Scalable production of human mesenchymal stem cells under xeno-free conditions for Cellular Therapy Rita Margarida de Sousa Costaa INTRODUCTION

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1 1 Scalable production of human mesenchymal stem cells under xeno-free conditions for Cellular Therapy Rita Margarida de Sousa Costa a a Master Student in Biotechnology at Instituto Superior Técnico Universidade Técnica de Lisboa, Av. Rovisco Pais, 1, Lisboa, Portugal Mesenchymal stem cells (MSCs) are promising candidates for Cell Therapy applications. For clinical-scale manufacturing, serum-free and xenogeneic-free formulations have been suggested as alternatives to fetal bovine serum (FBS)-supplemented media. In this work, when a virally-inactivated human platelet lysate (hpl) was used to pre-coat plastic microcarriers, with the objective of improving the initial MSC adhesion, an improvement on cell adhesion was observed (38±0% and 49±6% for BM MSCs and ASCs, respectively), resulting in higher proliferation rates. Furthermore, we also confirmed the ability to expand both BM MSCs and ASCs using culture media supplemented with 5 and 10% of hpl under static conditions. For both cases, cells cultivated using hpl, maintained their characteristic immunophenotype and multipotency. These results demonstrate the feasibility to further optimize a xenofree stirred culture system for human MSC cultivation to obtain clinically meaningful doses. INTRODUCTION Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM), but also from the umbilical cord matrix (UCM) and the adipose tissue (AT) [1-2]. There has been considerable interest in the application of these cells in Regenerative Medicine and Tissue Engineering due to their self-renewing capacity, extensive proliferative potential, the ability to differentiate and their immunomodulatory properties [3-5]. Clinical studies employing MSCs have been initiated for the treatment of diseases and injuries such as myocardial infarction, osteogenesis imperfecta, graftversus-host disease (GVHD), and Crohn s disease, multiple sclerosis, and diabetes [6]. Due to the large numbers required for MSCs clinical applications (cells doses up to 5 million MSC/kg body weight [7]), and due to the small number of MSCs in vivo, an efficient and Good Manufacturing Practices (GMP)-compliant ex-vivo expansion process is required [7-8]. Currently, most expansion protocols use medium supplemented with fetal bovine serum (FBS), which provides the cells with vital nutrients, attachment factors, and growth factors [9]. Nevertheless, the use of serum is associated with a risk of transmitting infectious agents and immunizing effects [7]. Replacement of FBS and other animal derived cell culture reagents by defined serum-free and xeno-free media formulations has been shown to enhance significantly the safety and quality of the transplanted stem cells [10]. These chemically-defined media are very advantageous for their precise chemical composition and the absence of microorganisms. It is known that the optimal defined serum-free and xeno-free media formulations, although very promising for clinical grade expansion of stem cells, allowing more reproducible results and contributing to a safer cellular product, may vary largely between and within cell types [11]. Due to this, a variety of human supplements have been tested as alternatives to FBS to provide nutrients, attachment factors, and also growth factors. These include autologous or allogeneic human serum, human plasma, cord blood serum, human platelet derivatives including platelet lysate, and platelet released factors [9, 11-12].

