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In traditional tissue engineering, synthetic or natural scaffolds are usually used as removable temporal support, which involves some biotechnology limitations. The concept of "scaffield" approach utilizing the physical fields instead of biomaterial scaffold has been proposed recently. In particular, a combination of intense magnetic and acoustic fields can enable rapid levitational bioassembly of complex-shaped 3D tissue constructs from tissue spheroids at low concentration of paramagnetic agent (gadolinium salt) in the medium. In the current study, the tissue spheroids from human bladder smooth muscle cells (myospheres) are used as building blocks for assembling the tubular 3D constructs. Levitational assembly is accomplished at low concentrations of gadolinium salts in the high magnetic field at 9.5 T. The biofabricated smooth muscle constructs demonstrate contraction after the addition of vasoconstrictive agent endothelin-1. Thus, hybrid magnetoacoustic levitational bioassembly is considered as a new technology platform in the emerging field of formative biofabrication. This novel technology of scaffold-free, nozzle-free, and label-free bioassembly opens a unique opportunity for rapid biofabrication of 3D tissue and organ constructs with complex geometry., (© 2020 Wiley-VCH GmbH.)

Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Objective: Chondrospheres represent a variant of tissue spheroids biofabricated from chondrocytes. They are already being used in clinical trials for cartilage repair; however, their biomechanical properties have not been systematically investigated yet. The aim of our study was to characterize chondrospheres in long-term in vitro culture conditions for morphometric changes, biomechanical integrity, and their fusion and spreading kinetics., Results: It has been demonstrated that the increase in chondrospheres secant modulus of elasticity is strongly associated with the synthesis and accumulation of extracellular matrix. Additionally, significant interplay has been found between biomechanical properties of tissue spheroids and their fusion kinetics in contrast to their spreading kinetics., Conclusions: Extracellular matrix is one of the main structural determinants of chondrospheres biomechanical properties during chondrogenic maturation in vitro . The estimation of tissue spheroids' physical behavior in vitro prior to operative treatment can be used to predict and potentially control fusogenic self-assembly process after implantation in vivo .

Magnetic levitational bioassembly of three-dimensional (3D) tissue constructs represents a rapidly emerging scaffold- and label-free approach and alternative conceptual advance in tissue engineering. The magnetic bioassembler has been designed, developed, and certified for life space research. To the best of our knowledge, 3D tissue constructs have been biofabricated for the first time in space under microgravity from tissue spheroids consisting of human chondrocytes. Bioassembly and sequential tissue spheroid fusion presented a good agreement with developed predictive mathematical models and computer simulations. Tissue constructs demonstrated good viability and advanced stages of tissue spheroid fusion process. Thus, our data strongly suggest that scaffold-free formative biofabrication using magnetic fields is a feasible alternative to traditional scaffold-based approaches, hinting a new perspective avenue of research that could significantly advance tissue engineering. Magnetic levitational bioassembly in space can also advance space life science and space regenerative medicine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Reproducible, scalable, and cost effective fabrication and versatile characterization of tissue spheroids (TS) is highly demanded by 3D bioprinting and drug discovery. Consistent geometry, defined mechanical properties, optimal viability, appropriate extracellular matrix/cell organization are required for cell aggregates aimed for application in these fields. A straightforward procedure for fabrication and systematic multiparametric characterization of TS with defined properties and uniform predictable geometry employing non-adhesive technology is suggested. Applying immortalized and primary cells, the reproducibility of spheroid generation, the strong correlation of ultimate spheroid diameter, and growth pattern with cell type and initial seeding concentration are demonstrated. Spheroids viability and mechanical properties are governed by cell derivation. In this study, a new decision procedure to apply for any cell type one starts to work with to prepare and typify TS meeting high quality standards in biofabrication and drug discovery is suggested., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden constructs made of pure collagen with or without incorporated growth supplements support engineered constructs persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D bioprinting.

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd 3+ ) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd 3+ . Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd 3+ . Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering.