GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment

Zaquer Suzana Munhoz Costa-Ferro; Gisele Vieira Rocha; Katia Nunes da Silva; Bruno Diaz Paredes; Erick Correia Loiola; Johnatas Dutra Silva; John Lenon de Souza Santos; Rosane Borges Dias; Cláudio Pereira Figueira; Camila Indiani de Oliveira; Ludmilla David de Moura; Lígia Nunes de Morais Ribeiro; Eneida de Paula; Dalila Lucíola Zanette; Clarissa Araújo Gurgel Rocha; Patricia Rieken Macedo Rocco; Bruno Solano de Freitas Souza

doi:10.1016/j.jcyt.2024.04.074

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment

Cytotherapy. 2024 May 1:S1465-3249(24)00686-8. doi: 10.1016/j.jcyt.2024.04.074. Online ahead of print.

Authors

Zaquer Suzana Munhoz Costa-Ferro¹, Gisele Vieira Rocha¹, Katia Nunes da Silva², Bruno Diaz Paredes¹, Erick Correia Loiola¹, Johnatas Dutra Silva³, John Lenon de Souza Santos⁴, Rosane Borges Dias⁵, Cláudio Pereira Figueira⁶, Camila Indiani de Oliveira⁶, Ludmilla David de Moura⁷, Lígia Nunes de Morais Ribeiro⁸, Eneida de Paula⁷, Dalila Lucíola Zanette⁹, Clarissa Araújo Gurgel Rocha¹⁰, Patricia Rieken Macedo Rocco³, Bruno Solano de Freitas Souza¹¹

Affiliations

¹ Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil; D'Or Institute for Research and Education (IDOR), Salvador, Brazil.
² Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil.
³ Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil; Rio de Janeiro Innovation Network in Nanosystems for Health-NanoSaúde, Research Support Foundation of the State of Rio de Janeiro, Rio de Janeiro, Brazil.
⁴ Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil; Gonçalo Moniz Institute, FIOCRUZ, Salvador, Brazil.
⁵ Gonçalo Moniz Institute, FIOCRUZ, Salvador, Brazil; Federal University of Bahia, UFBA, Salvador, Brazil.
⁶ Gonçalo Moniz Institute, FIOCRUZ, Salvador, Brazil.
⁷ Institute of Biology, University of Campinas, Campinas, São Paulo, Brazil.
⁸ Institute of Biology, University of Campinas, Campinas, São Paulo, Brazil; Institute of Biotechnology, Federal University of Uberlândia, Uberlândia, Brazil.
⁹ Carlos Chagas Institute, FIOCRUZ, Curitiba, Brazil.
¹⁰ Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil; D'Or Institute for Research and Education (IDOR), Salvador, Brazil; Gonçalo Moniz Institute, FIOCRUZ, Salvador, Brazil; Federal University of Bahia, UFBA, Salvador, Brazil.
¹¹ Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil; D'Or Institute for Research and Education (IDOR), Salvador, Brazil; Gonçalo Moniz Institute, FIOCRUZ, Salvador, Brazil. Electronic address: bruno.solano@fiocruz.br.

PMID: 38762805
DOI: 10.1016/j.jcyt.2024.04.074

Abstract

Background aims: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model.

Methods: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model.

Results: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury.

Conclusions: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

Keywords: ARDS; biodistribution; extracellular vesicles; mesenchymal stromal cells; pre-clinical; proteomics; toxicity.