Scalable production of human mesenchymal stromal cell-derived extracellular vesicles under serum-/xeno-free conditions in a microcarrier-based bioreactor culture system

fcell-08-553444 October 28, 2020 Time: 18:7 # 1 ORIGINAL RESEARCH published: 03 November 2020 doi: 10.3389/fcell.2020.553444 Edited by: Lindolfo da Silva Meirelles, Universidade Luterana do Brazil, Brazil Reviewed by: Laura Iop, University of Padua, Italy Milena Botelho Pereira Soares, Gonçalo Moniz Institute (IGM), Brazil *Correspondence: Nuno Bernardes [email protected] Cláudia Lobato da Silva [email protected] Specialty section: This article was submitted to Stem Cell Research, a section of the journal Frontiers in Cell and Developmental Biology Received: 18 April 2020 Accepted: 05 October 2020 Published: 03 November 2020 Citation: de Almeida Fuzeta M, Bernardes N, Oliveira FD, Costa AC, Fernandes-Platzgummer A, Farinha JP, Rodrigues CAV, Jung S, Tseng R-J, Milligan W, Lee B, Castanho MARB, Gaspar D, Cabral JMS and da Silva CL (2020) Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System. Front. Cell Dev. Biol. 8:553444. doi: 10.3389/fcell.2020.553444 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 Fuzeta1,2, Nuno Bernardes1*, Filipa D. Oliveira2, Ana Catarina Costa1, Ana Fernandes-Platzgummer1, José Paulo Farinha3, Carlos A. V. Rodrigues1, Sunghoon Jung4, Rong-Jeng Tseng5, William Milligan5, Brian Lee4, Miguel A. R. B. Castanho2, Diana Gaspar2, Joaquim M. S. Cabral1and Cláudia Lobato da Silva1* 1iBB-Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 2Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal, 3Centro de Química Estrutural and Department of Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 4PBS Biotech Inc., Camarillo, CA, United States, 5AventaCell Biomedical Corp., Atlanta, GA, United States 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 MSCderived 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 Frontiers in Cell and Developmental Biology | www.frontiersin.org 1November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 2 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors 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. Keywords: extracellular vesicles, mesenchymal stromal cells (MSC), scalable production, bioreactors, serum- /xenogeneic-free 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). MSC are able to inhibit apoptosis and scarring (fibrosis), promote angiogenesis and support growth and differentiation of progenitor cells into functional regenerative units (Caplan and Dennis, 2006;da Silva Meirelles et al., 2009). The array of beneficial effects attributed to MSC has made them one of the most studied cells in clinical trials (Heathman et al., 2015). The trophic activity of MSC relies greatly on the secretion of bioactive factors that assist in repair and regeneration processes through paracrine signaling (Caplan and Dennis, 2006;da Silva Meirelles et al., 2009). Recently, increasing evidence suggests that several MSCassociated paracrine therapeutic features are mediated by extracellular vesicles (EVs) (Bruno et al., 2009;Lai et al., 2010;Lener et al., 2015;Börger et al., 2017). 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, thus providing high biocompatibility to target cells (Alvarez-Erviti et al., 2011;El Andaloussi et al., 2013; Van Niel et al., 2018). EV can be used in therapeutic settings through two different approaches. On one hand, EVs are able to mediate some of the therapeutic effects from their cells of origin (Lai et al., 2010; Bruno et al., 2012). Therefore, EVs could be potentially used in substitution of their cell of origin, as a cell-free therapy triggering equivalent therapeutic effect. On the other hand, EVs can be used as drug delivery vehicles, by loading EVs with therapeutic cargo, as an alternative to synthetic drug delivery systems (Batrakova and Kim, 2015). MSC are particularly interesting for EV production for a number of reasons. MSC are considered immune evasive cells and the safety of their administration has already been confirmed in a number of clinical trials (Lalu et al., 2012). Therefore, it is reasonable to assume that MSC-derived EVs (MSC-EVs) are not prone to immune reaction from the host immune system (Mendt et al., 2018;Elahi et al., 2020), and promising for the development of allogeneic (i.e., off-the-shelf) therapeutic products. MSC are intrinsically therapeutic, with promising applications for multiple diseases and MSC-EVs convey similar benefits as well (Lener et al., 2015;Phinney and Pittenger, 2017). Finally, MSC show great ability for expansion when cultured ex vivo and robust expansion platforms have already been established (Rafiq et al., 2013;dos Santos et al., 2014;Schirmaier et al., 2014;Carmelo et al., 2015;Mizukami et al., 2016;Lawson et al., 2017). 