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 100 µm 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.

Bioengineering of a scaffold-less three-dimensional tissue using net mould.

Tissue engineering has attracted attention worldwide because of its application in regenerative medicine, drug screening, and cultured meat. Numerous biofabrication techniques for producing tissues have been developed, including various scaffold and printing methods. Here, we have proposed a novel tissue engineering method using a net metal mould without the use of a scaffold. Briefly, normal human dermal fibroblasts seeded on a dimple plate were subjected to static culture technique for several days to form spheroids. Spheroids of diameter ⩾200µm were poured into a net-shaped mould of gap ⩽100µm and subjected to shake-cultivation for several weeks, facilitating their fusion to form a three-dimensional (3D) tissue. Through this study, we successfully constructed a scaffold-free 3D tissue having strength that can be easily manipulated, which was difficult to construct using conventional tissue engineering methods. We also investigated the viability of the 3D tissue and found that the condition of the tissues was completely different depending on the culture media used. Collectively, this method allows scaffold-free culture of 3D tissues of unprecedented thickness, and may contribute largely to next-generation tissue engineering products.

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue.

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.

Measuring the Contractile Force of Multilayered Human Cardiac Cell Sheets.

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

A novel rat model of inflammatory bowel disease developed using a device created with a 3D printer.

OBJECTIVE: Inflammatory bowel disease (IBD) is an intractable condition. Existing models of experimental IBD are limited by their inability to create consistent ulcers between animals. The aim of this study was to develop a novel model of experimental colitis with ulcers of reproducible size. DESIGN: We used a 3D printer to fabricate a novel device containing a small window (10 × 10 mm) that could be inserted rectally to facilitate the creation of a localized ulcer in the rat intestinal mucosa. The mucosa within the window of the device was exposed to 2,4,6-trinitrobenzene sulfonic acid (TNBS) to generate ulceration. We evaluated the effects of conventional drug therapies (mesalazine and prednisolone) and local transplantation of allogeneic adipose-derived mesenchymal stem cells (ASCs) on ulcer size (measured from photographic images using image analysis software) and degree of inflammation (assessed histologically). RESULTS: The novel method produced localized, circular or elliptical ulcers that were highly reproducible in terms of size and depth. The pathological characteristics of the lesions were similar to those reported previously for conventional models of TNBS-induced colitis that show greater variation in ulcer size. Ulcer area was significantly reduced by the administration of mesalazine or prednisolone as an enema or localized injection of ASCs. CONCLUSION: The new model of TNBS-induced colitis, made with the aid of a device fabricated by 3D printing, generated ulcers that were reproducible in size. We anticipate that our new model of colitis will provide more reliable measures of treatment effects and prove useful in future studies of IBD therapies.