Neurodifferentiation and neuroprotection potential of mesenchymal stromal cell-derived secretome produced in different dynamic systems

Citation: Marques, C.R.; Fuzeta, M.d.A.; dos Santos Cunha, R.M.; Pereira-Sousa, J.; Silva, D.; Campos, J.; Teixeira-Castro, A.; Sousa, R.A.; Fernandes-Platzgummer, A.; da Silva, C.L.; et al. Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems. Biomedicines 2023, 11, 1240. https://doi.org/10.3390/ biomedicines11051240 Academic Editor: Andrei Surguchov Received: 31 January 2023 Revised: 30 March 2023 Accepted: 14 April 2023 Published: 22 April 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomedicines Article Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems Cláudia Raquel Marques 1,2, Miguel de Almeida Fuzeta 3,4 , Raquel Medina dos Santos Cunha 3,4, Joana Pereira-Sousa 1,2 , Deolinda Silva 1,2, Jonas Campos 1,2, Andreia Teixeira-Castro 1,2 , Rui Amandi Sousa 5, Ana Fernandes-Platzgummer 3,4, Cláudia L. da Silva 3,4,† and António JoséSalgado 1,2,*,† 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal 2ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal 3Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal 4Associate Laboratory i4HB-Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal 5Stemmatters, Biotecnologia e Medicina Regenerativa S.A., 4805-017 Barco, Portugal *Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein ( α -synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrowderived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel ™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α -synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNFβ ), osteopontin, nerve growth factor beta (NGF β ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD. Keywords: bioreactor; dynamic systems; mesenchymal stromal cells; Parkinson’s disease; secretome Biomedicines 2023,11, 1240. https://doi.org/10.3390/biomedicines11051240 https://www.mdpi.com/journal/biomedicines Biomedicines 2023,11, 1240 2 of 18 1. Introduction Mesenchymal stromal cells (MSC) are a population of multipotent progenitor cells with the ability to self-renew and the potential to differentiate into different cell lines, and that can be retrieved from different tissues [ 1 , 2 ]. Despite the advantages and limitations of each tissue source, bone marrow is still the gold standard and most widely used tissue source. Gathered evidence suggests that the beneficial effects of the secretome of MSC are equivalent to the effects of the administration of MSC and extend to different domains, including the hepatic, skeletal, cardiovascular, and nervous systems, among others (reviewed in [ 3 ]). The benefits of secretome have been studied for a large spectrum of conditions of the nervous system, including Parkinson’s disease (PD), where it showed to outperform MSC transplantation-related approaches [4–7]. PD is characterized by the decrement in dopamine (DA) levels due to the degeneration of dopaminergic cells present in the substantia nigra pars compacta (SNpc) [ 8 ]. The molecular pathways responsible for neurodegeneration in PD are not well clarified, but accumulated evidence suggests that mitochondrial dysfunction is highly linked to the development of the disease. This pathway leads to oxidative stress, accumulation of oxidized dopamine, which is correlated with lysosomal dysfunction, as well as alpha-synuclein ( α - synuclein) accumulation and aggregation [ 8 , 9 ]. Specifically, α -synuclein is a protein mainly located at the pre-synaptic terminal that is supposed to be involved in vesicular packaging, trafficking, and synaptic transmission [ 10 ]. However, the accumulation of α -synuclein and the presence of oligomerized or aggregated forms of α -synuclein can greatly impact different cellular mechanisms, leading to oxidative stress and neurodegeneration [11]. Different disease-modifying strategies for PD have been studied, including the use of secretome from MSC, due to its neuroprotective and neurodifferentiation potential. In fact, MSC-derived secretome has protected cultured rodent cortical neurons from death, a process that was dependent on the phosphoinositide 3-kinase/protein kinase B survival pathway. Brain-derived neurotrophic factor (BDNF) was defined as particularly responsible for the neuroprotective effect [ 12 ]. The same factor is required for the