Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system

Hélder Bandarra-Tavares; Teresa Franchi-Mendes; Cristiana Ulpiano; Sara Morini; Navjot Kaur; Abigail Harris-Becker; Mohan C Vemuri; Joaquim M S Cabral; Ana Fernandes-Platzgummer; Cláudia L da Silva

doi:10.1016/j.jcyt.2024.03.001

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system

Cytotherapy. 2024 Mar 11:S1465-3249(24)00088-4. doi: 10.1016/j.jcyt.2024.03.001. Online ahead of print.

Affiliations

¹ Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
² Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA.
³ Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal. Electronic address: claudia_lobato@tecnico.ulisboa.pt.

PMID: 38506771
DOI: 10.1016/j.jcyt.2024.03.001

Abstract

Background & aims: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium.

Methods: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 10⁶ cells per spinner flask, i.e., 2.8 × 10³ cells per cm² of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h.

Results: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1).

Conclusions: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

Keywords: dissolvable microcarriers; ex vivo expansion; extracellular vesicles; mesenchymal stromal cells; umbilical cord-derived Wharton's jelly.