**6. Conclusion**

and interleukin 8 (CXCL8) in the product, as published literature has identified the important biological activity of these growth factors, cytokines, and chemokines [40, 41]. MultiStem also successfully used an analogous in vitro angiogenic test of biological activity, and also showed that three factors—VEGF, CXCL8, and CXCL5—are the crucial factors for the angiogenic activity of the secretome. The depletion of any of them drops the angiogenic effect of the secretome. Concentrations of each of the factors can be used as a threshold for the lot release [42]. Based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for human adipose-derived MSC CM developed for tissue reparation and regeneration on "Specific activity." As the most important type of cells involved in tissue repair and regeneration after injury are fibroblasts, one of the models for MSC CM potency measurement was the assessment of human skin fibroblasts migration in the model of the scratch assay. In this regard that angiogenesis is also an indispensable process for the successful regeneration of tissues, we additionally used a model of human

**Figure 1.** Schematic reflecting multicomponent multi-MOA nature of MSC and effects of factors secreted by them. A detailed description is provided in the subsequent text. Abbreviations: MOA, mode of action; MSC, mesenchymal

Despite direct in vitro or in vivo biological activity tests seem to be more informative, they demonstrate several substantial disadvantages. The main challenge is to render some injuryspecific conditions in model objects. Many in vivo studies in the fields of oncology and adaptive immunity conducted on rodents have faced this problem. In accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and Directive 2010/63/EU on protection of animals used for scientific purposes, the 3R principles (replacement, reduction, and refinement) should be applied to production and control testing of medicinal products. [43]. Last but not

the least, it is rational to take into account a high cost of these types of potency test.

endothelial cells direct migration upon MSC CM gradient [26].

stromal cells.

56 Biopharmaceuticals

This chapter is dedicated to a novel class of biopharmaceuticals based on secretory components of MSC as products for regenerative medicine. CM-based cell-free therapeutics comprise multicomponent mixture with multiple targets and pleiotropic effects. These biopharmaceuticals reproduce many benefits of the rapidly developing cell therapy products. However, the use of cells might be complicated due to ectopic transplantation, tumorigenesis, and immune system reactions. MSC secretome is devoid of cell therapy side effects and has substantial advantages in manufacture, storage, and standardization making it a promising type of biopharmaceuticals.

We paid attention to safe and effective MSC CM-based cell-free therapeutics manufacturing conception. We have discussed several challenges concerning donor-associated variability, cell isolation procedure, optimal protocols for manufacturing and quality control, and lack of key regulatory decisions that must be overcome prior to the wide-scale clinical translation of such therapeutics. To reach the best safety and efficacy marks, developers could apply a system-wide approach to disease model analysis considering the principal mechanisms of tissue reparation and regeneration processes. In addition, it is rational to develop appropriate donor selection criteria that can help to involve only a suitable starting material to manufacturing process. Preventing the lot-to-lot variability together with improved efficacy could be facilitated by a relevant potency test development and validation required for the drug-quality control. The use of rational approach to the choice of MOA might help to relieve development and conduction of a potency test by transition to more simple surrogate approach. In addition, implementing of these approaches into practice would help to develop novel legal potency test guidelines for cell-free therapeutics with unambiguous rules and examples.