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. Similarly to the cell therapy context, where large cell numbers per dose are required (Ren et al., 2012;Golpanian et al., 2016; Wysoczynski et al., 2018), very large numbers of EVs are expected to be required for clinical use (e.g., each patient may require 0.5 – 1.4 ×1011 EVs, Kordelas et al., 2014). In order to achieve such large production capacities, robust and scalable manufacturing processes need to be developed. The development of cell-based therapies faces multiple challenges (recently reviewed de Almeida Fuzeta et al., 2019) and these also apply to manufacturing of EV products. One of these challenges is the use of appropriate cell culture medium. The most commonly used culture medium supplement in ex vivo expansion platforms of MSC is fetal bovine serum (FBS), which presents several disadvantages when considering the production of cell-based therapies for human use due to their animal origin. As an alternative to animal derived products, serum-/xenogeneicfree (S/XF) culture supplements have been developed, such as human platelet lysates (hPL). Another major challenge is determining the appropriate cell culture platform for scalable manufacturing of cell-based therapies (de Almeida Fuzeta et al., 2019). In order to achieve large product batches for clinical use, culture platforms require scalability as well as the ability to monitor and control culture parameters, which cannot be accomplished in traditional static culture systems. Multiple bioreactor configurations operating in dynamic culture conditions have been developed for this Frontiers in Cell and Developmental Biology | www.frontiersin.org 2November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 3 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors purpose (de Soure et al., 2016;de Almeida Fuzeta et al., 2019). Expansion of MSC immobilized on microcarriers has been explored in stirred-tank bioreactor configurations (de Soure et al., 2016;de Almeida Fuzeta et al., 2019). These bioreactors use an agitation system to maintain microcarriers in suspension and allow medium homogenization. However, agitation impacts cellular physiology due to increased shear stress. In order to improve agitation patterns in cell culture, PBS Biotech has developed scalable Vertical-WheelTM bioreactors (VWBR) that can provide gentle and uniform mixing with minimal shear stress. A vertically rotating wheel promotes radial and axial fluid flow and creates a more homogeneous hydrodynamic environment compared with traditional stirredtank bioreactors. In addition, the Vertical-WheelTM impeller can fully suspend microcarriers with minimal power input and thus minimize shear stress effects (Croughan et al., 2016). Moreover, this technology is scalable, being available at working volumes that range from 100 mL up to 500 L. Recently, VWBR have been successfully applied in microcarrier-based cell culture processes for the expansion of MSC from multiple sources (Sousa et al., 2015;de Sousa Pinto et al., 2019), as well as for human induced pluripotent stem cells (Rodrigues et al., 2018; Nogueira et al., 2019). In this work, EVs were produced by MSC isolated from different human tissue sources, namely bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM). A S/XF microcarrier-based culture system was implemented in a singleuse VWBR, employing a hPL culture supplement (UltraGROTMPURE), toward the production of MSC-EVs. When compared with traditional static culture systems (i.e., T-flasks), the bioreactor-based culture system allowed a substantial improvement in EV production. This culture system is expected to contribute to robustly manufacture human MSCEVs in a scalable manner, which can be applied as intrinsic medicines or as delivery vehicles in different therapeutic settings. MATERIALS AND METHODS MSC Isolation From Human Samples Human MSC used in this study are part of the cell bank available at the Stem Cell Engineering Research Group (SCERG), iBB-Institute for Bioengineering and Biosciences at Instituto Superior Técnico (IST). MSC were previously isolated/expanded according to protocols previously established at iBB-IST. UCM MSC were isolated in hPL-supplemented medium according to the protocol described by de Soure et al. (2017). BM MSC were isolated in hPL-supplemented medium by adapting the protocol for cell isolation using FBS-supplemented medium described by dos Santos et al. (2010). AT MSC were originally isolated in FBS-supplemented medium according to Oliveira et al. (2012), cryopreserved and later adapted for 1 or 2 passages to hPL-supplemented medium. Originally, human tissue samples were obtained from local hospitals under collaboration agreements with iBB-IST (bone marrow: Instituto Português de Oncologia Francisco Gentil, Lisboa; adipose tissue: Clínica de Todos-os-Santos, Lisboa; umbilical cord: Hospital São Francisco Xavier, Lisboa, Centro Hospitalar Lisboa Ocidental, Lisboa). All human samples were obtained from healthy donors after written informed consent according to the Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells (Portuguese Law 22/2007, June 29), with the approval of the Ethics Committee of the respective clinical institution. Human MSC from the different sources (BM, AT, and UCM) were cryopreserved in a liquid/vapor-phase nitrogen container. MSC Expansion in Static Conditions In general, MSC expansion in static conditions was performed as previously described (de Sousa Pinto et al., 