existence of an adequate number of DA neurons in the SNpc [ 13 ]. Moreover, our group has been showing the beneficial effects of secretomes from MSC on the survival of DA neurons and stimulation of neurodifferentiation in different models of the disease [6,14–16]. Bioreactors are an important platform to enable the establishment of dynamic cultures, that better recreate the microenvironment of MSC [ 17 ]. As already shown by our group, the production of secretomes in a bioreactor system can impact the expression of different factors and even enhance the expression of others, compared with secretomes produced in static conditions [ 15 , 18 ]. Bioreactors also provide the means to produce large volumes of secretome, fundamental for application in large groups of patients. Nevertheless, there is a wide range of bioreactor configurations available in the market, with different characteristics, and the effects of their particularities in the secretory profile of MSC are poorly understood. In this work, two different mechanically agitated systems were used to produce secretomes from bone marrow-derived MSC (BMSC). The widely known spinner flask (SP) system has a cylindrical shape and is harnessed with 90 ◦ paddles and a magnetic stir bar. Its agitation mechanism greatly contrasts with the Vertical-Wheel ™ bioreactor (VWBR) in which agitation is generated by a large vertical impeller and a U-shaped bottom, providing mixing and suspension of particles with low agitation speeds [ 19 ]. Both systems are scalable and are available in a single-use format, which could facilitate translation into clinical use. Indeed, single-use systems are important for biopharmaceutical manufacturing, to eliminate the need for cleaning and sterilization between runs, thus significantly reducing the contamination rates and production costs [ 20 ]. Herein, the capacity of MSCderived secretomes produced in both dynamic systems to induce neurodifferentiation and neuroprotection was assessed and compared. Biomedicines 2023,11, 1240 3 of 18 2. Materials and Methods 2.1. Human Bone Marrow Mesenchymal Stromal Cell (BMSC) Cultures BMSCs 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). MSCs were previously isolated/expanded according to protocols previously established at iBB-IST) [ 21 ]. Originally, BMSCs were isolated from bone marrow aspirates obtained from healthy donors after written informed consent at Instituto Português de Oncologia Francisco Gentil, Lisboa, Portugal, 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, 29 June), with the approval of the Ethics Committee of the respective clinical institution, according to the Portuguese Regulation (Law 21/2014, 16 April). MSCs were retrieved according to the established protocols as described by dos Santos and colleagues [ 21 ]. Cells from one donor with 4 and 5 passages were used. 2.2. BMSC Cultures under Static Conditions Cryopreserved BMSCs were thawed and plated at a cell density of 3000 cells · cm −2 , on T-75 flasks with low glucose (1 g · L −1 ) Dulbecco’s Modified Eagle Medium [DMEM, (Gibco, Thermo Fisher Scientific, New York, NY, USA) supplemented with 5% v/vhuman platelet lysate (hPL) UltraGRO ™ -PURE (AventaCell, Atlanta, GA, USA) and AntibioticAntimycotic (1 × ) (Gibco, Thermo Fisher Scientific) (DMEM/HPL). Alternatively, cells were plated at the same cell density in CELLstart ™ substrate (Gibco, Thermo Fisher Scientific) pre-coated T-75 flasks with StemPro ™ MSC SFM XenoFree medium (Gibco, Thermo Fisher Scientific) supplemented with Glutamax (1 × ) (Gibco, Thermo Fisher Scientific) and Antibiotic-Antimycotic (1%) (StemPro). Cells cultured in DMEM-HPL were grown in VWBR, whereas cells grown in SP were cultured in StemPro. At 70% cell confluence, MSCs were harvested with 1 × TrypLE ™ Select Enzyme solution (Gibco, Thermo Fisher Scientific) for 5 min at 37 ◦ C. Cell number and viability were determined using the Trypan Blue (Gibco, Thermo Fisher Scientific) exclusion method. 2.3. Microcarrier Preparation Animal product-free SoloHill plastic microcarriers [MCs (Sartorius, Gottingen, Germany)] of 360 cm 2· g −1 superficial area, were used for BMSC culture in both systems. The preparation of the MCs varied according to the culture medium used in each system. 