Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail

Abstract

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.

Human Osteochondral Explants as an Ex Vivo Model of Osteoarthritis for the Assessment of a Novel Class of Orthobiologics

“…Furthermore, multiple factors resulted/were enriched in the secretome-treated groups, suggesting their presence in the CM rather than an active release by the osteochondral tissues. Consistently, 21 molecules, among which are Dkk-1 and IGFBP-2, were recently quantified in several CM batches by our laboratory [14]. The levels of selected molecules were validated by multiplex assay, as shown in Supplementary S5.…”

“…Then, it was concentrated using 3 kDa molecular weight cut-off filter devices (Millipore, Burlington, MA, USA), aliquoted and stored at −80 • C. This protocol for CM production allows one to preserve both soluble factors and EVs. Product characterization was performed as follows: (i) quantification of total protein content by Bradford Protein Assay (Bio-Rad, Milan, Italy), (ii) Nanoparticle Tracking Analysis (NTA) by NanoSight NS300 (Malvern PANalytical, Salisbury, UK) [14], (iii) qualitative assessment of EV morphology by Transmission Electron Microscopy (TEM) at Unitech NOLIMITS facility, University of Milan and (iv) Western Blot analysis for the expression of the typical negative (Calnexin) and positive (HSP70, FLOT1, TSG101 and CD9) EV markers, following the procedure exhaustively described in [12,15]. Moreover, 3 of the 10 ASC-CM batches employed in this study were extensively characterized for the levels of 200 cytokines, chemokines, receptors, inflammatory mediators and growth factors, as reported in [14].…”

Lipidomics of Cell Secretome Combined with the Study of Selected Bioactive Lipids in an In Vitro Model of Osteoarthritis

“…Our recent research has identified key ingredients, including also lipids, 13 in ASC secretome that may be involved in its therapeutic action and whose consistent levels among different ASC-CM batches may represent promising quality control criteria. 27 In this work, our lipidomic MS developed methods 19 have demonstrated their usefulness in assessing a total of 9 lipid molecules in MSC- and DF-derived CM and EV samples. 2AG, PEA, OEA, SEA, DHEA belonging to EC/NAE and AA, EPA, DHA, PGE2 belonging to PUFA/eicosanoids were detected and quantified.…”

Functionalized Hydrogels for Cartilage Repair: The Value of Secretome-Instructive Signaling

“…The characterization of MSC secretome composition has been described by several groups [ 58 , 59 ]. Its composition varies according to the cell source, culture medium, culture time, and others.…”