2019). In summary, previously isolated BM, AT and UCM MSC were thawed and plated on T-flasks (Falcon), at a cell density between 3000–6000 cell/cm2. MSC were cultured in low glucose (1 g/L) Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Life Technologies), supplemented with 5% v/v of the human platelet lysate (hPL) UltraGROTM-PURE (AventaCell Biomedical) and AntibioticAntimycotic (1x) (Gibco, Life Technologies). Cells were maintained at 37◦C and 5% CO2in a humidified atmosphere and culture medium was changed every 3–4 days. At 70–80% cell confluence, MSC were detached from the flasks using the xeno-free cell detachment solution TrypLETM Select (1x) (Gibco, Life Technologies) for 7 min at 37◦C. Cell number and viability were determined using the Trypan Blue (Gibco, Life Technologies) exclusion method. After thawing, MSC were passaged at least once before either final inoculation in T-flasks for EV production under static conditions or inoculation in VWBR. MSC were always plated at 3000 cell/cm2. For each cell source, MSC from three independent donors (n= 3) in passages (P) from P4 to P5 were used to inoculate either the final T-flasks for EV production or the VWBR [specifically, BM1 (P4); BM2 (P5); BM3 (P4); AT1 (P4); AT2 (P4); AT3 (P5); UCM1 (P4); UCM2 (P4); UCM3 (P5)] (Figure 1). MSC-EV Production Under Static Conditions For the production of MSC-EVs under static conditions, previously cultured MSC were passaged to T-175 flasks, at 3000 cells/cm2. Cells were cultured in the same conditions described before for MSC expansion under static conditions. When maximum cell confluency in the flasks was achieved (90–100%), cells were washed once with basal DMEM low glucose (i.e., supplemented only with Antibiotic-Antimycotic) and subsequently cultured for 48 h in basal DMEM low glucose (20 mL per T-175), for medium conditioning. At the end of the 48 h period, the conditioned medium was recovered, centrifuged (360 ×g, 10 min) to remove cell debris and stored at 4◦C for less than 1 week until processing for EV isolation. After recovery of the conditioned medium, MSC were detached from the flasks and cell number was determined as previously described. Cells were re-suspended in Frontiers in Cell and Developmental Biology | www.frontiersin.org 3November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 4 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors FIGURE 1 | Workflow of the production and characterization of MSC-EVs in bioreactors and static systems. MSC were isolated from three different human tissue sources: BM, AT, and UCM. Firstly, MSC were expanded in static conditions (i.e., T-flasks) in hPL supplemented DMEM. These cells were subsequently used to inoculate a VWBR (5M cells; 100 mL final working volume), as well as to maintain a static culture in T-175 flasks. For each cell source, MSC from three independent donors (n= 3; BM1, 2, 3; AT1, 2, 3; UCM1, 2, 3) were used to inoculate either the final T-flasks for EV production or the VWBR, in passages from P4 to P5 [specifically, BM1 (P4); BM2 (P5); BM3 (P4); AT1 (P4); AT2 (P4); AT3 (P5); UCM1 (P4); UCM2 (P4); UCM3 (P5)]. Upon reaching stationary growth phase in VWBR or maximum confluency in static, the culture medium was changed for supplement-free culture medium and culture was maintained for 48 h. Over this period, culture medium was enriched in EVs secreted by cultured MSC. This conditioned culture medium was recovered and EVs were isolated by precipitation using a commercially available kit. Finally, EV production was quantified in both static and dynamic systems and samples were characterized using multiple techniques. MSC, mesenchymal stromal cells; EV, extracellular vesicles; VWBR, Vertical-WheelTM bioreactor; hPL, human platelet lysate; BM, bone marrow; AT, adipose tissue; UCM, umbilical cord matrix; NTA, nanoparticle tracking analysis; AFM, atomic force microscopy. The cells, T-flask and Eppendorf cartoons were obtained from Smart Servier Medical Art (https://smart.servier.com). phosphate-buffered saline (PBS) for pelleting and stored at −80◦C until further analysis (i.e., Western blots). MSC Expansion and MSC-EV Production in the Bioreactor Culture System Expansion of human MSC in VWBR was generally performed as previously described (de Sousa Pinto et al., 2019). In summary, previously isolated and expanded human MSC were inoculated in a PBS 0.1 MAG bioreactor (PBS Biotech Inc.) with a working volume of 100 mL. Animal productfree SoloHill plastic microcarriers (PALL) were used in order to provide a surface for MSC to adhere and proliferate. Inoculation in the VWBR was performed in 60 mL of the same culture medium used for static conditions (i.e., DMEM low glucose, 5% v/v UltraGROTM-PURE, Antibiotic-Antimycotic 1x), with an initial MSC number of 5 ×106and 2 g of microcarriers. The VWBR was placed at 37◦C and 5% CO2in a humidified atmosphere. After an initial intermittent agitation regime, a continuous agitation mode was set at 25 rpm, as previously described (de Sousa Pinto et al., 2019). This agitation rate was always maintained, except for AT MSC culture, which required an increment in the agitation rate to 30 rpm at