Scalable production of small extracellular vesicles (sEV) for immune therapy: integrating management of cellular stress in upstream processing
Résumé
Scalable production of small extracellular vesicles (sEV) for immune therapy: integrating management of cellular stress in upstream processing Laurence de Beaurepaire1, Thibaud Dauphin1, Claire Boursier1, Claire Nouguier1, Mathilde Laubert1, Mailys Le Devehat1, Lucie Grare1, Quentin Le Yondre 1, Quentin Pruvost 1, Eugénie Lahet 1, Dominique Jégou1, Philippe Courcoux2, Aurélien Dupont3, Julien Pichon4, Laurence Dubreil4, Mayeul Collot5, Blandine Lieubeau1, Grégoire Mignot1, Jean-Marie Bach1, Steffi Bosch1, and Mathilde Mosser1 1 IECM, Oniris, INRAE, USC1383, Nantes, France 2 StatSC, Oniris, INRAE, USC 1381, Nantes, France 3 MRic, Biosit, UMS3480 CNRS, University of Rennes 1, Rennes, France 4 PAnTher, INRAE, Oniris, F-44307 Nantes France 5 Laboratoire de Biophotonique et Pharmacologie, UMR CNRS 7213, Université de Strasbourg, Illkirch, France Contact: mathilde.mosser@oniris-nantes.fr Introduction. Current advances in the development of sEV-based therapeutics highlight the need for scalable production. Stirred tank bioreactors allow culturing adherent cells on microcarriers or in aggregates at large scale, but potentially engender cellular stress responses due to shearing forces and serum starvation that may affect sEV immune properties. Hence, alleviating cellular stress upstream is key to streamline robust manufacturing of therapeutic EV. Method. We here describe a generic framework for the identification and management of critical sources of stress in culture including the medium formulation (w/ or w/o serum, glucose control), the mode of culture (monolayer/ aggregates) and the process parameters (stirring, duration, cell density). The response variables were cell viability (Trypan blue, LDH), stress markers (CHOP, GRP94, XBP1), cell function (metabolic response) and sEV production. sEV were isolated by a method combing differential centrifugation, tangential flow filtration and size exclusion chromatography and compared for yield and purity. Results. First, our data show the potential of aggregate cultures to promote cell maturation, without changing the quantity of sEV produced or their size distribution. However, both stirring and serum starvation significantly increase the expression of cellular stress markers and decrease cell viability. As these conditions are both required to design a GMP scalable process, a Response Surface Methodology was conducted to minimize cellular stress by tuning the impellor speed, the seeding cell density and the duration of culture. Interestingly, this optimization step in stirred tank, allowed reaching a similar level of the cell stress that was measured in the static two-dimensional standard culture. Conclusion. The proposed workflow should instruct rational management of cellular stress upstream in sEV-manufacturing bioprocesses for therapy.
| Origine | Fichiers produits par l'(les) auteur(s) |
|---|---|
| Licence |