Research Article Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail Chiara Giannasi , 1,2 Stefania Niada , 2 Elena Della Morte, 2 Sara Casati , 1 Marica Orioli , 1 Alice Gualerzi , 3 and Anna Teresa Brini 1,2 1 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy 2 IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Applicazioni Biotecnologiche, Milan, Italy 3 IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milano, Italy Correspondence should be addressed to Chiara Giannasi; chiara.giannasi@unimi.it Received 1 June 2021; Revised 2 August 2021; Accepted 7 August 2021; Published 26 August 2021 Academic Editor: Vasilis Paspaliaris Copyright © 2021 Chiara Giannasi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC- CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs (1:0 × 10 9 ±1:1 × 10 8 particles per million donor ASCs) with a mean size of 190 ± 5:2 nm, suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications. 1. Introduction Over the years, the transplantation of autologous or alloge- neic stem cells, either naïve, differently primed, or geneti- cally manipulated, has paved the way to the successful clinical management of several diseases whose pharmacolog- ical need was previously unmet. In particular, mesenchymal stem/stromal cells (MSCs), thanks to their regenerative and immunomodulatory potential [1, 2], have gained popularity as cell therapy in disparate clinical scenarios, from immuno- logical diseases [3] to orthopedic conditions [4] and central nervous system injuries and disorders (e.g., traumatic brain injury, Parkinson’s disease, and ischemic stroke) [5]. Besides the overall promising results, MSC transplantation (as well as cell-based therapy in general) entails evident drawbacks, such as ethical controversies, concerns linked to ex vivo Hindawi Stem Cells International Volume 2021, Article ID 3086122, 13 pages https://doi.org/10.1155/2021/3086122