day 2 or 3 of culture and to 35 rpm at day 4 or 5, due to increased medium viscosity and the subsequent formation of cell aggregates. After 2 days of culture, 40 mL of fresh culture medium with a glucose pulse (3 g/L) was added to the VWBR, achieving a final working volume of 100 mL. From this day onward, 25% v/v of culture medium was exchanged every 24 h, with the addition of fresh culture medium supplemented with a glucose pulse (3 g/L). Cell growth and viability were assessed every day, as previously described (de Soure et al., 2017). Growth rate was determined by performing an exponential fitting to experimental data corresponding to the exponential growth phase. Cell visualization on microcarriers was performed by staining the cells with 40,6-diamidino-2phenylindole (DAPI, Sigma, 1.5 µg/mL in PBS), as previously described (de Soure et al., 2017). When MSC cultures reached stationary growth and the maximum cell concentration was achieved, the MSC expansion stage of the process was concluded and the EV production stage started. The culture medium was removed from the VWBR, after a 10 min sedimentation of cells attached to microcarriers inside the vessel. The VWBR was washed with 60 mL basal DMEM low glucose medium, at 30 rpm agitation, in order to remove hPL components. The cells on microcarriers were sedimented once again for 10 min and the washing medium was removed. MSC were kept in culture in the VWBR for 48 h in 60 mL basal DMEM low glucose medium, in the same conditions (i.e., agitation speed, temperature, O2and CO2concentrations) used for MSC expansion. At the end of the 48 h period, the whole culture volume was recovered from the VWBR and transferred to 50 mL tubes (Falcon), where cells on microcarriers were sedimented for 10 min. The MSC conditioned medium was recovered and centrifuged at 360 ×gfor 10 min, to remove remaining microcarriers, cells and cell debris. Conditioned medium was stored at 4◦C for less than 1 week until processing for EV isolation. After recovery of the conditioned medium, cells Frontiers in Cell and Developmental Biology | www.frontiersin.org 4November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 5 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors attached to microcarriers were re-suspended in PBS and stored at −80◦C for further analysis (i.e., Western blots). Isolation of EVs From MSC Cultures EV were isolated using the Total Exosome Isolation reagent (Invitrogen, Life Technologies), according to the manufacturer instructions. Briefly, MSC conditioned medium was centrifuged for 30 min at 2000 ×g, to remove cell debris and incubated overnight at 4◦C with the isolation reagent. This mixture was then centrifuged for 1 h at 10000 ×gand 4◦C. The supernatant was discarded and the EV fraction was recovered by thoroughly washing the walls of the centrifuge tube with PBS 1x (Invitrogen, Life Technologies) in UltraPureTM DNase/RNase-Free Distilled Water (Invitrogen, Life Technologies). EV samples were resuspended in a PBS volume corresponding to a concentration factor of 20x to 70x relatively to the processed conditioned medium volume. EV samples were frozen at −80◦C in aliquots (50–100 µL), in order to minimize freeze-thawing cycles. Comprehensive Characterization of Manufactured EVs Protein Quantification Total protein was quantified in EV samples using the PierceTM BCA Protein Assay Kit (Thermo Scientific), according to manufacturer instructions for the microplate procedure. Samples were quantified either undiluted or after a 2x dilution. Three replicates were quantified for each sample. Sample concentration was determined by applying a linear fit to the bovine serum albumin (BSA) standards and using the resulting equation to determine each sample concentration from its absorbance measurement. Nanoparticle Tracking Analysis EV size distribution profiles and concentration measurements were obtained by nanoparticle tracking analysis (NTA), using a NanoSight LM14c instrument equipped with a 405 nm laser (Malvern) and NTA software version 3.1 (Malvern). Silica 100 nm microspheres (Polysciences, Inc.) were routinely analyzed to check instrument performance (Gardiner et al., 2013). NTA acquisition and post-acquisition settings were optimized and kept constant for all samples. These settings were established using silica 100 nm microspheres (Gardiner et al., 2013) and subsequently adjusted for optimal detection of MSC-EVs. EV samples were diluted in 2 mL of PBS 1x in UltraPureTM DNase/RNase-Free Distilled Water, to obtain a final concentration in the range of 5 ×108to 3 ×109particles/mL. Samples were measured using a camera level of 13. Acquisition temperature was controlled and maintained at 20◦C. Each sample was recorded 10 times for 30 s, using fresh sample for each acquisition (by pushing the sample syringe). The detection chamber was thoroughly washed with PBS between each sample measurement. A threshold level of 7 was applied for video processing. Each video recording was analyzed to obtain the size and concentration of EVs. Western Blot Cells were lysed with Catenin lysis buffer (1% Triton X-100, Sigma, 1% Nonidet P-40, Sigma, in PBS) supplemented