Process Development for MSC-Derived Extracellular Vesicles: Revealing Critical Parameters in Small- and Large-scale Cultivations
Background
Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) hold great promise for therapy due to their immunomodulatory, regenerative, and angiogenic properties. However, standardized and scalable production methods are still lacking.
Methods
We investigated the effects of various process parameters on MSC growth and production of MSC-EVs and their functionality in a HaCaT cell migration assay. Using an immortalized cell line (hMSC-TERT) and chemically defined medium, we aimed to optimize both small- and large-scale processes.
Results
In small-scale (T-flask), our results suggest that several process parameters, which do not necessarily require complex and expensive equipment for control, can significantly increase EV production and functionality. These parameters include raw material selection, cell culture parameters such as seeding density, cell proliferation state, harvest time for MSC-EV, culture temperature and oxygen levels. Integrating these parameters into a standardized EV process development led to a growth-associated EV production and a potential correlation between EV production and glucose consumption, allowing the estimation of final MSC-EV yields.
For late clinical phases, scalable processes are essential. We first investigated dynamic cultivations in spinner and shaking flasks. Using non-porous microcarriers in spinner flasks increased cell-specific MSC-EV production six-fold. High particle production was associated with increased glucose consumption. In a 1-L stirred tank bioreactor, cell-specific EV production increased 24-fold, and functional MSC-EV concentration tripled. Additional stresses such as hypoxic conditions and high shear flow regimes did not alter EV production, but reduced cell-specific glucose consumption and consequently increased the EV yield per unit glucose.
Conclusion
Our results highlight the importance of optimizing key process parameters to improve MSC-EV production and functionality. The advancements lay the foundation for efficient and scalable MSC-EV manufacturing, essential for advancing cell therapy solutions to clinical applications.
Keywords
Mesenchymal stromal cell, extracellular vesicles, process development, large-scale MSC-EV production.
Authors
Jan Thorbow1, Peter Czermak1,2, Denise Salzig1 (Corresponding Author: denise.salzig[at]lse.thm[dot]de)