Consistent functional properties of MSC-exosomes from different batches revealed by comparative multi-omics, bioinformatics and functional tests

Mesenchymal stromal cell-derived exosomes (MSC-EXOs), also called Extracellular Vesicles (EVs), exhibit anti-inflammatory effects in various diseases and are being developed for clinical use. For instance, MSC-EXO promotes skin healing post-laser therapy and improves outcomes in severe COVID-19. Selecting an optimal cellular source is critical for the therapeutic development of MSC-EXO. It has been suggested that GMP-produced MSC-exosomes have slightly different molecular content. This study therefore aimed to compare adipose tissue-derived MSC-EXO (ASC-EXO) from three healthy donors, isolated and characterized under GMP conditions. Exosomes were analyzed by nano-tracking analysis (NTA), cryo-electron microscopy, tetraspanin profiling, and multi-omics (proteomics, lipidomics, small RNA sequencing), as well as bioinformatics. Functional assays quantified anti-inflammatory effects in RAW264.7 macrophages and collagen production by human dermal fibroblasts (HDF). In vivo efficacy of ASC-EXOs was evaluated in a house dust mite antigen-induced atopic dermatitis mouse model through histopathological evaluation of ear thickness and differential cell counting. No significant batch differences were observed in ASC-EXO yield or characteristics. Omics analyses revealed minor variations in protein, lipid, and small RNA cargo among the three batches, but GO-term bioinformatics indicated highly similar functional profiles. IL-6 suppression in RAW264.7 cells and cell proliferation and collagen production by HDF were similar among the ASC-EXO batches. CD73 enzymatic activity was consistent among batches. In vivo, the ASC-EXOs reduced skin thickness and eosinophilia in an atopic dermatitis model, with similar effects among the batches. In summary, ASC-EXOs from all batches demonstrated comparable anti-inflammatory and collagen-modulating effects in vitro, and similar inhibition of atopic dermatitis signs in vivo. We suggest that biological efficacy combined with bioinformatics analysis should guide MSC-EXO therapeutic quality control assays. These findings support the development of ASC-EXOs for clinical applications.