With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.

Our neuroblastoma cDNA project previously identified Src homology 2 domain containing F (Shf) as one of the genes expressed at high levels in favorable neuroblastoma. Shf is an adaptor protein containing four putative tyrosine phosphorylation sites and an SH2 domain. In this study, we found that Shf interacted with anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase in neuroblastoma. Real-time PCR analysis showed that Shf mRNA is highly expressed in non-metastatic neuroblastomas compared to metastatic tumor samples (P < 0.030, n = 106). Interestingly, patients showing high ALK and low Shf mRNA expressions showed poor prognosis, whereas low ALK and high Shf expressions were related to better prognosis (P < 0.023, n = 38). Overexpression of ALK and siRNA-mediated knockdown of Shf yielded similar results, such as an increase in cellular growth and phosphorylation of ALK, in addition to Erk1 ⁄ 2 and signal transducer and activator of transcription 3 (STAT3) that are downstream signals of the ALK-initiated phospho-transduction pathway. Knockdown of Shf also increased the cellular mobility and invasive capability of neuroblastoma cells. These results suggest that Shf interacts with ALK and negatively regulates the ALK-initiated signal transduction pathway in neuroblastoma. We thus propose that Shf inhibits phospho-transduction signals mediated by ALK, which is one of the major key players on neuroblastoma development, resulting in better prognosis of the tumor. (Cancer Sci 2013; 104: 563-572) N euroblastoma, a solid tumor that accounts for 15% of all pediatric cancer deaths, originates from the sympathoadrenal lineage derived from the neural crest. The clinical behavior of neuroblastoma is markedly heterogeneous. (1) Tumors found in patients under 1 year of age yield favorable prognosis frequently accompanied by spontaneous differentiation and regression, whereas those found in older patients grow aggressively, often resulting in fatal outcomes. (1) Despite the recent treatments and care that have been improved, neuroblastoma harboring the amplified MYCN oncogene in an advanced stage is closely correlated to poor outcome. (1,2) Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, originally identified as an oncogenic fusion protein nucleophosmin-ALK in anaplastic large cell lymphoma. (3)(4)(5) Such unique oncogenic fusion of the ALK gene due to chromosomal translocation is responsible for the activation of the ALK signaling pathway in many human cancers including non-smallcell lung cancer. (6)(7)(8)(9) Although the expression pattern of ALK in tissues strongly suggests that ALK plays a pivotal role in normal development of the nervous system, (10-12) the molecular mechanism underlying the signal transduction pathway oriented by ALK during neural development and carcinogenesis still remains unclear. Several point mutations that activate the ALK gene have been studied in both familial and sporadic cases of neuroblastoma. (13)(14)(15)(16)(17) Frequency of point mutations activating ALK i...

Construction of three-dimensional (3D) tissues with perfusable vascular networks remains a major challenge in the field of tissue engineering. Although various sacrificial templates have been employed to fabricate the vascular networks, there are some issues with respect to cytocompatibility, structural controllability, and degradability for the achievement of precisely controlled vasculatures without cytotoxicity. Here, we demonstrate a naturally occurring polysaccharide, gellan gum (GG), as a sacrificial template material due to its unique character. GG showed rapid gelation at 30–50 °C during the cooling process depending on the concentration of bivalent calcium ions by intermolecular ionic cross-linking and subsequent double-helix formation of GG molecules. Although chelate agents such as EDTA are generally effective in decomposing ionic cross-linking gels, e.g., alginate gel, they usually show cytotoxicity. In the case of GG gel, the gels could not be decomposed by the chelate agents but were easily decomposed by Tris–HCl buffer (pH = 7.4), which is a basic molecule with high cytocompatibility. We finally fabricated straight vascular tubes in 3D-gelatin gels and then demonstrated perfusion of human whole blood at 3.0 cm/s for 2 h. Since the complex vascular networks were constructed by 3D inkjet printing of the GG solution, GG would be useful as a structurally controllable and easily decomposable sacrificial material with cytocompatibility.