Extracellular Vesicles represent one of the most innovative alternative to cell therapy in regenerative medicine. However, the manufacturing methods of batches containing huge amount of extracellular vesicles are costly and time consuming. In collaboration with the Matière et Systèmes Complexes laboratory from Université de Paris/CNRS, EVerZom has developed a unique and patented technology to overcome these hurdles. Our process that can be adapted to large scale boosts extracellular vesicle release by exerting a controlled mechanical stimulation on cells during their three-dimensional culture in a turbulent flow in spinner-flasks or bioreactors.

Thus we grow adherent cells, like human mesenchymal stem cells, on microcarriers. These 200 μm polymer beads benefit from a high surface to volume ratio and can be handled easily. A few grams of these beads can correspond to the surface of tens of flasks. The cells anchored to microcarriers are contained in spinner-flasks or bioreactors that can be considered like bottles of media agitated by a central impeller. The two 3D setups differ : 1) spinner-flasks ranging from 100 mL to 8 L, are placed in incubators on magnetic agitators while bioreactors ranging from 1 L to 50L, are independent stirring devices where many parameters like temperature, pH or gas are controlled.

During the seeding or growing cellular phase, a gentle mixing homogeneously resuspends the beads. The medium which provides nutrients to the cells is changed regularly depending on their metabolic activity. When we have reached the desired confluence on microcarriers (less than a week), we trigger a massive extracellular vesicles release (few hours) with a precise mechanical stimulation in a serum-free medium. To do this, we calculate the right micro-scale vortices that stimulate cells by Kolmogorov’s equation considering parameters like the geometry of the system, kinematic viscosity and the impeller rotation speed. All along the vesiculation protocol, we control that this shear stress does not damage the cells and monitor the quantity and size of the extracellular vesicles released. The turbulence-generated extracellular vesicles conserve the properties of their parental cells are collected at the end of the process, submitted to purification and analyzed. The ambition of EVerZom technology is to produce clinical grade/GMP exosomes services and bring these new discoveries to the patient care.

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