2.3.1. Inoculation of the Vertical-Wheel Bioreactor (VWBR) Following sterilization by autoclaving (120 ◦ C, 20 min), the coating of MCs with a solution of DMEM supplemented with 50% hPL UltraGRO ™ -PURE was performed using a Thermomixer ® comfort (Eppendorf AG, Hamburg, Germany) following a protocol consisting of cycles of 2 min at 750 rpm agitation and 10 min without agitation, during 1 h. Prior to cell inoculation, MCs were resuspended in DMEM/HPL [22]. 2.3.2. Inoculation of the Spinner Flask (SP) After sterilization by autoclaving (120 ◦ C, 20 min), MCs were coated with a CELLstart ™ substrate (diluted 1:100 in 1 × PBS) for 1 h at 37 ◦ C, with an intermittent agitation (cycles of 2 min at 750 rpm, 8 min without agitation) using a Thermomixer ® comfort, and afterwards equilibrated in pre-warmed StemPro [23]. 2.4. BMSC Culture in VWBR Expansion of BMSC in VWBR was generally performed as previously described [ 22 ]. In summary, PBS MINI vertical-wheel bioreactor, namely PBS 0.1 (PBS Biotech, Camarillo, CA, USA) was operated at its full working volume (100 mL). Previously prepared MCs were used at a concentration of 20 g · L −1 . Following the introduction of the MCs into Biomedicines 2023,11, 1240 4 of 18 the bioreactor, BMSC previously expanded under static conditions for 2 passages, were transferred to the bioreactor (5 × 10 6 cells). DMEM/HPL was added to reach 60 mL of culture medium inside the bioreactor. The culture was maintained at 37 ◦ C and 5% CO 2 . During the first 6 h, the agitation was set to cycles of 25 rpm for 1 min, followed by 20 min without agitation. At the end of this regime, the agitation was set to 25 rpm. After 48 h, 40 mL of fresh culture medium with a glucose pulse (3 g/L) was added to the VWBR, to achieve a final volume of 100 mL. Feeding was performed from the third day of culture on a daily basis, by replacing 25% of volume with fresh culture medium supplemented with a glucose pulse (3 g/L). Daily sampling of the culture was performed to determine total cell number and metabolite concentration. Briefly, when the MCs settled, supernatant was harvested, centrifuged, transferred to a new tube, and stored at − 20 ◦ C until further analysis of glucose and lactate concentrations. Afterwards, a representative sample of the homogenized culture was collected, and the MCs were incubated with TrypLE ™ Select Enzyme solution at 37 ◦C for 7 min and 750 rpm, using a Thermomixer ® comfort. After stopping the reaction by diluting with culture medium, mechanical dissociation was performed by pipetting up and down and the mixture was then filtered using a 100 µ m cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) to remove the MCs. Cells were centrifuged at 350 × gfor 7 min, and total cell number and viability were determined throughout time using the Trypan Blue exclusion method. The determination of the specific growth rate was based on an exponential fitting to the experimental data that correspond to the exponential growth phase. The doubling time was calculated by the ratio between the natural logarithm of 2 and the growth rate. Adhesion efficiency to the MCs was calculated as the ratio between the total number of cells on day 1 and the number of inoculated cells. To monitor cell adhesion and growth within the culture, an additional sample was collected daily. Following cell fixation using 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA), MCs containing cells were incubated with 4-6-diamidino-2-phenylindoledihydrochloride (DAPI, Sigma-Aldrich) at 1.5 mg · L −1 for 10 min and observed in a fluorescence inverted microscope (Leica DMI 3000 B). 2.5. BMSC Culture in SP Bellco ® spinner flasks (Bellco Glass, Vineland, NJ, USA) offer a working volume of 100 mL but were operated at 80 mL. Prior to use, spinner flasks were autoclaved and treated with Sigmacote ® (Sigma-Aldrich) to prevent microcarrier adhesion to the surface of the flask. Notably, 20 g · L −1 MCs, prepared following the aforementioned protocol, and 4 × 10 6 MSC previously expanded under static conditions with StemPro for 2 passages, were added to the SP with a total volume of 40 mL and incubated at 37 ◦ C and 5% CO 2 . Following cell inoculation, agitation was set to 30 rpm. After 24 h, agitation was increased to 40 rpm. The necessary volume of medium to attain 80 mL was added 96 h post-inoculation. Feeding was performed from the fourth or fifth day of culture onwards, by replacing 25% of the volume with pre-warmed medium or by the addition of a glucose pulse to maintain the levels of glucose at a non-limiting concentration (superior to 1 mM) [24]. Daily sampling was performed according to the methodology followed for the culture in the VWBR. Adhesion, growth rate, and population doubling time were calculated for both cultures. 