2 2 Human platelet lysate (hpl) has been reported to efficiently substitute FBS in the in vitro expansion of MSCs, maintaining their immunophenotype and multipotential capacity [13-14]. Clinically-prepared hpl contains mitogenic and growth factors required for cell expansion such as platelet derived growth factors (PDGFs), transforming growth factor-b (TGF-b), epidermal growth factor (EGF) and fibroblast growth factor (FGF) [15]. In this work BM MSCs and ASCs were expanded using culture media supplemented with different concentrations of hpl (5% and 10%), in order assess it efficiency. Furthermore, and due the low cell adhesion to plastic microcarriers under serum-free conditions, hpl was also tested as a coating agent for the dynamic expansion of BM MSCs and ASCs in spinner flasks. MATERIALS AND METHODS Human Samples Human BM MSC cultures BM aspirates were obtained from healthy donors after informed consent at Instituto Portugueˆs de Oncologia Francisco Gentil, Lisboa, Portugal. MSCs were isolated according to the protocol described by Dos Santos et al. [16]. Cells from 5 different donors, at passages 3, 5 and 7, were used. Human ASC cultures Human ASC were isolated and characterized as described previously in the literature [17]. Cells from 5 different donors, at passages 1, 2, 3 and 5, were used. The ASC were obtained from healthy donors after informed consent under a protocol reviewed and approved by the Pennington Biomedical Research Center Institutional Review Board. Ex-vivo expansion of MSCs MSCs culture under static condition Cryopreserved MSC were thawed and plated, at a cell density of 3000 or 6000 cells/ cm 2 on T-flasks, changing the respective culture media every 3 days. At 70% cell confluence, MSC were detached from the flasks by adding TrypLE solution (Invitrogen) for 5 min at 37ºC. Cell number and viability were determined using the Trypan Blue exclusion method. The serumfree culture media used was StemPro MSC SFM XenoFree (Invitrogen) medium, and DMEM containing GlutaMAX, a stabilized form of glutamine, supplemented with 5 or 10% Human Platelet Lysate (hpl). In the StemPro MSC SFM XenoFree (Invitrogen) culture medium the surface where the cells were cultured was precoated with a commercial xeno-free substrates, CELLstart TM (Invitrogen) diluted 1:100 in PBS with Ca 2+ and Mg 2+. The antibiotics added to culture media (100x) were penicillin (0.025μg/mL) and streptomycin (0.025U/mL). The fold increase at each passage was calculated as the ratio between the number of viable cells at the end of each passage and the number of viable cells at the beginning (day 0). The population doublings (PD) were calculated by dividing the logarithm of the fold increase by the logarithm of 2. (1) Consequently, cumulative population doublings came as: Expansion of MSC in spinner flasks (2) In this work Bellco spinner flasks (Bellco Glass, Inc.) with a working volume of 80mL, equipped with 90º paddles (normal paddles) and a magnetic stir bar were used. The initial cell density used for MSCs was cells.ml -1. Plastic microcarriers (SoloHill Engineering, Inc.) were prepared according to manufacturer s instructions. For the experiments using StemPro MSC SFM XenoFree (Invitrogen) medium, plastic microcarriers were pre-coated with a CELLstart CTS (diluted 1:100 in PBS with Ca 2+ and Mg 2+ ) or hpl solution for 1 h at 37ºC, with an intermittent agitation (1min at 300 rpm, 10min non-agitated) using a Thermomixer confort (Eppendorf AG), and afterward equilibrated in the respective prewarmed medium. Human MSCs, previously expanded under static conditions for 2-5 passages, were seeded on 20 g/l of pre-coated plastic microcarriers in 15mL of the respective medium. Then, pre-warmed medium was added until reaching half of the final volume, and the cell suspension was transferred to the spinner flask. During the first day, an intermittent agitation regime n was set (18h at 25 rpm followed by 6 h non-agitated). After the initial 24