with protease inhibitor (Sigma) and phosphatase inhibitor (Sigma) for 10 min on ice and then centrifuged at 14000 ×gfor 10 min at 4◦C to remove insoluble material. Supernatants were recovered and used as whole cell lysates (WCL). For CD63 and CD81 detection, cells and EV samples were lysed with RIPA lysis buffer (150 mM NaCl, 25 mM Tris pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) and sonicated (three rounds of 5 s, at 50% intensity). Total protein content in WCL and EV samples was quantified using the BCA kit as previously described. Both WCL and EV samples were mixed with sample buffer in reducing conditions and heated to 100◦C for 10 min. For CD63 and CD81 detection, urea containing sample buffer was used. All samples were loaded (6–30 µg of total protein) in 4–12% Bis– Tris polyacrylamide gels (Invitrogen, Life Technologies), in equal protein content for each gel, and subjected to electrophoresis. Proteins were transferred into nitrocellulose membranes using a Power Blotter System (Invitrogen, Life Technologies). Membranes were blocked with 5% w/v non-fat dry milk solution in tris-buffered saline (TBS) Tween 20 buffer 1x (Thermo Fisher Scientific), for 1 h with mild orbital agitation at room temperature and incubated with primary antibodies overnight at 4◦C. For CD63 and CD81 detection, membranes were blocked with 5% BSA solution in TBS Tween 20 buffer 1x. Finally, membranes were incubated with HRP conjugated secondary antibodies for 1 h at room temperature and PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific) was applied for membrane revelation. Primary antibodies included anti-Calnexin (1:1000, BD), anti-Synthenin (1:1000, Abcam), anti-CD63 (1:1000, Genetex), anti-CD81 (1:500, Abcam) and anti-GAPDH (1:1000, Santa Cruz). Secondary antibodies included Goat anti-Mouse IgG (H +L) Cross-Adsorbed Secondary Antibody, HRP (1:5000, Invitrogen, Life Technologies) and Goat anti-Rabbit IgG HRPconjugated (1:1000, R&D Systems). Image acquisition was performed on iBrightTM CL1500 Imaging System (Invitrogen, Life Technologies). Atomic Force Microscopy Imaging EV samples were prepared for atomic force microscopy (AFM) imaging in freshly cleaved mica without any previous dilution. A volume ranging between 30–70 µL was used and samples were allowed to deposit during 30 min to 2 h. After this period, the samples were washed with filtered MilliQ water and air dried. AFM imaging was performed with a JPK Nano Wizard IV mounted on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss). The AFM head is equipped with a 15 µm z-range linearized piezoelectric scanner and an infrared laser. Uncoated silicon ACL cantilevers from AppNano were used, with resonance frequencies and spring constants ranging between 160–225 kHz and 36–90 N/m, respectively. Scan speeds were between 0.1 and 0.3 Hz. Total areas with 10 ×10 µm were scanned with a 512 ×512 pixel resolution, in AC mode. Height and error images were recorded, and line fitted. Image processing was performed on JPK SPM data processing software version spm-6.0.55. Frontiers in Cell and Developmental Biology | www.frontiersin.org 5November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 6 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors Zeta Potential EV samples were diluted to a final protein concentration of 25 µg/mL, in PBS. Samples were loaded into disposable zeta cells with gold electrodes and allowed to equilibrate for 15 min at 37◦C. Zeta potential measurements consisted in a set of 15 runs, each one resulting from an automatically defined number of subruns (ranging from 10 to 100) performed on the Zetasizer Nano ZS (Malvern), at a constant voltage of 40 V. Lactate Dehydrogenase Activity Measurements Cell culture medium samples from VWBR cultures were recovered daily and centrifuged at 360 ×gfor 10 min, to remove remaining microcarriers, cells and cell debris. Lactate dehydrogenase (LDH) activity was quantified in cell culture supernatants using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) by adapting the manufacturer instructions for the microplate procedure. The same procedure was applied to a positive control (1 µL LDH Positive Control in 10 mL of 10% BSA in PBS). Three replicates were quantified for each sample. The LDH activity was reported as the quotient between the LDH activity of each sample and the LDH activity of the positive control, according with the following equation. LDH activity(%)=LDHsample LDHpos.control ×100 Statistical Analysis Statistical analysis was performed using GraphPad Prism 8 Software. Results are presented as mean ±standard error of the mean (SEM) of the values obtained from different MSC donors (i.e., biological replicates) or as mean ±standard deviation (SD) of the values from technical replicates. Paired t-test was applied to evaluate the statistical significance of the differences in EV concentration and specific EV concentration in the conditioned medium from MSC cultures in static and VWBR systems. These data sets passed normality tests. P-values result from twotailed tests with a 95% confidence interval. Differences were considered significant at P<0.05 and