2.6. BMSC Conditioning and Secretome Collection BMSCs were cultured in both dynamic cultures for 24 h before the end of the dynamic cultures. The culture medium was totally replaced by fresh medium for conditioning. For that, the exhausted medium was removed, and cell-containing MC systems were washed once with 100 mL of Neurobasal-A (Gibco, Thermo Fisher Scientific) or AlphaMEM (Gibco, Thermo Fisher Scientific) culture medium. Afterwards, 100 mL for the VWBR or 80 mL for the SP of AlphaMEM (SP1 and VWBR1) or Neurobasal-A (SP2 and VWBR2) medium, with Biomedicines 2023,11, 1240 5 of 18 1% Antibiotic-Antimycotic were added to the culture systems. After 24 h, the medium was harvested and centrifuged at 300×gfor 10 min to remove cell debris. The secretome collected using AlphaMEM medium was intended to be used for C. elegans assays and was concentrated by centrifugation to 2 × using a Vivaspin 5 kDa cutoff concentrator (GE Healthcare, UK). The secretome collected in Neurobasal-A medium was used for the differentiation of human neural progenitor cells (hNPCs) and was not concentrated. Aliquots of secretome were flash-frozen with liquid nitrogen and stored at −80 ◦C. 2.7. Metabolite Analysis throughout Cultures The consumption of glucose and the production of lactate were monitored throughout the culture period. Upon medium exchange, the concentration of both metabolites was assessed before and after medium replacement. Glucose and lactate concentrations were analyzed using a YSI 7100MBS equipment (YSI Incorporated, Yellow Springs, OH, USA). The yield of lactate from glucose (YLac/Glc), was calculated for each day as the ratio between the specific metabolic rates, qmet (mol · day −1· cell −1 ), which corresponds to the production of lactate during that day and the consumption of glucose during the same period. These parameters were determined as described in the literature [25]. 2.8. Immunophenotypic Analysis of BMSC At the end of the culture, cells were harvested from the MCs and analyzed for the expression of specific surface antigens by flow cytometry. Approximately 1 × 10 5 cells were resuspended in 1 × PBS and incubated for 15 min in the dark with the antibodies. After a washing step with 1 × PBS, cells were fixed with 1% PFA and stored at 4 ◦ C until analysis using a BD FACSCaliburTM platform, equipped with the CellQuestTM software (BD Biosciences). The antibodies used were CD105 PE (phycoerythrin), CD90 FITC (Fluorescein isothiocyanate), CD80 PE, CD73 PE, CD45 FITC, CD34 FITC, CD14 PE, and HLA-DR PE. All of them were purchased from BioLegend (USA), except for CD105 PE (Thermo Fisher Scientific, USA). Non-stained cells were also prepared for every experiment. A minimum of 10,000 events were collected for each sample. Analysis was performed using FlowJo ™ v10 software (BD Biosciences). The employed gating strategy can be found in the supplementary data. 2.9. Multilineage Differentiation Assays of BMSC Upon dynamic culture, multilineage differentiation assays were performed for BMSC as previously described [ 26 ]. Briefly, BMSCs were retrieved from the MCs (as previously described) and cultured with appropriate medium [adipogenesis, osteogenesis, or chondrogenesis StemPro differentiation kits (Thermo Fisher Scientific)] for 22 days, in order to stimulate the differentiation into each one of three lineages. Differentiation toward an adipocytic phenotype was assessed through staining based on the Oil Red-O solution to visualize the existence of lipidic vacuoles. For osteogenic differentiation, cells were prepared for alkaline phosphatase (ALP) and von Kossa staining. ALP enables the visualization of osteogenic progenitors and von Kossa stains calcium deposits. For chondrogenic differentiation, Alcian Blue was used to stain the proteoglycan aggrecan (an indicator for cartilage formation), which is dark blue. 2.10. Expansion of Human Neural Progenitor Cells and Incubation with hBMSC Secretome The procedure for isolation of hNPCs was already described by our group and followed