title: A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.
abstract: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs.
  
  
  
  Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit.
  
  
  
  Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV.
  
  
  
  The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.
authors.affiliation: Microscopy Facility, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria. Electronic address: karin.pachler@pmu.ac.at.
authors.forename: Karin
authors.initials: K
authors.lastname: Pachler
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lener
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Streif
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Zsuzsanna A
authors.initials: ZA
authors.lastname: Dunai
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Desgeorges
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Feichtner
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Öller
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schallmoser
authors.affiliation: GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Rohde
authors.affiliation: Microscopy Facility, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria; University Clinic for Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Mario
authors.initials: M
authors.lastname: Gimona
mesh_terms: D050156:Adipogenesis / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000592:standards*; D002454:Cell Differentiation / Q000187:drug effects; D060846:Cell Engineering / Q000379:methods / Q000592:standards*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D000067128:Extracellular Vesicles / Q000637:transplantation*; D006801:Humans; D066192:Manufacturing Industry / Q000379:methods / Q000592:standards*; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology; D017410:Practice Guidelines as Topic / Q000592:standards; D012015:Reference Standards
chemical_list: D017077:Culture Media, Conditioned
keywords: EV depletion; Good Manufacturing Practice; exosomes; extracellular vesicles; human platelet lysate; mesenchymal stromal cells