Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV...

Area of Science:

• Regenerative Medicine
• Extracellular Vesicle Biology
• Biotechnology

Background:

• Mesenchymal stromal cells (MSCs) are utilized in regenerative medicine, with their therapeutic effects largely mediated by secreted extracellular vesicles (EVs).
• MSC-derived EVs (MSC-EVs) offer advantages over cellular therapies but exhibit significant product diversity due to varied manufacturing and characterization methods.
• Lack of standardized characterization hinders reliable inter-laboratory comparisons and clinical translation of MSC-EV therapeutics.

Purpose of the Study:

• To establish a common, standardized method for characterizing MSC-EV preparations to facilitate inter-laboratory comparisons.
• To identify a consistent panel of surface markers for the reliable identification of MSC-derived EVs.
• To assess the impact of manufacturing and culture conditions on MSC-EV surface marker profiles.

Main Methods:

• A novel multiplex bead-based EV flow cytometry assay panel was developed and validated.
• An inter-laboratory assessment was conducted involving 11 MSC-EV products from five different laboratories.
• MSC-EV preparations were analyzed for a range of cell surface markers, including established MSC markers and other relevant proteins.

Main Results:

• A consistent set of positive surface markers (CD44, CD73, CD105) and negative markers (CD11b, CD45, CD197) were identified across all tested MSC-EV preparations.
• Hierarchical clustering revealed distinct surface marker profiles correlated with specific preparation processes and laboratory conditions.
• Culture medium components, such as human platelet lysate, were found to influence EV surface marker expression.

Conclusions:

• The proposed standardized flow cytometry approach enables robust characterization of MSC-EV surface markers for quality control.
• CD73, CD105, and CD44 are proposed as reliable positive markers, while CD11b, CD14, CD19, CD45, and CD79 serve as reliable negative markers for MSC-EV identification.
• This standardization is crucial for enhancing consistency, reproducibility, and quality control in the emerging field of therapeutic MSC-EVs.