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Optimized operation of a controlled stirred tank reactor system for the production of mesenchymal stromal cells and their extracellular vesicles
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  • Ana Fernandes-Platzgummer,
  • Raquel Cunha,
  • Sara Morini,
  • Marta Carvalho,
  • Juan Moreno-Cid,
  • Joaquim Cabral,
  • Claudio Lobato da Silva
Ana Fernandes-Platzgummer
Universidade de Lisboa Instituto Superior Tecnico

Corresponding Author:[email protected]

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Raquel Cunha
Universidade de Lisboa Instituto Superior Tecnico
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Sara Morini
Universidade de Lisboa Instituto Superior Tecnico
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Marta Carvalho
Universidade de Lisboa Instituto Superior Tecnico
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Juan Moreno-Cid
Bionet Servicios Técnicos SL
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Joaquim Cabral
Universidade de Lisboa Instituto Superior Tecnico
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Claudio Lobato da Silva
Universidade de Lisboa Instituto Superior Tecnico
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The therapeutic effects of human mesenchymal stromal cells (MSC) have been attributed mostly to their paracrine activity, exerted through small-secreted extracellular vesicles (EVs) rather than their engraftment into injured tissues. Currently, the production of MSC-derived EVs (MSC-EVs) is performed in laborious static culture systems with limited manufacturing capacity using serum-containing media. In this work, a serum-/xenogeneic-free microcarrier-based culture system was successfully established for bone marrow-derived MSC cultivation and MSC-EV production using a 2 L-scale controlled stirred tank reactor (STR) operated under fed-batch (FB) or fed-batch combined with continuous perfusion (FB/CP). Overall, maximal cell numbers of (3.0±0.12)×10 8 and (5.3±0.32)×10 8 were attained at days 8 and 12 for FB and FB/CP cultures, respectively, and MSC(M) expanded under both conditions retained their immunophenotype. MSC-EVs were identified in the conditioned medium collected from all STR cultures by TEM, and EV protein markers were successfully identified by WB analysis. Overall, no significant differences were observed between EVs isolated from MSC expanded in STR operated under the two feeding approaches. EV mean sizes of 163±5.27 nm and 162±4.44 nm (P>0.05) and concentrations of (2.4±0.35)x10 11 EVs/mL and (3.0±0.48)x10 11 EVs/mL (P>0.05) were estimated by nanoparticle tracking analysis for FB and FB/CP cultures, respectively. The STR-based platform optimized herein represents a major contribution towards the development of human MSC- and MSC-EV-based products as promising therapeutic agents for Regenerative Medicine settings.
21 Dec 2022Submitted to Biotechnology and Bioengineering
21 Dec 2022Submission Checks Completed
21 Dec 2022Assigned to Editor
21 Dec 2022Review(s) Completed, Editorial Evaluation Pending
23 Dec 2022Reviewer(s) Assigned
20 Feb 2023Editorial Decision: Revise Major
24 Apr 20231st Revision Received
24 Apr 2023Submission Checks Completed
24 Apr 2023Assigned to Editor
24 Apr 2023Review(s) Completed, Editorial Evaluation Pending
24 Apr 2023Reviewer(s) Assigned
25 Apr 2023Editorial Decision: Accept