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 × 109 ± 1.1 × 108 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.
Highlights
Over the years, the transplantation of autologous or allogeneic stem cells, either naïve, differently primed, or genetically manipulated, has paved the way to the successful clinical management of several diseases whose pharmacological need was previously unmet
Our research focused on the investigation of the conditioned medium (CM) from adipose tissuederived mesenchymal stem/stromal cells (MSCs) (ASCs) in terms of biochemical composition [10,11,12,13] and therapeutic action, both in vitro [14, 15] and in vivo [16, 17]
CM samples were obtained, as previously described, from the culture medium harvested from confluent adipose-derived MSCs (ASCs) cultured for 3 days in serum-free conditions and concentrated by centrifugal filter devices of about 60 times
Summary
The transplantation of autologous or allogeneic stem cells, either naïve, differently primed, or genetically manipulated, has paved the way to the successful clinical management of several diseases whose pharmacological need was previously unmet. A critical search through ClinicalTrials.gov database, performed at the end of April 2021 using alternatively the keywords “secretome,” “conditioned medium,” or “extracellular vesicles” and the filter “interventional” as study type, lists a total of 14 studies based on MSC-secretome administration. Most of these protocols relied on the use of CM (n = 11 versus n = 3 studies using EVs) derived from allogeneic MSCs (n = 8 versus n = 1 study following an autologous setting). This picture allows us extrapolating some general considerations on the state of the art of MSC-based cell-free therapies:
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