statistical output was represented as ∗∗ <0.01. RESULTS MSC Expansion and Medium Conditioning for MSC-EV Production From Three Different Human Sources (BM, AT and UCM) Was Achieved in the Bioreactor Culture System Bioreactors have been implemented as scalable platforms for MSC manufacturing. Building on previous work from our group (de Sousa Pinto et al., 2019), a S/XF microcarrier-based culture system implemented in a VWBR originally targeting MSC expansion was adapted to the production of cell-derived products such as MSC-EVs and compared with traditional static culture systems (i.e., T-flasks) (Figure 1). BM, AT and UCM MSC were successfully expanded in the VWBR system (Figure 2A, upper panel). The expansion of BM MSC was the most heterogeneous among donors (n= 3), with final post-expansion cell numbers ranging between 12.0 ±3.6 ×106and 53.4 ±5.5 ×106, depending on BM donor. The expansion culture period also ranged from 7 to 11 days in BM MSC cultures. AT and UCM MSC expansion curves were more homogeneous, reaching an average of 29.2 ±1.7 ×106and 19.9 ±2.4 ×106cells, respectively, at the end of the expansion period. This expansion period was 7 days for AT MSC and 9–10 days for UCM MSC. Estimated adhesion efficiency of MSC to microcarriers after VWBR inoculation was higher for AT MSC (110 ±12%), followed by BM MSC (68 ±17%) and UCM MSC (55 ±4%) (Table 1). AT MSC adhered and started proliferating in less than 24 h, which resulted in estimated adhesion efficiencies higher than 100%. BM MSC showed the highest average growth rate (0.47 ±0.05 day−1), which was very similar to AT MSC (0.45 ±0.06 day−1), while UCM MSC showed the lowest growth rate (0.35 ±0.09 day−1), as a consequence of the lower initial adhesion efficiency observed. In general, BM and AT MSC maintained cell viability close to 100% throughout culture (Figure 2A, lower panel). Cell viability suffered more oscillations in UCM MSC cultures, especially in the first days of culture. Throughout the culture period, microcarrier colonization by cells increased progressively as MSC expanded (Figure 2B). The increasing microcarrier occupancy was followed by microcarrier aggregation, as MSC expansion reached higher cell numbers. We observed that cell expansion stopped when large microcarrier aggregates were formed, likely due to lack of surface available to attach and proliferate (Figures 2A,B). In some cultures, a significant decrease in cell number was observed at the start of the medium conditioning stage, immediately after the culture medium was changed from hPLsupplemented medium to supplement-free culture medium. This can be explained, at least partially, by a possible removal of microcarriers during medium change operation, resulting in a loss of cells from the vessel. Additionally, it should be noticed that microcarrier aggregation might affect our estimation of cell numbers at this stage. In the medium conditioning stage, MSC were cultured for 48 h in a supplement-free medium, which could be a stress factor for cell culture. Although a decrease in the cell number was occasionally observed during the 48 h medium conditioning period, this was an exception rather than the rule (Figure 2A). High cell viabilities were maintained (Figure 2A) and there were no visible differences in microcarrier occupancy during this stage (Figure 2B). Still, in order to thoroughly assess if MSC were experiencing induced cell stress, the levels of LDH activity in culture were monitored during the 48 h conditioning period. LDH activity can be used as a readout of cell stress, as this toxic compound is released to cell culture medium upon plasma membrane damage (Racher et al., 1990). LDH activity did not change significantly over this period for any of the MSC sources (Figure 2C). Therefore, there were no indications that MSC were experiencing significant stress in stirred culture due to the absence of hPL in the 48 h conditioning period. Frontiers in Cell and Developmental Biology | www.frontiersin.org 6November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 7 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors FIGURE 2 | MSC culture in the microcarrier-based bioreactor system. (A) Evolution of cell number (upper panel) and cell viability (lower panel) over culture period time, for MSC from three different human tissue sources (bone marrow, adipose tissue, and umbilical cord matrix). MSC from three different donors (i.e., three biological replicates) were used per tissue source, which are represented in three different shades of gray. Two data points are presented for the same day when the medium conditioning stage (i.e., EV production) started. Results are presented as mean ±SD of cell count for each time point. (B) Representative images of microcarrier occupation by MSC throughout culture. Cell nuclei were stained with DAPI and images were acquired using a fluorescence microscope. In this case, EV production started on day 9 of culture and finished on day 11. Scale bar = 100 µm. (C) LDH activity profile during the medium conditioning (i.e., EV production) stage in the VWBR system. Culture medium samples were taken at 0, 24, and 48 h after