Optimized operation of a controlled stirred tank reactor system for the production of mesenchymal stromal cells and their extracellular vesicles

Author:

Fernandes‐Platzgummer Ana12ORCID,Cunha Raquel12ORCID,Morini Sara12ORCID,Carvalho Marta12ORCID,Moreno‐Cid Juan3ORCID,García Carmen3,Cabral Joaquim M. S.12ORCID,da Silva Cláudia L.12ORCID

Affiliation:

1. Department of Bioengineering and iBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico Universidade de Lisboa Lisboa Portugal

2. Associate Laboratory i4HB ‐ Institute for Health and Bioeconomy, Instituto Superior Técnico Universidade de Lisboa Lisboa Portugal

3. Bionet Servicios Técnicos S.L., Avenida Azul, parcela 2.11.2, 30320 Parque Tecnológico de Fuente Álamo Murcia Spain

Abstract

AbstractThe 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) × 108 and (5.3 ± 0.32) × 108 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 transmission electron microscopy, and EV protein markers were successfully identified by Western blot 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) × 1011 EVs/mL and (3.0 ± 0.48) × 1011 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 toward the development of human MSC‐ and MSC‐EV‐based products as promising therapeutic agents for Regenerative Medicine settings.

Funder

Fundação para a Ciência e a Tecnologia

Publisher

Wiley

Subject

Applied Microbiology and Biotechnology,Bioengineering,Biotechnology

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