3 3 h, agitation was set at 40 rpm. At day 3 the other half of the culture medium was added up to 80 ml. Afterwards, 25% of the volume of the culture media was replaced every day. The cell growth in the spinner flask culture was evaluated daily by taking two samples of 0.5 ml from the homogeneous culture in the spinner flask. After microcarriers settled down, the medium was removed and 1mL of TrypLE Express (1 ) was added. Microcarrier suspension was then incubated at 37ºC for 7-10 min at rpm using ThermoMixer confort. Subsequently, 4mL of the corresponding medium was added to stop enzymatic activity and the cell plus microcarrier suspension was filtered using a 100mm Cell Strainer (BD Biosciences). Then the cells were centrifuged and ressupended in PBS. The number of viable and dead cells was determined by counting them in a hemocytometer under an optical microscope using the Trypan Blue exclusion method., Characterization of the expanded MSCs from different human sources Clonogenic potential To evaluate the capacity of a cell to form colonies (CFUs-F colony forming unitsfibroblast), the cells were plated at a very low density (10 cells/cm 2 ) on T-12.5 flasks (Falcon BD Biosciences ) in presence of DMEM + 10% FBS and maintained for 14 days at 37ºC and 5% CO 2 without medium change. After 14 days, the cells were washed once with PBS and incubated with 0.5% Crystal Violet solution in methanol (Sigma ) for 30 minutes at room temperature. The stained colonies were washed four times with PBS and once with distilled water. After drying at room temperature, the number of small (5 to 25 cells), medium (25 to 50 cells) and large (more than 50 cells) colonies was counted. The average number of CFUs-F was calculated for each cell passage. Immunophenotypic analysis Before and after the expansion cells were also analyzed by flow cytometry using a panel of mouse anti-human monoclonal antibodies, PE-conjugated, against: CD14, CD19, CD38, CD45, CD73, human leukocyte antigen (HLA)-DR (BectonDickinson Immunocytometry Syste), CD31, CD80, CD90, CD146 (Biolegend), CD105, CD106 and CD275 (BD Pharmingen); and FITC-conjugated against CD34 and CD45 (BectonDickinson Immunocytometry System). The cells were centrifuged for 7 minutes at 1250 rpm, ressupended in PBS and split onto FACS tubes (100 μl). The respective antibody (5μL) was added to each FACS tube and incubated for 15 minutes at room temperature in the dark. To remove the excess of antibody, 2 ml of PBS was added and the cells were centrifuged for 5 minutes at 1000 rpm. Then, the cells were ressupended and fixed in 2% paraformaldehyde (PFA) (Sigma ) and stored at 4ºC. Isotype controls were also prepared for every experiment. The cells were analyzed by flow cytometry (FACSCalibur equipment, Becton Dickinson ) that quantitatively determine the expression of each surface marker. A minimum of events was collected for each sample and the CellQuest software (Becton Dickinson) was used for acquisition and analysis. Phalloidin and DAPI staining At days 1,3 and 8, samples of cellcontaining beads were washed two times with PBS (500 μl), fixed with 500 μl of 2% paraformaldehyde (Sigma) for 20 min at room temperature and washed, once again, with PBS (500 μl). Then, the cells were permeabilized with 0.1% Triton-X100 in PBS for 1 min, and finally incubated in rhodamine phalloidin (Sigma- Aldrich), a high-affinity F-actin probe (red), at a concentration of 1 μl/ml for 60 min. After washing with PBS, cells were incubated in the dark with 500 μl of 4,6-diamino-2-phenylindole dilactate (DAPI, 1.5 µg/ml in PBS) for 5 min at room temperature and protected from light, washed three times with PBS (500 μl) and stained nuclei (blue) were visualized under a fluorescence microscope (Leica DMI 3000B, Germany) and photographed. Using the ImageJ program, it was possible to merge photographs resultant from different stainings, in this case phalloidin and DAPI staining. It was also possible to estimate different parameters such as cells area and roughness using the CellProfiler (r11710) program. Multilineage differentiation assays Osteogenic differentiation MSCs expanded in both static and stirred conditions were plated at 6000 cells/cm 2 on 12-well plates using DMEM supplemented with 10% FBS. At 80% cell confluency, osteogenesis was induced using StemPro Osteogenesis Differentation Kit (Invitrogen). The medium was changed twice a week for 14 days. After induction, cells were prepared for alkaline

