Michiya Matsusaki

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.

A novel cell-accumulation technique has been developed using highly biocompatible nanofilms by layer-by-layer assembly for the rapid construction of thick layered tissues with a well-controlled layer number and thickness. Furthermore, 3D tissues with highly developed blood capillary networks (over 1 cm2 of layered tissues) were also constructed by sandwiching endothelial cells between the layered tissues. Such a simple and rapid methodology would be useful for tissue engineering and drug assessment. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies. STATEMENT OF SIGNIFICANCE: Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e., fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo, resulting in a reduction of cancer cell sensitivity to the anticancer treatment.

Layer it on: Cellular multilayers were fabricated by preparing nanometer-sized extracellular matrix (ECM) films (6-nm thick) with fibronectin (FN) and gelatin on the surface of each cell layer. The four-layer cellular architecture was well organized and self-standing. Xenogenic human bilayer architectures similar to blood vessels were prepared by fabrication of the nanofilms on cell surfaces.

Biodegradable hollow capsules encapsulating proteins were prepared via layer-by-layer assembly of chitosan and dextran sulfate on protein-entrapping mesoporous silica particles and the subsequent removal of the silica. The enzymatic degradation of the capsules in the presence of chitosanase was explored by scanning electron microscopy (SEM). With increasing time, the chitosan component was degraded by chitosanase, and the capsules began to deform and were finally destroyed. Sustained release of the encapsulated proteins was attained by using the enzymatic degradation of the hollow capsules. The release behavior was successfully manipulated by altering the charge of capsule surface.