Review: MSCs and Exosomes Production

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www.pelobiotech.com This difference in functionality raises concerns about the suitability of iMSCs for certain applications and underscores the importance of further research to understand the implications of iMSC behaviour and characteristics. Moreover, the source of MSCs, whether primary, induced pluripotent stem cell (iPSC)-derived, or immortalized, can influence EVs production. While iPSC derived MSCs have shown promise for specific applications, primary MSCs are still preferred in many cases due to their superior supportive capabilities in co-culture systems 19 . The choice of MSC source is a critical consideration in EVs production, as it can impact the quantity and quality of EVs generated for therapeutic purposes. The culture media used for MSC expansion can also influence EVs production and functionality. The use of defined media has been suggested as advantageous for maintaining the desired characteristics of MSCs and their derived EVs 20 . Additionally, it may be necessary to add special lipid cocktails for high EVs production. The balance between high proliferation/expansion rates and EVs production is a critical consideration. While high proliferation rates are desirable for obtaining large quantities of MSCs, it may lead to competition for resources, such as lipids, which are essential for both proliferation and EVs biogenesis 21 . Studies have indicated that the efficiency of EVs production may inversely correlate with the developmental maturity of the MSC donor, further highlighting the importance of donor selection for optimal EVs yield 21 . Moreover, the choice of having serum in the cell culture, such as FBS, hPL, or AB serum, can introduce both variability in proliferation, and expansion. Utilizing defined media can help to overcome these batch-to-batch variabilities and ensure consistent functional EVs production 20 . In conclusion, the culture and expansion of MSCs for EVs production involve various factors that influence the quantity and quality of EVs. Careful consideration of expansion methods, culture media, cell source, proliferation rates, and serum choice is essential to optimize EVs production for therapeutic applications. Special lipid cocktails may be necessary to enhance EVs production efficiency and yield, further emphasizing the importance of optimizing culture conditions for successful EVs-based therapies. Isolation Techniques for MSC-Derived EVs Isolating EVs from MSCs is a noteworthy area of research, essential for obtaining pure EVs samples for applications ranging from therapeutic use, drug delivery, regenerative medicine, and tissue engineering. Various methods such as ultracentrifugation, differential ultracentrifugation, and tangential flow filtration are employed, each with its distinct advantages and limitations 4 . • Ultracentrifugation: Ultracentrifugation is one of the most traditional and widely used methods for Extracellular Vesicles isolation. This technique relies on the application of extremely high centrifugal forces, typically ranging from 100,000 to 200,000g to sediment EVs from MSC culture media or other biological fluids. The process involves multiple centrifugation steps at varying speeds and durations to progressively remove cells, cell debris, and larger vesicles, culminating in the sedimentation of EVs. o Advantages: Widely available: The equipment required for ultracentrifugation is available in most research laboratories. Tel: +49 (0) 89 517 286 59 0 Mail: info@pelobiotech.com
www.pelobiotech.com Scalable: It can be adapted for large-volume preparations, making it suitable for both research and clinical applications. o Limitations: Time-consuming: The process is labor-intensive and requires several hours to complete. Potential for contamination: Co-isolation of protein aggregates or other vesicles of similar density can occur. Sample integrity: The high forces applied can potentially damage the EVs or alter their functional properties. • Differential Ultracentrifugation Differential ultracentrifugation refines the basic ultracentrifugation process by employing a series of centrifugation steps at gradually increasing speeds. This method allows for more precise separation of EVs from other components based on their size and density. o o Advantages: Improved purity: By carefully adjusting the centrifugation parameters, it is possible to enhance the purity of the isolated EVs. Versatility: It can be used in conjunction with other purification steps to further increase the yield and purity of EVs. Limitations: Complexity: The process requires meticulous optimization of centrifugation speeds and times for each specific sample type. Sample loss: Each centrifugation step may lead to a loss of EVs yield. • Tangential Flow Filtration (TFF) 32 Tangential flow filtration is a more modern approach that utilizes a crossflow mechanism, where the sample fluid flows tangentially across the surface of a membrane filter. This method effectively separates EVs based on their size, allowing them to pass through the membrane while larger particles are retained. o o Advantages: Efficiency: TFF can process large volumes of samples in a relatively short amount of time. Gentle on samples: The technique is less likely to damage EVs compared to ultracentrifugation. Scalability and reproducibility: TFF is easily scalable and offers high reproducibility, making it suitable for clinical applications. Limitations: Equipment cost: The initial investment for TFF systems can be high. Membrane maintenance: Over time, the membrane may become clogged with particles, requiring regular maintenance or replacement. The choice of an EVs isolation technique depends on various factors, including the source of MSCs, the volume of the sample, the desired purity and yield of EVs, and the available Tel: +49 (0) 89 517 286 59 0 Mail: info@pelobiotech.com
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