Received: 9 September 2021 | Revised: 22 November 2021 | Accepted: 26 November 2021 DOI: 10.1111/jcmm.17112 C O M M E N TA R Y Clinical translation of mesenchymal stromal cell extracellular vesicles: Considerations on scientific rationale and production requisites Yvan Courageux1,2 | Marta Monguió-­Tortajada1,2,3 | Cristina Prat-­Vidal1,2,3,4 | Antoni Bayes-­Genis1,2,3,5,6 | Santiago Roura1,2,3,7,8 1 ICREC Research Program, Health Science Research Institute Germans Trias i Pujol, Badalona, Spain 2 Heart Institute (iCor), Germans Trias i Pujol University Hospital, Badalona, Spain 3 CIBERCV, Instituto de Salud Carlos III, Madrid, Spain 4 Servei de Teràpia Cel·lular, Banc de Sang i Teixits, Barcelona, Spain 5 Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Spain 6 Department of Medicine, Universitat Autonoma de Barcelona, Barcelona, Spain 7 Institut d’Investigació Biomèdica de Bellvitge-­IDIBELL, L´Hospitalet de Llobregat, Spain 8 Faculty of Medicine, University of Vic-­Central University of Catalonia, Vic, Spain Correspondence Santiago Roura, ICREC Research Program, Abstract Health Science Research Institute The present paper is a commentary to ‘Identification and characterization of hADSC-­ Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain. derived exosome proteins from different isolation methods’ (Huang et al. 2021; 10.1111/ Email: sroura@igtp.cat jcmm.16775). Given the enthusiasm for the potential of mesenchymal stromal cell-­ Funding information derived extracellular vesicles (MSC-­E Vs), some considerations deserve attention as This work was supported by grants they move through successive stages of research and application into humans. We from the Spanish Ministry of Economy and Competitiveness-­MINECO herein remark the prerequisite of generating that evidence ensuring a high consist- (SAF2017-­8 4324-­C2-­1-­R and PID-­ ency in safety, composition and biological activity of the intended MSC-­E V prepara- 2019-­11137RB-­I 00), Instituto de Salud Carlos III (PI17/01487, PI18/00256, tions, and the suitability of disparate isolation techniques to produce efficacious EV PIC18/00014, ICI19/00039, PI19/01788 preparations and fulfil requirements for standardized clinical-­grade biomanufacturing. and ICI20/00135) funds, Red de Terapia Celular-­TerCel (RD16/00111/0006), CIBER Cardiovascular (CB16/11/00403) KEYWORDS projects, as part of the Plan Nacional biomanufacturing, clinical translation, extracellular vesicles, mesenchymal stromal cells, size de I+D+I, and co-­funded by ISCIII-­ exclusion chromatography Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) by the Generalitat de Catalunya [SGR2017 00483, SLT002/16/00234], and the CERCA Programme/Generalitat de Catalunya, ‘la Caixa’ Banking Foundation, Sociedad Española de Cardiología and Societat Catalana de Cardiologia This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. J Cell Mol Med. 2022;26:937–939.  wileyonlinelibrary.com/journal/jcmm | 937 938 | COMMENTARY Available preclinical findings indicate that mesenchymal stromal to large-­scale production while meeting current good manufacturing cell-­derived extracellular vesicles (MSC-­E Vs) are highly versatile and practices and regulatory instructions?’. We and others agree with powerful biologicals in terms of regenerative and anti-­inflammatory the authors that the diversity in molecular contents specifically re- properties for the development of efficient cell-­free therapies.1 Data flects the wide range of biological functions of isolated MSC-­E Vs, point out that MSC-­E Vs trigger effects that are comparable to those as well as certain advantages and disadvantages of each method's of their parental cells. Also, they display better stability and handling impact on forthcoming uses, preferentially those aimed at patients.8 as well as reduced toxicity with no alloreactivity, while not being as- Regarding the methods that Huang et al. analysed, UC has generally sociated with tumorigenic nor thrombogenic risk after intravascular been considered as the most conventional method for MSC-­E V iso- administration, in addition of having a targetable biodistribution. lation, from either cells or biological fluids, but it is relatively time-­ Thus, much effort is currently focused on their effective clinical ap- consuming and requires large sample volume processing and access plication. In this context, Huang et al. characterized the differences to specialized laboratory equipment that are not always available in protein cargo from MSC-­E Vs after using ultracentrifugation (UC), to clinical researchers. Furthermore, UC has been demonstrated to size exclusion chromatography (SEC), ExoQuick-­TC precipitation and affect integrity of EVs, and pellet EVs together with protein aggre- 2 ExoQuick-­TC ULTRA as isolation methods. In our opinion, the pre- gates and other impurities present in the starting material. Thus, it is sented results lead to some crucial questions. difficult to discern if the resulting biological activity is predominantly On the question ‘Do we have enough evidence of the therapeu- associated with EVs instead of with co-­isolated soluble mediators. tic value of MSC-­E Vs to endorse their clinical application?’, many The same happens with alternative approaches based on adding pre- animal experimental studies in diverse disease in vivo models sug- cipitating agents such as ExoQuick-­TC, which more quickly and easily gest that MSC-­E V administration is safe and promotes beneficial recover EVs from low-­volume samples than UC, but also with other effects.3 However, despite the efforts for standardized character- non-­E V impurities. Moreover, there are some techniques allowing a ization agreed with International Society for