Pachler Karin, Lener Thomas, Streif Doris, Dunai Zsuzsanna A, Desgeorges Alexandre, Feichtner Martina, Öller Michaela, Schallmoser Katharina, Rohde Eva, Gimona Mario
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.
Cytotherapy. 2017 Apr;19(4):458-472. doi: 10.1016/j.jcyt.2017.01.001. Epub 2017 Feb 7.
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.
间充质基质细胞（MSC）释放的细胞外囊泡（EV）可能参与组织再生、免疫调节和神经保护等生物学过程。评估其治疗潜力并将其应用于未来的临床试验，需要在明确的培养基条件下对EV的含量和产生情况进行全面表征，且培养基中不含异种物质和血清来源的囊泡。针对这种生长培养基的明显需求，我们开发了一种基于人血小板裂解物（pHPL）的培养基配方，该配方不含动物来源的异种添加剂且去除了EV。
通过在120000g下离心3小时，从完全生长培养基中去除EV，这将含RNA的pHPL EV减少至检测限以下。
在这种培养基中培养的骨髓（BM）来源的MSC保留了特征性的表面标志物表达、细胞形态、活力以及体外成骨和成脂分化潜力。48小时后增殖率未受到显著影响，但96小时后降低了13%。从在去除EV的培养基中培养的BM-MSC收集的EV显示出与在标准含pHPL EV的培养基中产生的EV相似的RNA模式，但呈现出更清晰定义的EV特征性蛋白质模式。将pHPL含量从10%降至2%或无血清/无pHPL条件会强烈改变MSC特征和释放的EV的RNA含量。
基于10%pHPL的去除EV的培养基适用于纯化仅来源于人MSC的EV。通过这种符合药品生产质量管理规范（GMP）级别的方案，现在可以实现对MSC来源的EV的蛋白质和RNA谱进行表征和建立，以识别治疗性EV中的活性成分，用于未来的临床应用。