the strict ethical guidelines established and approved by the Conjoint Health Research Ethics Board (CHREB, University of Calgary, AB, ID: E-18786) [ 27 ]. hNPCs were thawed and placed onto a T-75 flask containing 10 mL of Complete NeuroCultTM-NS-A Proliferation Medium (STEMCELL Technologies, Canada). After two days, cells were harvested and mechanically dissociated into a single-cell suspension and plated at a density of 1 × 10 4 viable cells/cm −2 in new culture flasks. The feeding regime was performed every Biomedicines 2023,11, 1240 6 of 18 2 days, by adding 10% of fresh complete medium. After 10–12 days of culture, coverslips (Marienfeld, Germany) were placed inside a 24-well plate and were pre-coated with polyD-lysine hydrobromide (100 µ g/mL) (Sigma-Aldrich, USA) for 2 h at room temperature (RT) and laminin (10 µ g/mL) (Sigma-Aldrich) for 3 h at 37 ◦ C. hNPCs were mechanically dissociated and plated at a density of 5 × 10 4 cells/cm −2 . Cells were incubated at 37 ◦C , 5% CO 2 , 95% air, and 90% relative humidity for 5 days with secretome collected in NeurobasalA medium from each culture system (SP2 and VWBR2) supplemented with 1% GlutaMAX (Gibco). Neurobasal-A medium with 1% Antibiotic-Antimycotic and 1% GlutaMAX was used as a control. 2.11. Immunocytochemistry Analysis of Human Neural Progenitor Cells Following incubation with secretome, the induction of differentiation was evaluated. Therefore, cells were fixed with 4% PFA (PanReac, Barcelona), washed with 1 × PBS, and blocked as already described [ 28 ]. Following blocking, cells were incubated for 1 h at RT with the primary antibodies, namely, rabbit anti-doublecortin (DCX; 1:300, Abcam, USA), to detect immature neurons and mouse microtubule-associated protein-2 (MAP-2; 1:500, Sigma) and to detect mature neurons. After a washing step, cells were incubated for 1 h at RT with secondary antibodies (1:1000): Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific) and Alexa Fluor 594 goat anti-mouse (Thermo Fisher Scientific). Following this procedure, DAPI (Life Technologies) was added for 5 min. Coverslips were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Tokyo, Japan). Briefly, four coverslips per condition and ten representative fields per coverslip were analyzed, and the experiment was repeated independently four times. Results are presented as percentage of cells positive for MAP-2 or DCX markers divided by the total number of cells/field (DAPI-positive cells). 2.12. Nematode Strains and Culture Conditions All strains were kept in nematode growth media (NGM) containing agar plates seeded with Escherichia coli OP50 at 20 ◦ C, as previously described [ 29 ]. Strain BZ555 egIs1 (Pdat1::green fluorescent protein [GFP]) was acquired from the Caenorhabditis Genetics Center. Strain UA44 (baInl1; Pdat-1:: α -synuclein high, Pdat-1::GFP) was gently provided by Guy Caldwell (University of Alabama). Secretome collected in AlphaMEM medium was used for C. elegans assays. Briefly, three types of plates were prepared: two were seeded with 2x concentrated secretome in AlphaMEM from VWBR or SP diluted in inactivated OP50 (secretome final concentration = 1× ) and the other was seeded with 2 × concentrated AlphaMEM diluted in inactivated OP50 (AlphaMEM final concentration = 1 × ) as control. To prepare inactivated OP50, bacteria were grown overnight at 37 ◦ C and 150 rpm in Luria Broth medium, pelleted by centrifugation, inactivated by three cycles of freeze/thawing, frozen at − 80 ◦ C and then resuspended in S-medium supplemented with 25 U/mL PenStrep (Thermo Fisher Scientific) and 50 U/mL Nystatin (Sigma-Aldrich). 2.13. Quantitative Analysis of Dopaminergic Neuronal Loss Age-synchronized BZ555 and UA44 worms were obtained by egg-laying, by plating adult worms in freshly prepared plates followed by their removal from plates after 2 h (day 0). Worms born on the treated plates were maintained until day 10 and prepared for scoring of dopaminergic neurons according to the procedures described by Marques and colleagues [ 30 ]. Intact dopaminergic neurons were scored, and the experiment was repeated independently three times (n= 12 animals/condition). 