Cell-dense Tissues Research Articles

Perfusable vascular tree like construction in 3D cell-dense tissues using artificial vascular bed.

Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to introduce functional vascular networks observable as vascular tree, finely spaced at intervals of tens of micrometers as in living tissues, into a 3D cell-dense tissue. Herein, we propose a method for introducing numerous vascular networks that can be perfused with blood into 3D tissues constructed by cell sheet engineering. We devise an artificial vascular bed using a hydrogel that is barely deformed by cells, enabling perfusion of the culture medium directly beneath the cell sheets. Triple-layered cell sheets with an endothelial cell network prepared by fibroblast co-culture are transplanted onto the vascular bed and subjected to perfusion culture. We demonstrate that numerous vascular networks are formed with luminal structures in the cell sheets and can be perfused with India ink or blood after a five-day perfusion culture. Histological analysis also demonstrates that perfusable vascular structures are constructed at least 100μm intervals uniformly and densely within the tissues. The results suggest that our perfusion culture method enhances vascularization within the 3D cell-dense tissues and enables the introduction of functional vasculature macroscopically observable as vascular tree in vitro. In conclusion, this technology can be used to fabricate functional tissues and organs for regenerative therapies and in vitro experimental models.

Cell division in tissues enables macrophage infiltration.

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Fundamental Technologies and Recent Advances of Cell-Sheet-Based Tissue Engineering.

Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications.

A modular polymer microbead angiogenesis scaffold to characterize the effects of adhesion ligand density on angiogenic sprouting

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1–0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.

625 Fibroblast-specific overexpression of collagen XII leads to a delay in skin repair

FACIT collagen XII decorates large collagen fibrils within stiff connective tissues. While its mutation or loss causes disorders of connective tissues in humans, its overabundance was described in fibrotic lesions of scleroderma patients and corneal scarring, indicating a vital role of collagen XII in ECM homeostasis. We postulate that collagen XII has the potential to influence tissue properties by linking scaffolding molecules, transmitting signals to cells or by modulating availability of growth factors. We study the roles of dermal collagen XII during reconstitution of the skin following excisional wounding in mice with fibroblast-specific overexpression of collagen XII and controls. In controls, collagen XII was abundantly deposited into early granulation tissue and persisted at high levels throughout the healing process. The overexpressing mice showed delayed wound healing with enlarged, more cell dense granulation tissue. The number of macrophages was significantly increased. Characterization of macrophage subpopulations revealed that in the collagen XII overexpressing mice, the macrophages maintained an early wound healing (M1-like) phenotype, indicating that overall wound healing was delayed. In vitro interaction studies between macrophages and collagen XII showed that only M1, but not unstimulated or M2 polarized macrophages were able to adhere to collagen XII. These findings led to the hypothesis that overproduction of collagen XII favors selective adhesion and persistent presence of M1 polarized, pro-inflammatory macrophages causing a delay in the healing process and possibly more severe scarring. Our future work will identify the receptors mediating the selective adhesion of M1 polarized macrophages to collagen XII and characterize scar tissue maturity at late stages of the healing process.

Effective stacking and transplantation of stem cell sheets using exogenous ROS-producing film for accelerated wound healing

Extensive skin loss caused by burns or diabetic ulcers may lead to major disability or even death. Therefore, cell-based therapies that enhance skin regeneration are clinically needed. Previous approaches have been applied the injections of cell suspensions and the implantation of biodegradable three-dimensional scaffolds seeded cells. However, these treatments have limits due to poor localization of the injected cells and insufficient delivery of oxygen and nutrients to cells. Recently, cell sheet-based tissue engineering has been developed to transplant cell sheets, which are cell-dense tissues without scaffolds. Because cell density is one of the important factors for improving the therapeutic effect of cell transplantation, transplanting layered cell sheet constructs can promote the recovery of tissue function and tissue regeneration compared with a single cell sheet. Thus, this study designed ROS-induced cell sheet stacking method with newly fabricated hematoporphyrin-incorporated polyketone film (Hp-PK film) to enhance cell sheet delivery efficiency and application in wound healing. We have demonstrated the therapeutic effect of a multi-layered mesenchymal stem cell sheets onto a full-thickness wound defect in nude mice. Consequentially, three-layered cell sheets transplanted and stacked by ROS-induced method promoted angiogenesis and skin regeneration at the wound site. Thus, our strategy based on Hp-PK film, which allows for easy stacking and transplantation of cell sheets, could be applied to enhance tissue regeneration. Statement of SignificanceWe herein report exogenous ROS-induced cell sheet stacking method with newly fabricated hematoporphyrin-incorporated polyketone film (Hp-PK film) to enhance cell sheet transplantation efficiency and application in wound healing. Although there are several ways to stack-up cell sheets, all of these methods have limitations in transplanting the cell sheet directly to the target site. The method is simple and takes a relatively short time compared to previously reported methods for stacking and transplanting cell sheets. Thus, our study will provide a scientific impact because the method of applying exogenous ROS generated from Hp-PK film on cell detachment can transplant the cell sheet through a process of putting a cell sheet-cultured film on the lesion, irradiating with light, and then removing only the film.

