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Biofabrication of organ-like engineered 3D tissue through the assembly of magnetized 3D multi-cellular spheroids has been recently investigated in tissue engineering. However, the cytotoxicity of magnetic nanoparticles (MNPs) and contraction-induced structural deformation of the constructs have been major limitations. In this study, we developed a method to fabricate composite stem cell spheroids using MNP-coated fibers, alleviating MNP-mediated toxicity and controlling structural assembly under external magnetic stimuli. The MNP-coated synthetic fibers (MSFs) were prepared by coating various amounts of MNPs on the fibers via electrostatic interactions. The MSFs showed magnetic hysteresis and no cytotoxicity on 2D-cultured adipose-derived stem cells (ADSCs). The composite spheroids containing MSFs and ADSCs were rapidly formed in which the amount of impregnated MSFs modulated the spheroid size. The fusion of in vitro composite spheroids was then monitored at the contacting interface; the fused spheroids with over 10 μg of MSF showed minimal contraction after 7 d, retaining around 90% of total area ratio regardless of the number of cells, indicating that the presence of fibers within the composite spheroid supported its structural maintenance. The fusion of MSF spheroids was modulated by external magnetic stimulation, and the effect of magnetic force on the movement and fusion of the spheroids was investigated using COMSOL simulation. Finally, ring and lamellar structures were successfully assembled using remote-controlled MSF spheroids, showing limited deformation and high viability up to 50 d during in vitro culture. In addition, the MSFs demonstrated no adverse effects on ADSC osteochondral differentiation. Altogether, we envision that our magnetic assembly system would be a promising method for the tissue engineering of structurally controlled organ-like constructs.

Recently, stem cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stem cell spheroids in different sizes from micro-scaled cell sheets (μCS) using thermosensitive hydrogels and investigated their effects on stem cell function. Mesenchymal stem cells isolated from different tissues such as human turbinate tissue, bone marrow, and adipose tissue were adhered selectively to each micro-pattern (squares with widths of 100 and 400 μm) on the surface of the hydrogel and formed μCS. The diameters of the spheroids were modulated by the size of the patterns (45±5 and 129±4 μm in diameter for the 100 and 400 μm micro-patterns, respectively) and the seeding density (129±4, 149±6, and 163±6 μm for 5.0, 10.0, and 15.0 × 10alt;supagt;4alt;/supagt; cells/cmalt;supagt;2alt;/supagt;, respectively, on 400-μm micro-pattern). In addition, the spheroids were successfully fabricated regardless of stem cell origin, and the diameter of the spheroids was also affected by cell spreading area on a cell culture dish. Stemness markers were highly expressed in the spheroids regardless of the spheroid size. Furthermore, an increase in E-cadherin and decrease in N-cadherin gene expression showed the stable formation of spheroids of different sizes. Gene expression levels of hypoxia inducible factors and secretion of vascular endothelial growth factor (VEGF) were increased (13.2±1.4, 325±83.4 and 534.3±121.5 pg/ng DNA in a monolayer, and 100- and 400-μm micro-patterned spheroids, respectively) proportional to the diameters of the spheroids. The size of spheroids were maintained even after injection, cryopreservation and 7 days of suspension culture with high viability (~90%). In conclusion, this novel technique to fabricate spheroids with controlled size could be widely applied in various applications that require a controlled size in regenerative medicine.