2.14. Membrane Antibody Arrays Some soluble factors present in all produced secretomes were identified and quantitatively compared using Human Cytokine Antibody Array 5 (AAH-CYT-5, RayBiotech, Peachtree Corners, GA, USA) and Human Neuro Discovery Array C1 (AAH-NEU-1, Biomedicines 2023,11, 1240 7 of 18 RayBiotech). Each antibody array matrix can simultaneously detect 80 cytokines and 20 neurologically relevant molecules, respectively. Briefly, antibody arrays were incubated with all four secretomes overnight at 4 ◦ C. Membranes were then processed according to the manufacturer’s instructions. Relative expression levels were evaluated by comparing signal intensities, which were obtained with the Sapphire Biomolecular Imager (Azure Biosystems, Dublin, OH, USA) and quantified by densitometry. Membranes present a positive control that was used to normalize the results from the different membranes being compared, which resulted in a normalized intensity value corresponding to each factor. 2.15. Statistics Statistical analysis was performed using the IBM SPSS statistics 25 software. The normal distribution of continuous variables was analyzed according to Shapiro-Wilk or Kolmogorov-Smirnov normality tests. Homogeneity of variances was assessed using Levene’s test. When both assumptions were not met, a robust ANOVA with Welch correction and bootstrap with BCA were performed. Bootstrap sampling was followed by one-way ANOVA with Sidak post hoc test with bias correction (for neurodifferentiation and neuroprotection assays). Pearson’s chi-squared test was used for proportion analysis, with the follow-up z-test for independent proportions with the Bonferroni correction (neuroprotection assay). Appropriate effect size measures were used for each test ( ω 2p for ANOVA with Welch correction and V for Pearson’s chi-square test). Detailed statistics are available in Supplementary Table S1. 3. Results 3.1. BMSCs Were Successfully Expanded in Both Dynamic Systems In this study, we employed each one of the culture systems, making use of previously established and optimized culture conditions aimed at maximizing cell expansion [22,23,31–33] . The cells were successfully expanded in both systems (Figure 1A,B). The percentage of cells that adhered to the MCs within the first 24 h was also determined. Both the maximum and the minimum values for cell adhesion efficiency were obtained with VWBR, but the average of both systems is similar (Table 1). The highest fold expansion (4.19 ± 0.81) was obtained with the VWBR (VWBR1). MC colonization increased over time with MSC expansion, and the higher occupancy of MCs translated into a boost in MC aggregation (Figure 1C). The highest specific growth rates and the lowest doubling times were attained in both cultures with SP; the VWBR2 culture had the lowest growth rate, probably as a result of the low initial adhesion efficiency (31% versus 76% for VWBR1, 48% for SP1, and 55% for SP2). 3.2. Glycolitic Metabolism Is Kept Consistent among Culturing Systems The determination of nutrient consumption and production of metabolites is a relevant procedure to ascertain the availability of nutrients or accumulation of waste products. Therefore, the concentration of glucose and lactate was monitored daily (Figure 2A–D). Glucose and lactate profiles were similar between both cultures in each system. The lowest values for glucose concentration were detected in SP cultures (Figure 2A,C). The levels of glucose in all cultures were kept within non-limiting (over 1 mM) levels for cell proliferation (Figure 2A,C). [ 24 ]. For all cultures, the highest values for lactate concentration were registered during the last days before the conditioning day and were always below 35 mM, described in the literature as a threshold inhibitory lactate concentration (Figure 2B,D) [ 24 ]. The evaluation of the consumption of glucose and production of lactate enables the determination of the average yield of lactate from glucose (Y Lac/Glc ). For all cultures, the value of Y Lac/Glc was approximately 2 mol lactate · mol −1 glucose ( Supplementary Figure S1 ), which is a typical value when cells rely on glycolysis for energy metabolism [34]. Biomedicines 2023,11, 1240 8 of 18 Biomedicines 2023, 11, x FOR PEER REVIEW 9 of 20 Figure 1. Ex vivo expansion of BMSC in the two dynamic culture systems. Representation of cell growth for cultures in the (A) SP and in the (B) VWBR. SP1 and VWBR1 represent the secretomes collected in AlphaMEM medium and SP2 and VWBR2 represent the secretomes collected in Neurobasal-A