4 4 phosphatase (ALP). Briefly, cells were washed in cold PBS and fixed in 10% cold neutralbuffered formalin (Sigma) for 15 min. After fixing, cells were washed and kept in distilled water for 15 min. Cells were incubated with a 0.1M solution of Tris-HCl (Sigma-Aldrich) containing Naphtol AS MX-PO4 (0.1mg.mL -1 ) (Sigma) in dimethylformamide (Fischer Scientific) and 0.6mg.mL - 1 of Red Violet LB salt (Sigma) for 45 min and washed four times with distilled water. Then cells were stained with 2.5% (w/w) silver nitrate (Sigma ) for 30 minutes at room temperature and washed three times with distilled water. Finally cells were observed under the microscope (Leica Microsystems) for ALP and Von Kossa staining, as a result of osteogenic commitment. Adipogenic differentiation Cells retrieved from microcarriers were plated at 3000 cells/cm2 on CELLstart CTSprecoated 12-well plates using StemPro MSC SFM XenoFree. The adipogenic differentiation was induced at 80% cell confluence after culture for 14 days, using StemPro Adipogenesis Differentiation Kit (Invitrogen). The medium was changed twice a week for 14 days. The assessment of differentiation toward an adipocytic phenotype was performed based on the accumulation of lipids, using Oil Red-O stain. Cells were washed with cold PBS and fixed in 2% formaldehyde for 30 min. After fixation, cells were then washed with distilled water and incubated with Oil Red-O solution (Sigma) (0.3% in isopropanol) at room temperature for 1 h. Chondrogenic differentiation Expanded BM MSC and ASC were plated as small droplets (5 10 ml) with high cell densities (*2 107 cells/ml) on ultra low attachment culture plates (Corning). After 30 min, StemPro Chondrogenesis Differentation Kit (Invitrogen) was added. The medium was changed twice a week for 14 days. The assessment of differentiation toward a chondrocytic phenotype was performed based on the synthesis of proteoglycans by chondrocytes, using Alcian Blue stain. Cells were washed with cold PBS and fixed in 2% formaldehyde for 30 min. After fixation, cells were then washed with distilled water and incubated with 1% Alcian Blue solution (Sigma- Aldrich) at room temperature for 1 h. RESULTS AND DISCUSSION Expansion of human MSCs in culture medium supplemented with 5 and 10% of hpl under static conditions For investigation of the growthpromoting effect of hpl, static cell culture experiments were performed using human BM MSCs and ASCs. In order to account for donor variability, two different donors at different passages were used for each source. The media used consisted of DMEM, supplemented either with + 10% FBS or 5% or 10% hpl. Cumulative population doublings were calculated throughout all passages, as shown in Figure 1. Relatively to ASCs, the present results clearly revealed that, for both donors and different passages, the growthpromoting effect of 5% and 10% of hpl is almost identical to that of 10% FBS. Those cells revealed a more similar behavior (Figure 1 A and B), when comparing expansion in the three different culture media, than BM MSCs (Figure 1 C and D). Furthermore, when comparing cell proliferation between different passages it was noticed that BM MSCs from the lower passage showed higher proliferation; however this did not happened for ASCs. Moreover, for the first donor (Figure 1 C), BM MSCs revealed similar proliferation in the three culture media, while in the second donor (Figure 1 D) FBS exhibit a higher cell expansion. It is then possible to say that different passages and donors can strongly influence cell proliferation in the culture media.

