Bioprinting is a technology for constructing bioartificial tissue or organs of complex three-dimensional (3-D) structure with high-precision spatial shape forming ability in larger scale than conventional tissue engineering methods and simultaneous multiple components composition ability. It utilizes computer-controlled 3-D printer mechanism or solid free-form fabrication technologies. In this study, sodium alginate hydrogel that can be utilized for large-dimension tissue fabrication with its fast gelation property was studied regarding material-specific printing technique and printing parameters using a multinozzle bioprinting system developed by the authors. A sodium alginate solution was prepared with a concentration of 1% (wt/vol), and 1% CaCl(2) solution was used as cross-linker for the gelation. The two materials were loaded in each of two nozzles in the multinozzle bioprinting system that has a total of four nozzles of which the injection speed can be independently controlled. A 3-D alginate structure was fabricated through layer-by-layer printing. Each layer was formed through two phases of printing, the first phase with the sodium alginate solution and the second phase with the calcium chloride solution, in identical printing pattern and speed condition. The target patterns were lattice shaped with 2-mm spacing and two different line widths. The nozzle moving speed was 6.67 mm/s, and the injection head speed was 10 µm/s. For the two different line widths, two injection needles with inner diameters of 260 and 410 µm were used. The number of layers accumulated was five in this experiment. By varying the nozzle moving speed and the injection speed, various pattern widths could be achieved. The feasibility of sodium alginate hydrogel free-form formation by alternate printing of alginate solution and sodium chloride solution was confirmed in the developed multinozzle bioprinting system.

The purpose of this study was to demonstrate self‐organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long‐term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro‐wells and the sustainability of the endothelial cell‐layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long‐term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.

Bioprinting is an emerging technology for constructing tissue or bioartificial organs with complex three-dimensional (3D) structures. It provides high-precision spatial shape forming ability on a larger scale than conventional tissue engineering methods, and simultaneous multiple components composition ability. Bioprinting utilizes a computer-controlled 3D printer mechanism for 3D biological structure construction. To implement minimal pattern width in a hydrogel-based bioprinting system, a study on printing characteristics was performed by varying printer control parameters. The experimental results showed that printing pattern width depends on associated printer control parameters such as printing flow rate, nozzle diameter, and nozzle velocity. The system under development showed acceptable feasibility of potential use for accurate printing pattern implementation in tissue engineering applications and is another example of novel techniques for regenerative medicine based on computer-aided biofabrication system.

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A major hurdle in engineering thick and laminated tissues such as skin is how to vascularize the tissue. This study introduces a promising strategy for generating multi-layering engineered tissue sheets consisting of fibroblasts and endothelial cells co-seeded on highly micro-fibrous, biodegradable polycaprolactone membrane. Analysis of the conditions for induction of the vessels in vivo showed that addition of endothelial cell sheets into the laminated structure increases the number of incorporated cells and promotes primitive endothelial vessel growth. In vivo analysis of 11-layered constructs showed that seeding a high number of endothelial cells resulted in better cell survival and vascularization 4 weeks after implantation. Within one week after implantation in vivo, red blood cells were detected in the middle section of three-layered engineered tissue sheets composed of polycaprolactone/collagen membranes. Our engineered tissue sheets have several advantages, such as easy handling for cell seeding, manipulation by stacking each layer, a flexible number of cells for next-step applications and versatile tissue regeneration, and automated thick tissue generation with proper vascularization.

The vascularization of three-dimensional (3D) tissue constructs is necessary for transporting nutrients and oxygen to the component cells. In this study, a vacuum forming method was applied to emboss a vascular pattern on an electrospun membrane so that guided vascular structures could develop within the construct. Two- or six-layer constructs of electrospun membranes seeded with endothelial cells and pericytes were stacked and subcutaneously implanted into mice. Blood vessel formation in the implanted constructs with six alternating layers of flat membranes and membranes embossed with a blood vessel pattern was observed after two weeks of implantation. The formation of blood vessels was observed along the embossed blood vessel pattern in the structure of the embossed membrane laminated at four weeks and eight weeks. Vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) were highly expressed in the vascularized structures. Therefore, we demonstrated that a structure capable of producing a desired blood vessel shape with electrospun membranes embossed with a blood vessel pattern can be manufactured, and that a variety of structures can be manufactured using electrospun membranes in the tissue engineering era.

The aggregation of multiple cells, such as mesenchymal condensation, is an important biological process in skeletal muscle development, osteogenesis, and adipogenesis. Due to limited in vivo study model systems, a simple and effective in vitro three-dimensional (3D) aggregation system is required to study the mechanisms of multicellular aggregation and its applications. We first generated controlled mesenchymal stem cell (MSC) aggregates using a bioprinting technique to monitor their aggregation and sprouting. We induced the angiogenic potential of the MSCs through chemical inhibition of the Rho/Rho-associated protein kinase (ROCK) pathway, which led to hairy sprouting in the aggregates. The angiogenic potential of this 3D construct was then tested by subcutaneously implanting the Matrigel with 3D MSC aggregates in a rat. Treatment of 3D MSCs with the ROCK inhibitor, Y27632, increased their angiogenic activity in vivo. The gene expressions and histological staining indicated that angiogenesis and neovascularization were mainly regulated by the paracrine factors secreted from human 3D MSC constructs. Our results demonstrate the enhancement of the angiogenic potential of the MSC constructs through the secretion of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) by the inhibition of the Rho/ROCK pathway.

Asinibacterium lactis gen. nov., sp. nov., a member of the family Chitinophagaceae, isolated from donkey (Equus asinus) milk A novel bacterial strain, designated LCJ02 T , was isolated on R2A agar from donkey (Equus asinus) milk powder and subjected to a taxonomic study using a polyphasic approach. Strain LCJ02 T showed a Gram-negative reaction, was non-motile, non-spore-forming and possessed rod-shaped cells and yellow-pigmented colonies. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolate formed a cluster with several uncultured bacterial clones and with cultured members of the genera Hydrotalea, Sediminibacterium and Lacibacter (family Chitinophagaceae, phylum Bacteroidetes). The gene sequence similarities with respect to the type strains of recognized species from the above genera and other phylogenetic neighbours ranged from 89.3 to 92.9 %. The G+C content of the genomic DNA was 49.2 mol%, the only isoprenoid quinone was MK-7 and the major fatty acids were iso-C 15 : 0 , iso-C 17 : 0 3-OH, iso-C 15 : 1 G and summed feature 3 (C 16 : 1 v7c and/or iso-C 15 : 0 2-OH). The major polar lipids of strain LCJ02 T were phosphatidylethanolamine, two unidentified aminophospholipids, one unidentified aminolipid and five unidentified lipids. The results of physiological and biochemical tests allowed phenotypic differentiation of strain LCJ02 T from its closest phylogenetic neighbours. On the basis of the evidence of this polyphasic study, isolate LCJ02 T represents a novel genus and species in the family Chitinophagaceae for which the name Asinibacterium lactis gen. nov., sp. nov. is proposed. The type strain is LCJ02 T (5KCCM 90108 T 5JCM 18484 T ).