Thermally-triggered fabrication of cell sheets for tissue engineering and regenerative medicine.

Cell transplantation is a promising approach for promoting tissue regeneration in the treatment of damaged tissues or organs. Although cells have conventionally been delivered by direct injection to damaged tissues, cell injection has limited efficiency to deliver therapeutic cells to the target sites. Progress in tissue engineering has moved scaffold-based cell/tissue delivery into the mainstream of tissue regeneration. A variety of scaffolds can be fabricated from natural or synthetic polymers to provide the appropriate culture conditions for cell growth and achieve in-vitro tissue formation. Tissue engineering has now become the primary approach for cell-based therapies. However, there are still serious limitations, particularly for engineering of cell-dense tissues. "Cell sheet engineering" is a scaffold-free tissue technology that holds even greater promise in the field of tissue engineering and regenerative medicine. Thermoresponsive poly(N-isopropylacrylamide)-grafted surfaces allow the fabrication of a tissue-like cell monolayer, a "cell sheet", and efficiently delivers this cell-dense tissue to damaged sites without the use of scaffolds. At present, this unique approach has been applied to human clinical studies in regenerative medicine. Furthermore, this thermally triggered cell manipulation system allows us to produce various types of 3D tissue models not only for regenerative medicine but also for tissue modeling, which can be used for drug discovery. Here, new cell sheet-based technologies are described including vascularization for scaled-up 3D tissue constructs, induced pluripotent stem (iPS) cell technology for human cell sheet fabrication and microfabrication for arranging tissue microstructures, all of which are expected to produce more complex tissues based on cell sheet tissue engineering.

Rapid creation system of morphologically and functionally communicative three-dimensional cell-dense tissue by centrifugation.

This study reports a rapid fabrication system of a morphologically and functionally communicative three-dimensional (3D) cell-dense tissue without scaffolds by centrifugation. The tight adhesion between C2C12 myoblasts and culture surface was accelerated without significant cell damage by centrifugation (80 x g, 37 °C, 30 min). A thicker tissue created on a temperature-responsive culture surface was harvested by decreasing temperature. The 3D myoblast tissues having approximately 200 μm-thickness were created at 1.5 h [centrifugation (80 x g, 37 °C) for 30 min and tissue harvest for 1 h]. However, in the case of without centrifugation, the myoblast tissues had fragile parts even at 7.5 h after the incubation. Additionally, electrically/functionally communicative and thicker human induced pluripotent stem (iPS) cell-derived cardiac tissues were created rapidly by the centrifugation and cultivation at 37 °C. We report a centrifugation system that significantly shortens the creation time of 3D tissues. We envision that this procedure will contribute to the field of tissue engineering and regenerative medicine. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1447-1453, 2018.

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet-Based Regenerative Therapy and 3D Tissue Fabrication.

This chapter describes the concept of "cell sheet engineering" for the creation of transplantable cellular tissues and organs. In contrast to scaffold-based tissue engineering, cell sheet engineering facilitates the reconstruction of scaffold-free, cell-dense tissues. Cell sheets were harvested by changing the temperature of thermoresponsive cell culture surfaces modified with poly(N-isopropylacrylamide) (PIPAAm) with a thickness on the nanometer scale. The transplantation of 2D cell sheet tissues has been used in clinical settings. Although 3D tissues were formed simply by layering 2D cell sheets, issues related to vascularization within 3D tissues and the large-scale production of cells must be addressed to create thick and large 3D tissues and organs.