5 Cumulative Population Doublings Cumulative Population Doublings Cumulative Population Doublings Cumulative Population Doublings 5 A B AT (P1-P3) AT (P2-P5) C % FBS 5% hpl 10% hpl D BM (P3-P6) BM (P5-P8) Figure 1. Ex-vivo expansion of MSCs under static conditions in three different media, DMEM supplemented with 10% FBS (blue), 5% hpl (red) and 10% hpl (green). A- Expansion of ASCs from passage 1 to 3 (Donor 1). B- Expansion of ASCs from passage 2 to 5 (Donor 2). C- Expansion of BM MSCs from passage 3 to 6 (Donor 1). D- Expansion of BM MSCs from passage 5 to 8 (Donor 2). Results are presented as n=1. In general it is possible to conclude that medium supplemented with hpl can efficiently replace FBS. However it is necessary to repeat these studies with more donors using the same passages. In what matters to batch-to-batch variations, these were not considered since both lots of hpl used had the exactly same composition. After efficient expansion of BM MSCs and ASCs under static conditions, those cells were seeded in plastic microcarriers and transferred to spinner flasks, in order to perform a large-scale expansion. However, all the spinners, with different MSCs, donors and passages, failed (data not shown). Since BM MSCs and ASCs used in this thesis where previously isolated in DMEM supplemented with FBS, it was hypothesized that expansion results could be influenced by the sudden change in culture medium. So, in order to compare with these results and see if there was any variation on MSCs proliferation when gradually exposed to media with hpl, an adaptation protocol was implemented. Firstly BM MSCs and ASCs were plated and expanded for two passages in DMEM with 10% FBS, then passed to a medium with half volume of DMEM with 10% FBS and half of DMEM with 10% hpl, followed by a passage to DMEM with 5% hpl medium, and finally a passage to DMEM with 10% hpl (data not shown). It was possible to see that in the last three passages there was a decrease in cell proliferation for both BM MSCs and ASCs. These results probably are related with the fact that consecutive passaging under the different culture conditions might have accelerated MSCs senescence, the typical

6 6 Hayflick phenomenon, since it was observed that cells started losing their adherence and typical morphology and reduced/stopped cell division. [18-20].. However, there are other approaches to take into account that could reduce the shock imposed to cells when transferred to medium with hpl. The first one is to use the same method of adaptation described above with the particularity that at each medium change, a higher amount of hpl is gradually added. The second hypothesis and probably the most efficient would be to directly isolate MSCs in culture medium supplemented with hpl. Morphology of MSCs in terms of cells morphology as can be seen in Figure 2 for ASCs (similar results were obtained for BM MSCs). When cultured in medium supplemented with FBS, the MSCs-like cells presented rougher and longer fibroblastoid morphology than the ones cultured in media supplemented with hpl. Cells expanded in the presence of hpl were smaller, sharper and had a more defined spindleshaped morphology, generating dense and more organized colonies. However, it was observed that when a higher confluence is reached, the layer of MSCs in hpl appear with many spaces between the cells in comparison with standard medium. The expansion of MSCs in different culture media resulted in slight differences FBS 5% hpl 10% hpl Adaptation Figure 3. Morphology of ASCs of one representative donor after culture in DEMEM supplemented with FBS, 5% hpl and 10% hpl, and after a gradual adaptation to culture media with hpl. Magnification 100. Table 2. Comparison between BM MSCs and ASCs morphology parameters when cultured in different culture media (DMEM supplemented with 10% FBS, 5% hpl and 10% hpl) for 19 days and 2 passages. Values obtained using CellProfiler program. Results represented as ± SEM. Mean Area (pixels) Form Factor (FF) Eccentricity (Ecc) BM MSCs ASCs FBS 5150± ± ±0.01 5% hpl 4290± ± ± % hpl 4400± ± ±0.02 FBS 4710± ± ±0.01 5% hpl 4050± ± ± % hpl 3040± ± ±0.01

