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The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~10 13 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cardiac explant-derived cells (cEDC), also referred as cardiac progenitors cells (CPC) (Barile et al., Cardiovasc Res 103(4):530-541, 2014; Barile et al., Cardiovasc Res 114(7):992-1005, 2018), represent promising candidates for the development of cell-based therapies, a novel and interesting treatment for cardioprotective strategy in heart failure (Kreke et al., Expert Rev Cardiovasc Ther 10(9):1185-1194, 2012). CPC have been tested in a preclinical setting for direct cell transplantation and tissue engineering or as a source for production of extracellular vesicles (EV) (Oh et al., J Cardiol 68(5):361-367, 2016; Barile et al., Eur Heart J 38(18):1372-1379, 2017; Rosen et al., J Am Coll Cardiol 64(9):922-937, 2014). CPC cultured as cardiospheres derived cells went through favorable Phase 1 and 2 studies demonstrating safety and possible efficacy (Makkar et al., Lancet 379(9819):895-904, 2012; Ishigami et al., Circ Res 120(7):1162-1173, 2017; Ishigami et al., Circ Res 116 (4):653-664, 2015; Tarui et al., J Thorac Cardiovasc Surg 150(5):1198-1207, 1208 e1191-1192, 2015). In this context and in view of clinical applications, cells have to be prepared and released according to Good Manufacturing Practices (GMP) (EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part I-basic requirements for medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ; EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part IV-guidelines on good manufacturing practices specific to advanced therapy medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ). This chapter describes GMP-grade methods for production and testing of a CPC Master Cell Bank (MCB), consisting of frozen aliquots of cells that may be used either as a therapeutic product or as source for the manufacturing of Exo for clinical trials.The MCB production method has been designed to isolate and expand CPC from human cardiac tissue in xeno-free conditions (Andriolo et al., Front Physiol 9:1169, 2018). The quality control (QC) methods have been implemented to assess the safety (sterility, endotoxin, mycoplasma, cell senescence, tumorigenicity) and identity/potency/purity (cell count and viability, RT-PCR, immunophenotype) of the cells (Andriolo et al., Front Physiol 9:1169, 2018).

Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 .

The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.