategies for the complete repair and regeneration of full-thickness skin defects, and provides a material with great potential for tissue regeneration in clinical applications.

Effects of Myostatin on Nuclear Morphology at the Myotendinous Junction.

Myostatin (Myo) is known to suppress skeletal muscle growth, and was recently reported to control tendon homeostasis. The purpose of the present study was to investigate the regulatory involvement of Myo in the myotendinous junction (MTJ) in vivo and in vitro. After Achilles tendon injury in mice, we identified unexpected cell accumulation on the tendon side of the MTJ. At postoperative day 7 (POD7), the nuclei had an egg-like profile, whereas at POD28 they were spindle-shaped. The aspect ratio of nuclei on the tendon side of the MTJ differed significantly between POD7 and POD28 (p = 4.67 × 10-34). We then investigated Myo expression in the injured Achilles tendon. At the MTJ, Myo expression was significantly increased at POD28 relative to POD7 (p = 0.0309). To investigate the action of Myo in vitro, we then prepared laminated sheets of myoblasts (C2C12) and fibroblasts (NIH3T3) (a pseudo MTJ model). Myo did not affect the expression of Pax7 and desmin (markers of muscle development), scleraxis and temonodulin (markers of tendon development), or Sox9 (a common marker of muscle and tendon development) in the cell sheets. However, Myo changed the nuclear morphology of scleraxis-positive cells arrayed at the boundary between the myoblast sheet and the fibroblast sheet (aspect ratio of the cell nuclei, myostatin(+) vs. myostatin(-): p = 0.000134). Myo may strengthen the connection at the MTJ in the initial stages of growth and wound healing.

"Fuzzy hair" promotes cell sheet detachment from thermoresponsive brushes already above their volume phase transition temperature.

Thermoresponsive poly(glycidyl ether) (PGE) brushes have shown to be viable substrates for the culture and temperature-triggered detachment of confluent cell sheets. Surface-tethered PGEs with a cloud point temperature (TCP) around ~30°C exhibit phase transitions well-centered within the physiological range (20-37°C), which makes them ideal candidates for cell sheet fabrication. However, PGEs with TCPs at ~20°C also afford the detachment of various types of cell sheets, even at room temperature (20-23°C), i.e., above the polymers' TCPs. In this study, we investigate the phase transition of PGE brushes tethered to polystyrene (PS) culture substrates with varying grafting density and TCP to arrive at a mechanistic understanding of their functionality in cell sheet fabrication. Using quartz crystal microbalance with dissipation (QCM-D) monitoring, we demonstrate that brushes fabricated from PGEs with TCPs at ~20°C display volume phase transition temperatures (VPTTs) well below room temperature. Although the investigated coatings obviously do not exhibit marked thermal switching in terms of brush hydration and layer thickness, their physical properties at the brush-water interface, as ascertained by QCM-D and AFM measurements, undergo subtle changes upon cooling from 37°C to room temperature which is sufficient to promote cell sheet detachment. Thus, it appears that discreet rehydration of the outmost brush layer, resembling "fuzzy hair" at the brush-water interface, renders the surfaces less protein- and cell-adhesive at room temperature. This minor structural change of the interface allows for the reliable detachment of human dermal fibroblast sheets already at 20°C well above the VPTT of the brushes.

In vitro circulation model driven by tissue-engineered dome-shaped cardiac tissue

The heart is an essential organ for animals and humans. With the increased availability of pluripotent stem cells, the use of three-dimensional cardiac tissues consisting of cultured cardiomyocytes in in vitro drug evaluation has been widely studied. Several models have been proposed for the realization of the pump function, which is the original function of the heart. However, there are no models that simulate the human circulatory system using cultured cardiac tissue. This study shows that a dome-shaped cardiac tissue fabricated using the cell sheet stacking technique can achieve a heart-like pump function and circulate culture medium, there by mimicking the human circulatory system. Firstly, human induced pluripotent stem cells were differentiated into autonomously beating cardiomyocytes, and cardiomyocyte cell sheets were created using temperature-responsive culture dishes. A cardiomyocyte sheet and a human dermal fibroblast sheet were stacked using a cell sheet manipulator. This two-layered cell sheet was then inflated to create a dome-shaped cardiac tissue with a base diameter of 8 mm. The volume of the dome-shaped cardiac tissue changed according to the autonomous beating. The stroke volume increased with the culture period and reached 21 ± 8.9 μl (n = 6) on day 21. It also responded to β-stimulant and extracellular calcium concentrations. Internal pressure fluctuations were also recorded under isovolumetric conditions by dedicated culture devices. The peak heights of pulsatile pressure were 0.33 ± 0.048 mmHg (n = 3) under a basal pressure of 0.5 mmHg on day 19. When the tissue was connected to a flow path that had check valves applied, it drove a directional flow with an average flow rate of approximately 1 μl s−1. Furthermore, pressure–volume (P–V) diagrams were created from the simultaneous measurement of changes in pressure and volume under three conditions of fluidic resistance. In conclusion, this cardiac model can potentially be used for biological pumps that drive multi-organ chips and for more accurate in vitro drug evaluation using P–V diagrams.