Extracellular Vesicles stringent EV separation, either based on gradient centrifugations or 4 recommendations, the lack of consensus in a common EV isolation immunoaffinity isolation, which allow distinction of subpopulations strategy together with the frequent coarse description of EV prepa- but would be not suited at all for large-­scale production due to ei- rations and the discrepancies in doses used in disease models make ther their intrinsic workflow or high time-­consuming and costs. SEC, it difficult to compare data from overly heterogeneous EV prepa- an isolation methodology based on size separation using prepacked 5 rations. This leads to a non-­negligible bias as well as noteworthy chromatography columns, is superior in isolating well-­defined EVs limitations and difficulties in extrapolating results from a plethora achieving effects that resemble those of parental MSCs.9 This tech- of scientific literature on the potential of MSC-­E Vs. For instance, nique is cost-­efficient, greatly reducing the amount of impurities in Huang et al. highlight the different compositions of EV samples the EV preparation, and it can be easily combined with tangential depending on the type of isolation technique used, as others have flow filtration for clarification and upstream/downstream concen- highlighted.6 At the same time, although Huang et al. used electron tration to obtain pure EVs from large-­scale productions. While it has microscopy to characterize the obtained preparations, higher set- been described that serum-­derived lipoproteins may remain,10 there tings for both image size and quality are more recommendable to be are approximations combining SEC with anionic affinity isolation or applied to undoubtedly discriminate whole double-­membrane EVs using different sizes of SEC matrix if they interfere with EV func- from non-­intact vesicles, highly electron-­dense non-­vesicular parti- tion and thus their presence needs to be avoided. To achieve clinical cles or simply protein aggregates to evaluate EV integrity and pres- translation, in addition to meeting the aimed scale range, production ence of gross impurities. In parallel, the use of Nanoparticle Tracking must necessarily warrant high reproducibility as well as an unequiv- Analysis for establishing the yield/recovery of EVs comparing differ- ocal safety profile associated with therapeutic benefit. In this sense, ent isolation techniques has a limited reliability, as while being one the implementation of a scalable MSC-­E V production is essential. To of the currently available EV quantification tools it can overestimate that end, growing attention is also given to the use of bioreactors the number of particles when EV preparations are not pure enough. and chemically defined medium which promise scalability, better These are just examples of the issues that need a rigorous evalua- reproducibility and less safety concerns. Additionally, specific pre- tion to ensure trustworthy conclusions and continue pursuing the conditioning of parental MSCs could stimulate the production of EVs clinical application of MSC-­E V products. Therefore, the scientific displaying modified cargoes and enhanced therapeutic potential. community, working back-­to-­back with official regulatory agencies, should focus on generating data from robust and well-­characterized C O N FL I C T S O F I N T E R E S T MSC-­E V samples, with safety and efficacy at the centre of the re- The authors confirm that there are no conflicts of interest. search, and anticipating relevant experiments and models to retrieve clues on the mechanisms of action.7 AU T H O R C O N T R I B U T I O N Given today's different in-­house EV separation strategies that Yvan Courageux: Writing –­original draft (equal); Writing –­review & result in varied degrees of yield, purity and composition as afore- editing (equal). Marta Monguió-­Tortajada: Writing –­ review & edit- mentioned, the second question is as follows: ‘Which of the techni- ing (equal). Cristina Prat-­Vidal: Writing –­ review & editing (equal). cal procedures shows the greatest applicability and ability to adapt Antoni Bayes-­Genis: Funding acquisition (equal); Writing –­ review COMMENTARY | 939 5. Gupta D, Zickler AM, El Andaloussi S. Dosing extracellular vesicles. Adv & editing (equal). Santiago Roura: Conceptualization (lead); Funding Drug Deliv Rev. 2021;178:113961. doi:10.1016/j.addr.2021.113961 acquisition (lead); Writing –­original draft (equal); Writing –­review & 6. Veerman RE, Teeuwen L, Czarnewski P, et al. Molecular evaluation editing (equal). of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J Extracell Vesicles. 2021;10(9):e12128. doi:10.1002/jev2.12128 DATA AVA I L A B I L I T Y S TAT E M E N T 7. Reiner AT, Witwer KW, van Balkom BWM, et al. Concise review: Data sharing not applicable –­ no new data generated, or the article developing best-­practice models for the therapeutic use of ex- describes entirely theoretical research tracellular vesicles. Stem Cells Transl Med. 2017;6(8):1730-­1739. doi:10.1002/sctm.17-­0 055 8. Taylor DD, Sahil SS. Methods of isolating extracellular vesicles im- ORCID pact down-­stream analyses of their cargoes. Methods. 2015;87:3-­ Santiago Roura https://orcid.org/0000-0003-4063-9661 10. doi:10.1016/j.ymeth.2015.02.019 9. Monguió-­Tortajada M, Roura S, Gálvez-­Montón C, et al. Nanosized REFERENCES UCMSC-­derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: 1. Johnson J, Shojaee M, Mitchell Crow J, Khanabdali R. From mes- implications for nanomedicine. Theranostics. 2017;7(2):270-­284. enchymal stromal cells to engineered extracellular vesicles: a doi:10.7150/thno.16154 new therapeutic paradigm. 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