Karin Pachler1, Thomas Lener2, Doris Streif3, Zsuzsanna A Dunai3, Alexandre Desgeorges3, Martina Feichtner3, Michaela Öller2, Katharina Schallmoser2, Eva Rohde2, Mario Gimona4. 1. 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. 2. 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. 3. GMP Laboratory, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria. 4. 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.
Abstract
BACKGROUND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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: Evelyn Kendall Williams; José R García; Robert G Mannino; Rebecca S Schneider; Wilbur A Lam; Andrés J García Journal: Integr Biol (Camb) Date: 2019-04-01 Impact factor: 2.192
Authors: Oscar P B Wiklander; Meadhbh Á Brennan; Jan Lötvall; Xandra O Breakefield; Samir El Andaloussi Journal: Sci Transl Med Date: 2019-05-15 Impact factor: 17.956
Authors: Athanasia Warnecke; Nils Prenzler; Jennifer Harre; Ulrike Köhl; Lutz Gärtner; Thomas Lenarz; Sandra Laner-Plamberger; Georg Wietzorrek; Hinrich Staecker; Teresa Lassacher; Julia Hollerweger; Mario Gimona; Eva Rohde Journal: J Extracell Vesicles Date: 2021-06-04