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell’s own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract’s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.

Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model.

Simple SummaryFulminant myocarditis (FM) is a serious inflammatory lesion of the myocardium accompanied by cardiac dysfunction, transitioning to end-stage heart failure. Due to such a difficult pathology, a therapeutic strategy that exerts a steadfast effect has yet to be developed. Blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. The aim of this study was to investigate whether transplantation therapy with hybrid cell sheets of fibroblasts and QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM) induced by purified porcine cardiac myosin. The transplanted hybrid cell sheet exerts cardioprotective effects against EAM, resulting in limited left ventricular remodeling and partially improved cardiac functions due to revascularization, anti-inflammation, and anti-fibrosis. Thus, tissue engineering using hybrid cell sheets of fibroblasts constructed with QQMNCs is expected to provide an effective therapeutic option for patients with severe FM.Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure–volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM.

Repair of peripheral nerve injuries using a prevascularized cell-based tissue-engineered nerve conduit

One of the major challenges in the development of a larger and longer nerve conduit for peripheral nerve repair is the limitation in oxygen and nutrient diffusion within the tissue after transplantation preventing Schwann cell and axonal migration. This restriction is due to the slow neovascularization process of the graft starting from both nerve endings. To overcome this limitation, we propose the design of a living tissue-engineered nerve conduit made of an internal tube with a three-dimensional structure supporting axonal migration, which is inserted inside a hollow external tube that plays the role of an epineurium and is strong enough to be stitched to the severed nerve stumps. The internal tube is made of a rolled living fibroblast sheet and can be seeded with endothelial cells to promote the formation of a network containing capillary-like structures which allow rapid inosculation with the host nerve microvasculature after grafting. Human nerve conduits were grafted in immunodeficient rats to bridge a 15 mm sciatic nerve gap. Human capillaries within the pre-vascularized nerve conduit successfully connected to the host circulation 2 weeks after grafting. Twenty-two weeks after surgery, rats transplanted with the nerve conduits had a similar motor function recovery compared to the autograft group. By promoting rapid vascularization of the internal nerve tube from both ends of the nerve stumps, this endothelialized nerve conduit model displays a favorable environment to enhance axonal migration in both larger caliber and longer nerve grafts.

Freezing of cell sheets using a 3D freezer produces high cell viability after thawing

In cell therapy, transplanting an appropriate number of cells to the target site is crucial. One way to achieve this is to transplant cell sheets. Transplantation of cell sheets has already been utilized for various diseases in clinical practice. However, reducing the cost of cell sheet utilization is essential so as to facilitate the spread of regenerative medicine. Several ways to reduce costs are available, one of which is the use of allogenic cells. Another alternative is the use of cell sheets, which necessitates the development of methods for freezing cell sheets. This is the first study to report the use of a 3D Freezer for freezing cells. 3D Freezers have been used in the field of food processing and technology for a long time. The 3D Freezer freezes objects using cold air at a uniform temperature from all directions. In this study, we analyzed the cooling speed of human fibroblast sheets in 11 cell preservation solutions using a 3D Freezer and a Program Freezer. The cooling speed was −2 °C per min in the 3D Freezer. Supercooling in 10 cell preservation solutions was lower in the 3D Freezer than in the Program Freezer. Cell viability after freeze–thaw of the cell sheets using 3D Freezer was more than 70% in five cell preservation solutions. The levels of hepatocyte growth factor and transforming growth factor-β1 were the same not only in the fibroblast sheets frozen using the five cell preservation solutions but also in the non-frozen fibroblast sheets. These results suggest that the 3D Freezer can freeze implantable cell sheets immediately after thawing.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

