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Multipotent mesenchymal stromal cells (MSC) are considered as a promising tool for regenerative medicine. Recent studies revealed the poor local engrafting and survival of implanted cells and indicated that the benefits of MSC therapy was mediated mostly by producing multiple bioactive components, including soluble factors (SF) and extracellular vesicles (EV), stimulating angiogenesis, neurogenesis, activating tissue-resident stem cells and modulating immune reactions. It highlighted the role of MSC as key regulators of reparation and regeneration processes in damaged tissues. MSC secretome, as conditioned media (MSC- CM) or its fractions, may have considerable advantages over cell therapy for biosafety, manufacturing and storage with comparable regenerative potential. We have developed the optimized MSC-CM production protocol based on dynamics of key factors secreted by human adipose-derived MSC during long-term conditioning in different xeno-free, chemically defined media. Computational prediction modelling was used to overcome the high donor-dependent variability of MSC-CM. We showed that MSC-CM stimulated survival and migration of endothelial cells and human fibroblasts in vitro. Both SF and EV of MSC-CM exhibited antifibrotic effects preventing and even reversing TGFb- induced fibroblast differentiation into myofibroblasts. Regenerative effects of MSC-CM in vivo were analyzed by their ability to restore spermatogonial stem cell niche and functionalize the tissue engineering constructs like collagen membrane for bladder wall defect reconstruction in rabbits. Using the rat cryptorchidism model of spermatogenesis failure, we revealed that MSC-CM injected under tunica albuginea recovered testes hypotrophy, prevent seminiferous tubules fibrosis, normalized spermatogonial stem cell differentiation and function of Sertoli and Leydig cells and resulted in increase of total and moving spermatozoa count. These effects were comparable to cell therapy with MSC from the same donors. Collagen membrane functionalized by MSC-CM significantly better restored injured bladder function by engaging smooth muscle cells, stimulating angiogenesis and modulating immune reactions. Our findings provide strong basis to develop cell-free therapeutic approaches for regenerative medicine. Funding Source: The study was conducted using biomaterials collected and preserved in the frame of RSF grant #14-50-00029 using the equipment purchased as a part of Lomonosov MSU Program of Development and supported by RFBR grant #18-015-00525.