Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System.

Miguel de Almeida Fuzeta,Diana Gaspar,Nuno Bernardes,Ana Fernandes-Platzgummer,Cláudia Lobato da Silva,Joaquim M. S. Cabral,Filipa D. Oliveira,Sunghoon Jung,William Milligan,Miguel A. R. B. Castanho,Rong-Jeng Tseng,Ana Catarina Costa,José Paulo Farinha,Carlos A. V. Rodrigues,Brian Lee

doi:10.3389/fcell.2020.553444

Abstract

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between −15.5 ± 1.6 mV and −19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

Highlights

Mesenchymal stromal cells (MSC) exhibit multilineage differentiation ability, as well as intrinsic immunomodulatory and trophic activities, standing as promising candidates for tissue engineering and cell-based therapies (Caplan and Dennis, 2006; da Silva Meirelles et al, 2009)
Building on previous work from our group, a S/XF microcarrier-based culture system implemented in a Vertical-WheelTM bioreactors (VWBR) originally targeting MSC expansion was adapted to the production of cell-derived products such as MSC-extracellular vesicles (EVs) and compared with traditional static culture systems (i.e., T-flasks) (Figure 1)
bone marrow (BM), adipose tissue (AT) and umbilical cord matrix (UCM) MSC were successfully expanded in the VWBR system (Figure 2A, upper panel)

Summary

Introduction

Mesenchymal stromal cells (MSC) exhibit multilineage differentiation ability, as well as intrinsic immunomodulatory and trophic activities, standing as promising candidates for tissue engineering and cell-based therapies (Caplan and Dennis, 2006; da Silva Meirelles et al, 2009). EVs, such as exosomes and microvesicles, are lipid membrane enclosed structures actively secreted by cells These vesicles have emerged as relevant mediators of intercellular communication, through the transfer of a cargo of proteins and RNA (i.e., microRNA and mRNA), which trigger alterations on host cells (Raposo et al, 1996; Ratajczak et al, 2006; Valadi et al, 2007). Their small size (generally 50 – 1000 nm) and resemblance to the cell membrane makes EVs ideal candidates to cross biological barriers, providing high biocompatibility to target cells (Alvarez-Erviti et al, 2011; El Andaloussi et al, 2013; Van Niel et al, 2018)

Methods

Results

Discussion

Conclusion