Effects of cell packing on chemoattractant distribution within a tissue

Diffusible signals provide critical information to cells in biological systems, often in a concentration-dependent manner. In animal development, such signals can determine different cell fates or guide motile cells to their proper locations. It is well-known that migrating cells respond to graded chemoattractant cues by moving toward areas of higher concentrations. However, it is not clear how cell-dense animal tissues impact the distribution of chemoattractants in three dimensions. We leverage the simple architecture of the Drosophila egg chamber to explore this idea. In this context, sixteen large germline cells are packed together, enveloped by a somatic epithelium. A small set of epithelial cells, the border cells, form a motile cell cluster and respond to guidance signals by moving across the egg chamber during oogenesis. We created a geometrically-realistic model of the egg chamber and determined the distribution of the chemoattractants through that domain using a reaction-diffusion system. We used this information to determine reasonable biophysical parameters of chemoattractant that would facilitate gradient formation in the appropriate developmental time, and to explore the effects of different secretion locations in the egg chamber. Our model revealed several interesting features: The chemoattractant is more concentrated and the gradient sets up more quickly in a cell-packed space, and cell packing creates dips in the concentration and changes in gradient along the migratory path. We simulated migration with our calculated chemoattractant gradient and compared it to that with a constant gradient. We found that with our calculated gradient, migration was slower initially than in the constant gradient, which could be due to the exponential nature of the gradient or other variation in signal due to the heterogeneous domain. Given the many situations in which cell migration occurs in complex spatio-temporal environments, including development, immune response, and cancer metastasis, we believe modeling chemoattractant distribution in heterogeneous domains is widely relevant.

Cell sheet engineering for integrating functional tissue in vivo: Successes and challenges

Abstract

Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue.

Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germ line-derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.

Bladder reconstruction using scaffold-less autologous smooth muscle cell sheet engineering: early histological outcomes for autoaugmentation cystoplasty.

To investigate the feasibility of a new approach for cystoplasty using autologous smooth muscle cell (SMC) sheet and scaffold-less bladder tissue engineering with the main focus on histological outcomes in a rabbit model. In all, 24 rabbits were randomly divided into two groups. In the experimental group, SMCs were obtained from the bladder muscular layer, labelled with PKH-26, and seeded on temperature-responsive culture dishes. Contiguous cell sheets were noninvasively harvested by reducing the temperature and triple-layer cell-dense tissues were constructed. After partial detrusorectomy, the engineered tissue was transplanted onto the urothelial diverticulum. The control group underwent partial detrusorectomy followed by peritoneal fat coverage. At 2, 4, and 12 weeks the rabbits were humanely killed and haematoxylin and eosin, Masson's trichrome, cluster of differentiation 34 (CD34), CD31, CD3, CD68, α-smooth muscle actin (α-SMA), picrosirius red, and pentachrome staining were used to evaluate bladder reconstruction. At 2 weeks after SMC-sheet grafting, PKH-26 labelled SMCs were evident in the muscular layer. At 4 weeks, 79.1% of the cells in the muscular layer were PKH-positive cells. The portion of the muscular layer increased in the experimental group during the follow-up and was similar to normal bladder tissue after 12 weeks. α-SMA staining showed well organised muscle at 4 and 12 weeks. CD34+ endothelial progenitor cells and CD31+ microvessels increased continuously and peaked 4 and 12 weeks after grafting, respectively. In the present study, we show that autologous SMC-sheet grafting has the potential for reliable bladder reconstruction and is technically feasible with a favourable evolution over the 12 weeks following implantation. Our findings could pave the way toward future bladder tissue engineering using the SMC-sheet technique.

Thermoresponsive Nanofabricated Substratum for the Engineering of Three-Dimensional Tissues with Layer-by-Layer Architectural Control

Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels

Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200–700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.

Endothelial colony-forming cells for preparing prevascular three-dimensional cell-dense tissues using cell-sheet engineering

Vascular-derived endothelial cell (EC) network prefabrication in three-dimensional (3D) tissue constructs before transplantation is useful for inducing functional anastomosis with the host vasculature. However, the clinical application of ECs is limited by cell isolation from the existing vasculature, because of the requirement for invasive biopsies and difficulty in obtaining a sufficient number of cells. Endothelial colony-forming cells (ECFCs), which are a subtype of endothelial progenitor cells in the blood, have a strong proliferative and vasculogenic potential. This study attempted to fabricate prevascular 3D cell-dense tissue constructs using cord blood-derived ECFCs and evaluate the in vivo angiogenic potential of these constructs. Human umbilical vascular endothelial cells (HUVECs) were also used in comparison with ECFCs, which were sandwiched between two human dermal-derived fibroblast (FB) sheets using a fibrin-coated cell-sheet manipulator. The inserted ECFCs in double-layered FB sheets were cultured for 3 days, resulting in the formation of network structures similar to those of HUVECs. Additionally, when ECFCs were sandwiched with three FB sheets, a lumen structure was found in the triple-layered cell-sheet constructs at 3 days after co-culture. These constructs containing ECFCs were transplanted into the subcutaneous tissue of immune-deficient rats. One week after transplantation, ECFC-lined functional microvessels containing rat erythrocytes were observed in the same manner as transplanted HUVEC-positive grafts. These results suggest that ECFCs might become an alternative cell source for fabricating a prevascular structure in 3D cell-dense tissue constructs for clinical application. Copyright © 2013 John Wiley & Sons, Ltd.