7 7 Concerning cell adhesion to plastic surfaces, MSCs cultured in hpl-containing medium demonstrated a higher adhesion capacity when compared to FBS. When using FBS-containing medium, cells detached from the plastic surface after 5-6 minutes of enzymatic action, whereas cells cultured using hpl, namely cells cultured with 10% hpl, needed additional 5 minutes. This fact must be a direct consequence of the presence of different proteins in hpl formulation, which improve cell adhesion. Relatively to MSCs morphology after the adaptation protocol, it can be seen that during all the passages under different culture medium conditions, cells lost their spindle-like shape and assumed a more flattened, spherical morphology throughout long-term culture. Besides this, cells started detaching by them self, losing their adherence. These results confirm the loss of cell quality during passages. These abnormalities resultant from long-term culture and from enzymatic action at each passage (trypsinization), are typical of the Hayflick model of cellular aging in cultured human fibroblasts. The cells varied in size and shape, the cytoplasm began to be granular with many cell inclusions, and debris was formed in the medium [20]. In order to get more information about those morphological differences BM MSCs and ASCs, expanded under the different culture conditions, were stained with phalloidin (for actin filaments) and DAPI (a nuclear stain), and with CellProfiler program different parameters were compared namely MSCs area, form factor (FF), which give us information about the circularity of the cell (for reference, 1 is a perfect circle), and eccentricity (Ecc), which is used to calculate cells elongation (for reference, 0 would be a perfect circle and 1 a straight line) [21]. The results are summarized in Table 2. Here it is possible to confirm that BM MSCs are larger than ASCs when taking into account the mean area obtained in different culture media. Furthermore it is also possible to verify that for both BM MSCs and ASCs are smaller when cultured in media supplemented with hpl. Relatively to MSCs shape, ASCs show higher FF values and lower values of Ecc, meaning that these cells are more irregular and have a less fibroblastoid morphology, as previously suggested by the microscopic analysis. When comparing MSCs cultured using FBS or hplf, it was observed that the ones with the hpl had a relatively more defined spindle-shaped morphology (higher values of Ecc). Clonogenic efficiency was accessed through the capacity of MSCs to form colonies (CFUs-F). The CFU-F frequency of MSCs cultured using hpl determined did not differ significantly from that of FBS, with values ranging from 40 to 56 CFU-F per 1000 cells (data not shown). This suggests that MSCs maintain the same ability to form colonies. However, although hpl did not modified CFU-F numbers, colonies differed in size: MSCs in hpl supplemented medium appear slightly larger, with a higher number of cells, than in control medium. Flow cytometric characterization was accessed by using the surface antigens proposed by the International Society of Cellular Therapy typical of adult MSCs [22]. Both ASCs and BM MSCs culture in different culture media were positive (expression >80%) for specific MSCs markers, CD73, CD90 and CD105 and negative (expression <2%) for HLA-DR, CD45 or the co-stimulatory molecule CD80. In general it is possible to conclude that no major differences were detected in MSCs immunophenotype, when cultured in media supplemented with hpl (data not shown). In this study it was also possible to see that MSCs maintained their multipotency. Ex-vivo expansion of MSCs on plastic microcarriers pre-coated with hpl In this study BM MSCs and ASCs where expanded in spinner flasks (80mL) using plastic microcarriers coated with hpl, with StemPro MSC SFM XenoFree medium (Figure 4 A). After 24h of culture, the percentage of adherent cells was 38% ± 0% for BM MSCs and 49% ± 6% for ASC. These values were higher than the ones already reported at SCBL-RM for the expansion of BM MSCs and ASC on plastic

