Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media.

This research article investigates the impact of three-dimensional (3-D) culture and different media on the yield of extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) sourced from horse bone marrow. The study found that while the overall yield of EVs wasn’t improved by 3-D culture or the used media, the number of EVs produced per MSC was enhanced under 3-D conditions with a specific commercial media.

Objective

The researchers aimed to explore the effects of 3-D versus 2-D culture and the impact of two types of media on the yield of EVs. These vesicles are seen as a promising biotherapeutic aid for osteoarthritis, but their mass production using traditional 2-D culture is problematic.

Methodology

• The study used MSCs obtained from the bone marrow of six healthy, young horses.
• Four types of microcarriers were tested in static and bioreactor cultures, with the best microcarrier chosen for further experimentation.
• The MSCs were then inoculated into a bioreactor with selected microcarriers or onto culture dishes and cultured in either Dulbecco modified Eagle medium or a commercially available medium (CM).
• Later, the supernatants were collected for analysing the pH and metabolites.
• After either attaining confluence (2-D culture) or post 7 days (3-D culture), a 48-hour EV collection period was started using media depleted of EVs.
• The obtained EVs were then isolated and characterised using nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification techniques.
• Finally, the MSCs were harvested, quantified, and analysed for the presence of stemness markers.

Results

• The experiments showed that neither the 3-D culture system nor the commercial medium significantly enhanced the total yield of EVs.
• Nevertheless, there was an increase in the number of EVs produced per MSC in the 3-D system when the commercial medium was used.
• The 3-D culture system also showed a decrease in glucose levels and accumulation of lactate and ammonium.
• Interestingly, the surface markers indicating stemness showed reduced activity in the 3-D culture.

Conclusion

The researchers concluded that refining their 3-D culture methods could lead to an improved expansion of MSCs, which could potentially enhance the overall yield of EVs. Further research is needed to understand the significance of the reduced expression of stemness markers and how it influences the therapeutic potential of EVs.