One of the key problems of regenerative medicine are avail-able autologous cell sources with huge differentiation poten-tial for cell therapy. Mesenchymal stem cells (MSCs) are one of the sources of myoblasts during muscle tissue regeneration. Differentiation potential of MSCs is elevated under 3D culture conditions. The aim of this work was to study cell products for therapy of musculoskeletal dysfunction from human gingival MSCs (MSCs-g) cultured in 2D and 3D (spheroids) conditions. Biopsies of gingiva were collected from healthy donors (n=10) after their informed consent. Mucosa fragments were treated in collagenase type II (200 u/ml, 120min, 37оС, 5% СО2). We used cultures at passages 3-4. Growth culture medium consist-ed of DMEM/F12 and 20% FBS (HyClone, USA), induction medium contained DMEM low glucose and 2% Horse Serum (BioInd, USA). Spheroids were formed on agarose plates with micro-wells made using 3D Petri Dishes (Microtissue, USA). Appearance of myotubes was monitored via live time-lapse microscopy in Cell-IQ device (CM Technologies, Finland). Se-cretome profile from 2D and 3D cultures was analyzed with 41plex kit (xMap technology, Millipore, USA). 2D and 3D cul-tures were fixed in 4% paraformaldehyde for analysis of MyoD and sarcomeric alfa-actin expression.In 2D culture after induction and in 40% cases spontaneous myogenic differentiation took place. Multinucleated myotubes in 2D culture expressed only MyoD, a marker of early stages of myogenesis. Expression of sarcomeric alfa-actin, which is characteristic of differentiated muscle cells, was absent. In 3D culture differentiation was more effective. Differentiated sphe-roids did not contain early progenitor cells, but well-formed myofibrils with characteristic peripheral nuclei arrangement and cross-striation, marked by antibodies against sarcomeric alfa-actin. Moreover, level of proangiogenic factor VEGF in the media conditioned by spheroids significantly increased. On the model of calf muscle injury suspension of MSC-g only reduced size of scar, whereas spheroids promoted full organotypic re-covery of muscle tissue.3D culturing of MSCs-g in form of spheroids stimulates ef-fective myogenic differentiation. Prevascularized microtissues from 3D cultures of accessible MSCs-g can find its place in personalized medicine for musculoskeletal dysfunction tissue replacement therapy in vivo. The study was financially sup-ported by Russian Science Foundation (grant № 17-75-30066). Keywords: Prevascularized Microtissue, 3D Culture, Spheroids, Mesenchymal Stem Cells, Gingiva, Muscle Regeneration