Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics

Abstract

Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.

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

“…An aliquot of the CM was concentrated about 40-50 times by centrifuging at 4000 g for 90 minutes at 4°C in AmiconUltra-15 Centrifugal Filter Devices with 3 kDa molecular weight cutoff (Merck Millipore, Burlington, MA, USA). This procedure allows the retention of the vesicular component of cell secretome, as previously demonstrated in the studies of Carlomagno et al, Niada et al, and Giannasi et al 13 , 14 , 18 In parallel, EV were isolated starting from CM through differential centrifugation at 100 000 g (L7-65; Rotor 55.2 Ti; Beckman Coulter, Brea, CA, USA), 4°C for 70 minutes). 14 , 21 The resulting CM and EV were kept at −80°C until use.…”

“… 12 In recent years, our research focused on the characterization of the CM from adipose-derived mesenchymal stem/stromal cells (ASC) and DF in terms of both soluble factors and vesicular components, through different approaches (ie, Raman spectroscopy and proteomic analysis), highlighting substantial differences in the total lipid content and a clear distinction between the two derivatives, also considering inflammatory molecules. 13 , 14 For this reason, a solid understanding of the individual bioactive factors secreted by MSC and DF, including lipids, and the mechanisms underlying their effect are necessary to refine secretome-based therapies in several pathological processes, such as inflammation. 7 , 15 , 16 Moreover, we evaluated both ASC-CM and ASC-EV action in an in vitro model of human articular chondrocytes (CH) induced toward an osteoarthritis (OA)-like phenotype by the inflammatory cytokine tumor necrosis factor alpha (TNFα).…”

Secretomes as an emerging class of bioactive ingredients for enhanced cosmeceutical applications

“…to characterize the main constituents of secretomes via the genetic screening using next-generation metabolomics, and high throughput chemical analysis through a combination of Raman spectroscopy and mass spectrometry techniques. 35,36 Keen knowledge of secretome constituents may in turn offer an opportunity for standardization through concentrations of notable factors depending on the application. Indeed, good manufacturing practice (GMP)-compliant manufacture and pooling of secretomes originating from many individuals could pave the way towards standardization of secretome composition and enable adherence to tighter quality specifications to conserve the secretome-based formulations from batch-to-batch.…”

“…The presence of exosomes and extracellular vesicles also presents added challenges as they can regulate and affect various inflammatory signalling pathways 34 . Nevertheless, efforts have been made to characterize the main constituents of secretomes via the genetic screening using next‐generation metabolomics, and high throughput chemical analysis through a combination of Raman spectroscopy and mass spectrometry techniques 35,36 . Keen knowledge of secretome constituents may in turn offer an opportunity for standardization through concentrations of notable factors depending on the application.…”

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

“…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].…”