medium conditioning started. Results from one experiment for each MSC source (BM, AT, and UCM). Results are presented as mean ±SD (n= 3). LDH, lactate dehydrogenase; VWBR, Vertical-WheelTM bioreactor; BM, bone marrow; AT, adipose tissue; UCM, umbilical cord matrix. Characterization of MSC-EVs Reveals Improved Properties Upon Bioreactor Manufacturing EV were successfully isolated from the conditioned medium of MSC cultures. We were able to identify the presence of EVs from static and bioreactor cultures of MSC, from the 3 different sources (i.e., BM, AT and UCM) through AFM (Figure 3A and Supplementary Figure 1). Individual vesicles of different sizes were observed, as well as vesicle aggregates. The formation of aggregates and collapsed vesicles may be caused by sample processing techniques, which involve sample dehydration. Larger vesicles were observed for AT MSC (Figure 3A). These vesicles may have a higher tendency to aggregate or even fuse together due to the higher medium viscosity observed in AT MSC cultures. The production of EVs was also confirmed by Western blot analysis (Figure 3B and Supplementary Figure 2). The EV protein markers synthenin, CD63 and CD81 were successfully detected in EV samples, while the negative EV protein marker calnexin (a protein from the endoplasmic reticulum) was present in cells, but absent in EV samples, as expected (Figure 3Bi). In general, synthenin and CD63 presence were verified for Frontiers in Cell and Developmental Biology | www.frontiersin.org 7November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 8 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors TABLE 1 | Parameters from cultures of MSC from three different human sources (BM, AT, and UCM) in bioreactors. Adhesion efficiency Growth rate (day−1) Duplication time (day) BM 68 ±17% 0.47 ±0.05 1.49 ±0.13 AT 110 ±12% 0.45 ±0.06 1.60 ±0.19 UCM 55 ±4% 0.35 ±0.09 2.30 ±0.61 Average initial cell adhesion efficiency, growth rate and duplication time for each MSC source. Adhesion efficiency was estimated by dividing the total cell number 24 h after inoculation (day 1) by the cell number used in bioreactor inoculation (day 0). Three biological replicates (i.e., MSC from three different human donors) were used for each MSC source (n = 3). Results are presented as mean ±SEM. MSC-EVs obtained from both static and bioreactor systems, using MSC from the 3 different tissue sources (Figure 3Bii). Interestingly, both synthenin and CD63 presence were increased when EVs were obtained from bioreactors. Contrarily to EVs, cells showed higher synthenin expression under static conditions compared to the bioreactor. CD81 was detected in EVs obtained from BM and AT MSC obtained from both static and bioreactor systems, but not from UCM MSC. CD81 was detected in higher quantity in EVs obtained from AT MSC cultured in bioreactors, compared with static conditions. The surface charge of MSC-EVs was also quantified. MSCEVs presented a negative surface charge, as determined through zeta potential measurements (Figure 3C). Overall, no significant differences were observed in the zeta potential between samples obtained from static or bioreactor platforms, neither between different MSC tissue sources. The zeta potentials ranged between −15.5 ±1.6 mV and – 19.4 ±1.4 mV. The size distribution of MSC-EVs was determined by NTA. In general, MSC-EV samples showed a size distribution profile mostly enriched in small EVs (<200 nm) (Figures 4A,B). Although EVs derived from AT MSC showed a more homogeneous size distribution when obtained from the bioreactor compared to static cultures, no significant difference was observed for other MSC sources. The sizes of EVs produced from AT MSC in the static platform were significantly larger, possibly due to vesicle aggregation or fusion. Therefore, the bioreactor system reveals potential to produce EVs with lower size dispersity, as observed for AT MSC-EVs. Bioreactor Culture Improves the Production of MSC-EVs MSC-EVs produced in the bioreactor system were quantified by NTA after EV isolation and compared with MSC-EVs obtained from static cultures. When EVs were produced in the bioreactor system, their concentration was significantly increased (Figure 5A), at an overall fold increase of 5.7 ±0.9 (Table 2). When analyzed individually, we observed a fold increase of 4.0 ±0.6 for BM MSC, 4.4 ±1.2 for AT MSC and 8.8 ±3.8 for UCM MSC, when EVs were produced in the bioreactor system (Table 2). Bioreactor cultured UCM MSC yielded the highest average EV concentration in the conditioned medium (6.9 ±1.7 ×109particles/mL) (Figure 5A). The average EV concentration in bioreactor cultures was similar for BM and AT MSC (4.6 ±0.2 ×109and 5.1 ±2.1 ×109particles/mL, respectively), although the latter presented higher heterogeneity between experiments. In order to evaluate if the conditions in the bioreactor might modulate the intrinsic capacity of cultured MSC for the production of EVs compared to static conditions, we estimated the EV productivity (i.e., specific EV concentration, per cell) by dividing the concentration of EVs (from NTA) by the cell concentration at the beginning of the conditioning