Osteogenic Response to Polysaccharide Nanogel Sheets of Human Fibroblasts After Conversion Into Functional Osteoblasts by Direct Phenotypic Cell Reprogramming.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-β signal) on freeze-dried nanogel-cross-linked porous (FD-NanoClip) polysaccharide sheets or fibers. Then, the ability of these directly converted osteoblasts (dOBs) to produce calcified substrates and the expression of osteoblast genes were analyzed in comparison with osteoblasts converted by exactly the same procedure but seeded onto a conventional atelocollagen scaffold. dOBs exposed to FD-NanoClip in both sheet and fiber morphologies produced a significantly higher concentration of calcium deposits as compared to a control cell sample (i.e., unconverted fibroblasts), while there was no statistically significant difference in calcification level between dOBs exposed to atelocollagen sheets and the control group. The observed differences in osteogenic behaviors were interpreted according to Raman spectroscopic analyses comparing different polysaccharide scaffolds and Fourier transform infrared spectroscopy analyses of dOB cultures. This study substantiates a possible new path to repair large bone defects through a simplified transplantation procedure using FD-NanoClip sheets with better osteogenic outputs as compared to the existing atelocollagen scaffolding material.

Delivery of a spheroids-incorporated human dermal fibroblast sheet increases angiogenesis and M2 polarization for wound healing

Low cell engraftment is a major problem in tissue engineering. Although various methods related with cell sheets have been attempted to resolve the issue, low cell viability due to oxygen and nutrient depletion remains an obstacle toward advanced therapeutic applications. Cell therapy using fibroblasts is thought of as a good alternative due to the short doubling times of fibroblasts together with their immunomodulatory properties. Furthermore, three-dimensional (3D) fibroblasts exhibit unique angiogenic and inflammation-manipulating properties that are not present in two-dimensional (2D) forms. However, the therapeutic effect of 3D fibroblasts in tissue regeneration has not been fully elucidated. Macrophage polarization has been widely studied, as it stimulates the transition from the inflammation to the proliferation phase of wound healing. Although numerous strategies have been developed to achieve better polarization of macrophages, the low efficacy of these strategies and safety issues remain problematic. To this end, we introduced a biocompatible flat patch with specifically designed holes that form a spheroids-incorporated human dermal fibroblast sheet (SIS) to mediate the activity of inflammatory cytokines for M2 polarization and increase angiogenic efficacy. We further confirmed in vivo enhancement of wound healing with an SIS-laden skin patch (SISS) compared to conventional cell therapy.

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.

Autologous Multilayered Fibroblast Sheets Can Reinforce Bronchial Stump in a Rat Model

Bronchopleural fistula is one of the most serious postoperative complications caused by the incomplete healing of a bronchial stump. Fibroblasts play an important role in wound healing by facilitating connective tissue formation and inducing angiogenesis. We developed a method for production of multilayered fibroblast sheets that secreted some growth factors and promoted wound healing. The present study aimed to assess the treatment effect of multilayered fibroblast sheets on bronchial stump healing. In this rat model, left pneumonectomy was performed, and multilayered fibroblast sheets derived from autologous oral mucosal tissues were transplanted to the bronchial stump. The changes in the bronchial stump were examined macroscopically, histologically, and mechanically. The fibroblast sheets promoted the formation of thick connective tissues around the bronchial stump. The formed connective tissues were accompanied by new blood vessels, and fibrosis was observed over time. Then, 7 days after the transplantation of the fibroblast sheets, the bronchial wall became significantly thicker, and the area of the blood vessels for the bronchial wall tissues was significantly larger in the experimental group than in the control group. In addition, the burst pressure in the bronchial stump was significantly higher in the experimental group than in the control group. Bronchial stumps were reinforced by the transplantation of multilayered fibroblast sheets derived from autologous oral mucosal tissues.

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.