A New Age of Regenerative Medicine: Fusion of Tissue Engineering and Stem Cell Research

After the biotech medicine era, symbolized by the progress of genetic engineering and the developments of physiologically active substances such as proteins and cytokines as medicine, the regenerative medicine era, when cells and tissues become medicine, is going to start and getting popular in recent years in the world. In particular, regenerative medicine is highly expected as curative-therapeutic treatments that can cure patients with obstinate diseases and physically impaired function, while the conventional symptomatic treatments are unable to do so. For supporting and enhancing the progress of regenerative medicine, remarkable advancements in regenerative medical sciences, and cell and tissue engineering are desired worldwide. Furthermore, to achieve new treatments based on regenerative medicine, multidisciplinary approaches from various fields such as molecular biology, cell biology, science and engineering, and pharmaceutical and medical sciences are essential. Regenerative medicine should be recognized as a completely new interdisciplinary academic discipline, which is unable to be obtained from the extrapolations of vertically connected and conventional academic disciplines. Therefore, for realizing regenerative medicine, not only the reconstruction of conventional medical science but also the establishment of new academic field, which integrates and assimilates scientific and engineering technologies, and biotechnology, become important. For example, technology controlling the expansion and differentiations of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is essential for obtaining the sufficient numbers of the cells allowing desired medical treatments to be realized. For separating remaining small amounts of undifferentiated ES and iPS cells from the differentiated cells with a high accuracy, an interdisciplinary research project having the horizontally integration of science, engineering, medicine should be considered. Further, the developments of technologies, which can efficiently transplant somatic cells in the body, are more important than those of cell sources, and new cooperative and integrative systems having various academic disciplines including medicine, science, engineering, and pharmacy are extremely important. For example, harvested cells, which are obtained from culture dishes by treating them with enzymes including trypsin or dispase after being cultured and expanded, are damaged in their structures and functions by enzymatically cleaving their cell-membrane proteins. Upon the direct injection of suspension containing damaged cells following the enzyme treatments to the tissue or organ, approximately 95% of the cells fail to stay in the target, and only less than several percent of the cells transplanted in the organ is speculated to be effective (Hofmann et al., 2005). The unwelcome fact that possible therapeutic effects are expected with a small number of transplanted cells should be noticed seriously. Tissue engineering is extremely important in terms of the more efficient engraftment of cell transplantation and is expected to proceed further with the integration of the technology of biomaterials contacting cell directly and bioand medical-technologies. One of the tissue engineering approaches is biodegradable polymer scaffold-based methods (Langer and Vacanti, 1993). It is important to promote research that investigates how the shape and function of cells cultured in the scaffolds are maintained after transplantation. In this special issue, the recent progress of tissue engineering approaches using biodegradable polymer scaffolds are reviewed by Rui Reis and his colleagues on periodontal tissue, Charles Vacanti and Koji Kojima on trachea, Toshiharu Shinoka and his colleagues on vasculature, Dietmar Hutmacher and his colleagues on osteochondral tissue, and Stephan Badylak and his colleagues on skeletal muscle. For maintaining cell-dense thicker tissue viability, sufficient amounts of oxygen and nutrients should be supplied into the tissue. However, scaffoldbased tissue has a heavy burden to have supplies of oxygen and nutrients from the scaffold surface by diffusion, which is hampered with increasing cell density. The developments of new technologies to break through this problem would be desired. Another approach for tissue engineering is cell sheet-based methods (Yang et al., 2007). Dense monolayered cell sheets that are harvested from temperature responsive culture dishes are fully viable and almost all of the cells are engrafted and keep their function after transplantation. In this special issue, the cutting edge research using cell sheet-based tissue engineering methods are reviewed by Teruo Okano and Kazuo Ohashi on liver and islets, Tatsuya Shimizu and Katsuhisa Matsuura on cardiac tissue, Masayuki Yamato and his colleagues on periodontal tissue, and Masato Sato and his colleagues on articular cartilage. In the cornea,

Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas

Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings.

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.

Cardiac Cell Sheet Transplantation Improves Damaged Heart Function via Superior Cell Survival in Comparison with Dissociated Cell Injection

Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature capillaries were also observed in the cardiac cell sheet transplantation. Additionally, the numbers of apoptotic cells with deterioration of integrin-mediated attachment were significantly lower after cardiac cell sheet transplantation. In accordance with increased cell survival, cardiac function was significantly improved after cardiac cell sheet transplantation in comparison to cell injection. Cell sheet transplantation can repair damaged hearts through improved cell survival and should become a promising therapy in cardiovascular regenerative medicine.