Dr. Fausto Gueths Gomes (Salzburg/ AT), Prof. Dr. Katharina Schallmoser (Salzburg/ AT), Dr. André Cronemberger Andrade (Salzburg/ AT), Dr. Martin Wolf (Salzburg/ AT), Dr. Sarah Hochmann (Salzburg/ AT), Dr. Linda Krisch (Salzburg/ AT), Christof Regl (Salzburg/ AT), Dr. Rodolphe Poupardin (Salzburg/ AT), Dr. Patricia Ebner-Peking (Salzburg/ AT), Prof. Dr. Christian Huber (Salzburg/ AT), Prof. Dr. Nicole Meisner-Kober (Salzburg/ AT), Prof. Dr. Dirk Strunk (Salzburg/ AT)
Background
Human platelet lysate (hPL) is currently used as efficient substitute for fetal bovine serum for clinical cell manufacturing. Furthermore, we have previoulsy observed accelerated skin organoid formation and in vivo wound healing by hPL. As shown by others and us, EVs bear a biologically active protein corona, depending on the mode of preparation and the protein milieu.
Methods
In this study we asked whether hPL-derived EVs or platelet-derived soluble factors mediate these trophic effects of hPL. We separated EVs from soluble factors of hPL to understand the mode of action during skin organoid formation and immunomodulation as model systems for tissue regeneration. EVs were concentrated from hPL by tangential-flow filtration (TFF-EVs) and further purified by size-exclusion chromatography (TSEC-EVs) separating EVs from (lipo-) protein-rich soluble factors (TSEC-sol.F). Samples were characterized by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy, and functionally tested during organoid formation and immunomodulation.
Results
We identified three major protein clusters by proteomic principle component analysis separating TSEC-EVs from hPL clustering with TFF-sol.F and TFF-EVs clustering with TSEC-sol.F. TFF-EVs induced significantly improved skin-organoid formation and inhibition of T-cell proliferation, compared to TSEC-EVs or to TSEC-sol.F. Reconstituting the corona on TSEC-EVs with TSEC-sol.F re-established functionality comparable to TFF-EVs. Zeta potential and super-resolution imaging confirmed corona formation.
Conclusion
TFF is a permissive technology enabling scalable enrichment and separation of functional corona-bearing EVs and soluble factors. Depletion of the TFF-EV corona by SEC or ultracentrifugation abrogated functionality indicating a novel mode of action. The corona could be artificially reconstituted in add-back of sol.F showing similar effects compared to TFF-EVs. This enables EV engineering with selected corona proteins for specific purposes and therapeutic applications.
Offenlegung Interessenkonflikt:
Kein Interessenskonflikt