Amniotic Stem Cells Cultured on Thermoresponsive Polymers Allow Obtaining a Full Cell Sheet

Amniotic stem cells promote adhesion and migration of epithelial cells. Obtaining a full sheet containing amniotic stem cells seems to be the best solution for the treatment of burn wounds. The main advantage of this method is obtaining a full sheet of cells by lowering the temperature below the transition temperature, which does not affect extracellular matrix. The purpose of this work was to produce a skin substitute—a full sheet consisting of amniotic mesenchymal stem cells—and compare with well-known fibroblast sheet. Amniotic membrane cells revealed better tendency to full sheet detachment than fibroblasts. Confluence after 24 hours was always higher on polymer-coated dishes than on normal polypropylene dishes. Also viability was higher than on the control culture dish, while the number of apoptotic cells was always highest on polypropylene (control). Ile-Lys-Val-ala-Val (IKVAV) 0.28 addition to poly (poly [ethylene glycol] ethyl methacrylate) (PTEGMA) caused best cell confluence and highest percentage of cells in mitosis phase of cell cycle, but also worst cell detachment was observed in both cell types on PTEGMA IKVAV 0.28. Viability of cells transferred in cell sheet form onto a new culture dish was higher than when detached as suspension enzymatically. Additionally, percentage of apoptotic cells transferred in cell sheet form onto a new culture dish was always lower than when detached as suspension enzymatically. Culturing of PTEGMA, PTEGMA IKVAV 0.28 and PTEGMA IKVAV 0.14 have a stimulating effect on number of cells in mitosis in amniotic cell culture even after cell sheet transfer onto a new dish, whereas such effect with fibroblast was not observed.

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.

A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice.

The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.

Off-the-Shelf Cell Sheets as a Pleural Substitute for Closing Visceral Pleural Injuries.

During pulmonary resections, removal of visceral pleura is frequently required, resulting in lung air leakage (LAL) and bleeding. Especially persistent LAL after pulmonary surgery has negative consequences. Current surgical procedures are ineffective in closing these visceral pleural injuries. Previously, the authors' laboratory has developed a novel and effective LAL sealant using tissue-engineered cell sheets harvested from temperature-responsive culture dishes. The clinical application of fresh fibroblast sheets (FSs) is limited by several problems related to the cell culture period, mass production, preservation, and transportation. Therefore, cryopreservation of FSs and feasibility of off-the-shelf FSs for repairing visceral pleural defects were investigated. Over 3 to 6 months, harvested skin-derived FSs in Dulbecco's modified Eagle's medium supplemented with 10% dimethyl sulfoxide were stored in an atmosphere of liquid nitrogen. The amounts of cytokines (basic fibroblast growth factor [bFGF] and vascular endothelial growth factor) released from frozen-thawed FSs were determined. bFGF levels were significantly elevated in frozen-thawed FSs compared with fresh FSs. After a visceral pleural injury model was created, a frozen-thawed skin-derived FS was transplanted directly to the defect. One month after transplantation, the frozen-thawed FS remained on the pleural surface, resulting in permanent closure, suggesting that cells in the off-the-shelf FS had the ability to proliferate and release various cytokines. Frozen-thawed FSs were useful for closing LALs during pulmonary surgery as an off-the-shelf technique and would be used as a pleural substitute.

Engineering stem cell cardiac patch with microvascular features representative of native myocardium.

The natural myocardium is a highly aligned tissue with an oriented vasculature. Its characteristic cellular as well as nanoscale extracellular matrix (ECM) organization along with an oriented vascular network ensures appropriate blood supply and functional performance. Although significant efforts have been made to develop anisotropic cardiac structure, currently neither an ideal biomaterial nor an effective vascularization strategy to engineer oriented and high-density capillary-like microvessels has been achieved for clinical cardiovascular therapies. A naturally derived oriented ECM nanofibrous scaffold mimics the physiological structure and components of tissue ECM and guides neovascular network formation. The objective of this study was to create an oriented and dense microvessel network with physiological myocardial microvascular features.Methods: Highly aligned decellularized human dermal fibroblast sheets were used as ECM scaffold to regulate physiological alignment of microvascular networks by co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs). The influence of topographical features on hMSC and EC interaction was investigated to understand underlying mechanisms of neovasculature formation.Results: Results demonstrate that the ECM topography can be translated to ECs via CD166 tracks and significantly improved hMSC-EC crosstalk and vascular network formation. The aligned ECM nanofibers enhanced structure, length, and density of microvascular networks compared to randomly organized nanofibrous ECM. Moreover, hMSC-EC co-culture promoted secretion of pro-angiogenic growth factors and matrix remodeling via metalloprotease-2 (MMP-2) activation, which resulted in highly dense vascular network formation with intercapillary distance (20 μm) similar to the native myocardium.Conclusion: HMSC-EC co-culture on the highly aligned ECM generates physiologically oriented and dense microvascular network, which holds great potential for cardiac tissue engineering.