medium. Results are presented as mean ± SD of cell count for each time point. (C) Representative images of BMSC attached to MCs at 2, 6, and 10 days of culture in SP and VWBR. Cell nuclei were stained with DAPI and images were acquired using a fluorescence microscope. Scale bar = 100 µm. SP, Spinner flask system; VWBR, Vertical-Wheel™ bioreactor; SD, standard deviation. Table 1. Characteristics of BMSC expansion in both dynamic systems. Dynamic System Culture Medium Used for Cell Expansion Culture Medium Used for Conditioning Agitation Rate (rpm) Cell Adhesion Efficiency (%) Specific Growth Rate (Day −1 ) Fold Expansion Doubling Time (Day) VWBR1 DMEM 5% UltraGRO™-PURE AlphaMEM 30 76 0.31 4.19 2.24 VWBR2 DMEM 5% UltraGRO™-PURE Neurobasal-A 25 31 0.24 2.58 2.90 SP1 StemPro MSC SFM AlphaMEM 40 48 0.36 2.93 1.92 SP2 StemPro MSC SFM Neurobasal-A 40 55 0.31 2.45 2.23 Figure 1. Ex vivo expansion of BMSC in the two dynamic culture systems. Representation of cell growth for cultures in the ( A ) SP and in the ( B ) VWBR. SP1 and VWBR1 represent the secretomes collected in AlphaMEM medium and SP2 and VWBR2 represent the secretomes collected in Neurobasal-A medium. Results are presented as mean ± SD of cell count for each time point. (C) Representative images of BMSC attached to MCs at 2, 6, and 10 days of culture in SP and VWBR. Cell nuclei were stained with DAPI and images were acquired using a fluorescence microscope. Scale bar = 100 µm . SP, Spinner flask system; VWBR, Vertical-Wheel ™ bioreactor; SD, standard deviation. Table 1. Characteristics of BMSC expansion in both dynamic systems. Dynamic System Culture Medium Used for Cell Expansion Culture Medium Used for Conditioning Agitation Rate (rpm) Cell Adhesion Efficiency (%) Specific Growth Rate (Day−1) Fold Expansion Doubling Time (Day) VWBR1 DMEM 5% UltraGRO™-PURE AlphaMEM 30 76 0.31 4.19 2.24 VWBR2 DMEM 5% UltraGRO™-PURE Neurobasal-A 25 31 0.24 2.58 2.90 SP1 StemPro MSC SFM AlphaMEM 40 48 0.36 2.93 1.92 SP2 StemPro MSC SFM Neurobasal-A 40 55 0.31 2.45 2.23 3.3. Cells Expanded in Both Systems Retain MSC Phenotype and Are Capable of Multilineage Differentiation Following BMSC expansion, immunophenotypic assays were performed in order to guarantee that their immunophenotype was not affected by the culture in each of the dynamic systems (Figures 3A and S2) [ 35 ]. Following culture in SP, CD73, CD90, and CD105 biomarkers were expressed in more than 95% of the cells ( Figures 3A and S2 ). Regarding MSC “negative” markers, we observed that a considerable percentage of cells expressed CD14 and CD80, especially cells from the first culture in SP. Overall, cells from VWBR1 kept the characteristic MSC immunophenotype after culture in the VWBR system Biomedicines 2023,11, 1240 9 of 18 ( Figures 3B and S2 ). Moreover, cells cultured in both systems preserved the multilineage differentiation ability toward adipogenic (Figure 3C), osteogenic (Figure 3D), and chondrogenic (Figure 3E) lineages. Biomedicines 2023, 11, x FOR PEER REVIEW 10 of 20 3.2. Glycolitic Metabolism Is Kept Consistent among Culturing Systems The determination of nutrient consumption and production of metabolites is a relevant procedure to ascertain the availability of nutrients or accumulation of waste products. Therefore, the concentration of glucose and lactate was monitored daily (Figure 2A– D). Glucose and lactate profiles were similar between both cultures in each system. The lowest values for glucose concentration were detected in SP cultures (Figure 2A,C). The levels of glucose in all cultures were kept within non-limiting (over 1 mM) levels for cell proliferation (Figure 2A,C). [24]. For all cultures, the highest values for lactate concentration were registered during the last days before the conditioning day and were always below 35 mM, described in the literature as a threshold inhibitory lactate concentration (Figure 2B,D) [24]. The evaluation of the consumption of glucose and production of lactate enables the determination of the average yield of lactate from glucose (Y Lac/Glc ). For all cultures, the value of Y Lac/Glc was approximately 2 mol lactate·mol −1 glucose (Supplementary Figure S1), which is a typical value when cells rely on glycolysis for energy metabolism [34]. Figure 2. Metabolic analyses of the expansion of BMSC in the SP and VWBR. Concentration profiles of glucose and lactate during the culture in (A,B) SP and in (C,D) VWBR. SP1 and VWBR1 represent the secretomes collected in AlphaMEM medium and SP2 and VWBR2 represent the secretomes collected in Neurobasal-A medium. Results are presented as mean ± SD of two independent readings. 