Comparison of msc-evs manufatured in 2D versus scalable 3D bioreactor systems

Abstract

Background & Aim There have been over 800 registered clinical trials using mesenchymal stem/stromal cells (MSCs) for therapeutic applications. Due to their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. It was recently found that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (Phinney, Pittenger 2017). Hence, most indications would require an MSC production lot size that is hardly achievable in 2D planar cell culture; larger scalable bioreactor systems will be crucial to generate enough EVs for clinical doses. Therefore, this study compared the yield and characteristics of MSC-derived EVs generated in typical 2D culture with those from scalable 3D bioreactor systems. Methods, Results & Conclusion Human bone marrow-derived MSCs were cultured on microcarriers in 3L and 15L bioreactors. After initial cell inoculation, a bioreactor feed was added on Day 3, and cultures were switched to an EV collection media on Day 4, then cultured for 3 additional days. Samples of the conditioned media were analyzed for particle size and concentration. The collected MSC-EVs were evaluated for protein and RNA expression and wound healing capability. MSC-EVs were compared between bioreactor scales and with a 2D control cultured in a 10-layer cell stack. Higher cell densities were achieved in 3D compared to 2D culture, with similar cell densities in the 3L and 15L bioreactors. The conditioned media was analyzed after the 3-day EV collection period. Particle size distributions were similar across culture systems. The particle concentration in the conditioned media (particles/L media) was similar between the 3L and 15L bioreactors, with the 3D cultures about 3x higher than 2D culture. When particle concentration was normalized to cell number, the productivity (particles/cell) was comparable in the two bioreactor cultures, with 3D cultures greater than 2D. Optimizing EV yield will become increasingly important as EVs are used in the clinic. We have developed a scalable process for MSC-EV generation in a bioreactor. The comparison between 2D and bioreactor cultures demonstrates that a greater EV yield can be accomplished in 3D culture. We intend to continue to scale this process up to production scale (80L) bioreactors, which is critical to generating lot sizes for clinically relevant doses of MSC-EVs.