period. When EVs were produced in the bioreactor system, EV productivity increased compared with static culture (Figure 5B) at an overall fold increase of 3.0 ±0.5 (Table 2). Although this difference was not statistically significant (which is likely due to the heterogeneities between the different tissue sources and donors used), the bioreactor system allowed an improved productivity of MSC-EVs for most of the MSC donors used (i.e., in six out of eight MSC donors). EV productivity increased in the bioreactor by a fold increase of 1.4 ±0.3 for BM MSC, 3.7 ±1.0 for AT MSC and 3.9 ±1.4 for UCM MSC (Table 2), compared with static conditions. Bioreactor cultured UCM MSC yielded the highest average EV productivity (2.7 ±0.6 ×104particles/cell) (Figure 5B). The average EV productivity in bioreactor cultures was similar for BM and AT MSC (1.6 ±0.5 ×104and 1.7 ±0.6 ×104 particles/cell, respectively). A particle to protein ratio (PPR) was also determined by dividing the EV concentration (determined by NTA) by the total protein concentration in the same sample (determined through BCA protein assay). The PPR can be used to assess the purity of an EV sample, as the higher is this ratio, the lower is the amount of co-isolated protein contaminants, thus the higher is the sample purity (Webber and Clayton, 2013). EV samples from BM and UCM MSC cultures presented a more homogeneous PPR in the bioreactor system than in static conditions (Figure 5C). EV samples from AT MSC cultures presented a homogeneous PPR for both culture platforms, but the average PPR was slightly higher in the bioreactor. Overall, the PPR was relatively constant in the bioreactor system, ranging between 1.63 ×108 and 3.40 ×108particles/µg protein (Figure 5C). PPR was much more heterogeneous in static conditions (i.e., T-flasks), ranging between 3.47 ×107and 9.88 ×108particles/µg protein. Additionally, the median PPR was higher for the EVs produced in the bioreactor system. DISCUSSION MSC hold great promise for the development of cell-based therapies for a variety of disorders. MSC-derived products such as MSC-EVs offer the opportunity to develop new therapeutic products benefiting from MSC regenerative properties in cell-free formulations. These cell-free therapies are expected to present significant advantages, obviating the complexity and safety issues in utilizing cells themselves as therapeutic systems in a clinical context (Batrakova and Kim, 2015;Conlan et al., 2017). MSC-EVs can be used as intrinsically therapeutic products, by mediating some of the effects conveyed by MSC. MSC-EVs present therapeutic properties for neurological, cardiovascular, Frontiers in Cell and Developmental Biology | www.frontiersin.org 8November 2020 | Volume 8 | Article 553444 fcell-08-553444 October 28, 2020 Time: 18:7 # 9 de Almeida Fuzeta et al. MSC-Derived EV Production in Bioreactors FIGURE 3 | Characterization of MSC-EVs. (A) Representative AFM images of MSC-EVs obtained in the VWBR system, using MSC from three different human tissue sources (bone marrow, adipose tissue, and umbilical cord matrix). AFM height images (top) and respective 3D projections (bottom), capturing a total area of 10 ×10 µm. A close-up image focusing on a single EV is presented for each AFM height image. (B) Western blots of MSC lysates and MSC-EV samples. (i) Representative Western blot images of synthenin, CD63, CD81 and calnexin detection in MSC-EVs and corresponding WCL (i.e., cells) obtained from VWBR cultures. (ii) Western blot detection of synthenin, CD63 and CD81 in MSC-EV samples and corresponding WCL (i.e., cells), obtained from BM, AT and UCM MSC after EV production in static and VWBR systems. Detection of the housekeeping protein GAPDH in the same WCL preparations. 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Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications. J. Extracell. Vesicles 8:1609206. doi: 10.1080/20013078.2019.1609206 Wysoczynski, M., Khan, A., and Bolli, R. (2018). New paradigms in cell therapy: repeated dosing, intravenous delivery, immunomodulatory actions, and new cell types. Circ. Res. 123, 138–158. doi: 10.1161/circresaha.118.313251 Zhang, B., Wang, M., Gong, A., Zhang, X., Wu, X., Zhu, Y., et al. (2015). HucMSCexosome mediated-Wnt4 signaling is required for cutaneous wound healing. Stem Cells 33, 2158–2168. doi: 10.1002/stem.1771 Conflict of Interest: SJ is employee of PBS Biotech, Inc. BL is CEO and co-founder of PBS Biotech, Inc. These collaborating authors participated in the development of the bioreactor systems used in the manuscript. R-JT and WM are employees of AventaCell Biomedical Corp. These collaborating authors participated in the development of the culture medium supplement used in the manuscript. This does not alter the authors adherence to all the policies of the journal on sharing data and materials. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2020 de Almeida Fuzeta, Bernardes, Oliveira, Costa, FernandesPlatzgummer, Farinha, Rodrigues, Jung, Tseng, Milligan, Lee, Castanho, Gaspar, Cabral and da Silva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 17 November 2020 | Volume 8 | Article 553444