3.3. Cells Expanded in Both Systems Retain MSC Phenotype and Are Capable of Multilineage Differentiation Following BMSC expansion, immunophenotypic assays were performed in order to guarantee that their immunophenotype was not affected by the culture in each of the dynamic systems (Figure 3A and S2) [35]. Following culture in SP, CD73, CD90, and CD105 biomarkers were expressed in more than 95% of the cells (Figure 3A and S2). Regarding MSC “negative” markers, we observed that a considerable percentage of cells expressed CD14 and CD80, especially cells from the first culture in SP. Overall, cells from VWBR1 kept the characteristic MSC immunophenotype after culture in the VWBR system (Figure Figure 2. Metabolic analyses of the expansion of BMSC in the SP and VWBR. Concentration profiles of glucose and lactate during the culture in ( A , B ) SP and in ( C , D ) VWBR. SP1 and VWBR1 represent the secretomes collected in AlphaMEM medium and SP2 and VWBR2 represent the secretomes collected in Neurobasal-A medium. Results are presented as mean ±SD of two independent readings. Biomedicines 2023, 11, x FOR PEER REVIEW 11 of 20 3B and S2). Moreover, cells cultured in both systems preserved the multilineage differentiation ability toward adipogenic (Figure 3C), osteogenic (Figure 3D), and chondrogenic (Figure 3E) lineages. Figure 3. Immunophenotypic analysis and multilineage differentiation potential of BMSC after expansion under dynamic conditions. The percentage of expression of each surface antigen (CD14, CD19, CD34, CD45, CD73, CD80, CD90, CD105, and HLA-DR), analyzed by flow cytometry is represented for one of the cultures in the (A) SP and (B) VWBR. Representative images of multipotency characterization of BMSC cultured in SP and VWBR through multilineage differentiation assays, upon 22 days under (C) Oil red-O for adipogenic, (D) alkaline phosphatase (ALP) and von Kossa staining for osteogenic, and (E) Alcian Blue for chondrogenic differentiating conditions. Scale bar = 100 µm. SP, Spinner flask system; VWBR, Vertical-Wheel™ bioreactor. 3.4. Secretomes Produced in Both Dynamic Culture Systems Were Able to Induce Neurodifferentiation The capacity of SP2 and VWBR2 secretomes to induce neurodifferentiation was explored by incubating hNPCs with both secretomes, followed by the analysis of the expression of DCX and MAP-2. After the incubation period, both secretomes induced cells toward a differentiated state, which was confirmed by the expression of both markers (Figure 4A,B). We observed that the SP2 secretome had a superior effect regarding the expression of DCX, an early neuronal marker, compared with the VWBR2 secretome and the positive control [36]. The same was not seen for MAP-2 expression because both secretomes induced the same level of MAP-2 expression (a characteristic marker of mature neurons) [37]. Figure 3. Immunophenotypic analysis and multilineage differentiation potential of BMSC after expansion under dynamic conditions. The percentage of expression of each surface antigen (CD14, CD19, CD34, CD45, CD73, CD80, CD90, CD105, and HLA-DR), analyzed by flow cytometry is represented for one of the cultures in the ( A ) SP and ( B ) VWBR. Representative images of multipotency characterization of BMSC cultured in SP and VWBR through multilineage differentiation assays, upon 22 days under ( C ) Oil red-O for adipogenic, ( D ) alkaline phosphatase (ALP) and von Kossa staining for osteogenic, and ( E ) Alcian Blue for chondrogenic differentiating conditions. Scale bar = 100 µm . SP, Spinner flask system; VWBR, Vertical-Wheel™ bioreactor. Biomedicines 2023,11, 1240 16 of 18 6. Teixeira, F.G.; Carvalho, M.M.; Panchalingam, K.M.; Rodrigues, A.J.; Mendes-Pinheiro, B.; Anjo, S.; Manadas, B.; Behie, L.A.; Sousa, N.; Salgado, A.J. Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson’s Disease. Stem Cells Transl. Med. 2017,6, 634–646. [CrossRef] 7. Chudickova, M.; Vackova, I.; Machova Urdzikova, L.; Jancova, P.; Kekulova, K.; Rehorova, M.; Turnovcova, K.; Jendelova, P.; Kubinova, S. 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