8 CD45 CD73 CD80 CD90 CD105 HLA-DR % Expression CD45 CD73 CD80 CD90 CD105 HLA-DR % Expression Cells/mL Fold increase 8 microcarriers pre-coated with the CEllStart solution (23% ± 3% for BM MSC and 22% ± 4% for ASC) [7]. Accordingly, higher average specific growth rates were determined for BM MSCs and ASCs 0.51±0.02 and 0.63 ±0.20, respectively) when compared to the ones obtained for MSCs expanded in microcarriers precoated with CELLstart (0.40 ± 0.09 and 0.34 ±0.01 day - 1, for BM MSCs and ASCs respectively). At day 8, BM MSCs reached a cell density of (2.3 ± 0.40) 10 5 cells/ml, whereas ASCs expanded to a density of (1.7 ± 0.75) 10 5 cells/ml, being both values higher than the ones obtained by Santos and colleagues at day 14 of culture using plastic microcarriers coated with CEllStart solution ((2.0±0.2) 10 5 cell/ml and (1.4±0.5) 10 5 cells/ml, respectively) [7]. Relatively to the fold increase values, maximum values of 12±0.2 and 7.1±1.7 for BM MSCs and ASCs, respectively (Figure 4 B). A 14 B 3.0E E E E E E E+00 ASCs coated with hpl BM-MSCs coated with hpl C Adipogenesis Osteogenesis Chodrogenesis D a) BM day 0 BM day b) AT day 0 AT day 8 0 Figure 4. (A) Cell density and (B) Fold increase of BM MSCs and ASCs expanded in StemPro MSC SFM XenoFree medium using plastic microcarriers pre-coated with hpl solution. (C) Multilineage differentiative potential of BM MSCs (a) and ASCs (b), after expansion. Cell differentiation was induced for 14 days and was assessed by staining for osteogenesis (alkaline phosphatase and von Kossa), adipogenesis (Oil Red-O), and chondrogenesis (Alcian blue). (D)Immunophenotype Analysis of BM MSCs and ASCs, before and after culture. (n = 2 for BM MSCs (donor 1 passage 5-11, and donor 2 passage 7-13 ) and ASCs (donor 1 passage 5-11,donor 2 passage 3-9)). This improvement in initial cell adhesion under xeno-free conditions and the higher proliferation rates obtained using hpl as a coating solution will represent a crucial advance toward a faster and more productive MSC expansion process. However, it still remains necessary to further optimize bead pre-coating protocol under xeno-free conditions in order to reach values closer to the 90% obtained for

9 9 cells cultured in serum-supplemented media [7]. Relatively to the multilineage differentiation of BM MSC and ASC after the spinner flask expansion, was evidence that cells maintained their mesodermal progenitor properties intact. Additionally, MSC characteristic immunophenotype was well maintained after dynamic expansion in different culture media for both BM MSC and ASC (Figure 4 C). A slightly lower percentage of CD90-positive cells for ASC after the expansion may be attributed to longer enzymatic cell detachment times or to an agitation effect, which are known to affect cell surface markers expression [23]. CONCLUSION In this work BM MSCs and ASCs were firstly expanded in static conditions using culture media supplemented with different concentrations (5 and 10%) of virally inactivated hpl. This culture media supported growth and proliferation of MSCs, being able to efficiently substitute FBS. However, some differences between cell donors and passages were noticed, being necessary in the future to replicate this experiment using the same conditions, since it is known that different passages and donors can strongly influence cell proliferation performance. Due to the low cell adhesion to plastic microcarriers observed in serum-free culture media several efforts have been made to optimize cell attachment and growth, such as surface coatings using different ECM proteins and small molecules [24]. In this study, it was tested the ability of hpl as a coating agent of plastic microcarriers for the dynamic culture of BM MSCs and ASCs in spinner flasks. The improvement observed in initial cell adhesion under serum-free xeno-free conditions and the higher proliferation rates obtained using hpl as a coating solution bring results in a faster and more productive MSC expansion process, for further application in Regenerative Medicine. However, it still remains necessary to further optimize the coating protocol for the microcarriers, as well as the inoculation step using serum-free xeno-free culture media and replicate these results using MSCs from UCM [7]. Overall, this thesis demonstrates the feasibility to further optimize a xeno-free stirred culture system for human MSC manufacturing, using only human-derived products, towards the development and optimization of more robust and efficient platforms to achieve larger numbers of MSCs meeting the needs of the allogeneic off-the-shelf MSCs therapy sector. REFERENCES 1. Kern, S., et al., Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, (5): p Wagner, W., et al., Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol, (11): p Bexell, D., S. Scheding, and J. Bengzon, Toward Brain Tumor Gene Therapy Using Multipotent Mesenchymal Stromal Cell Vectors. Mol Ther, (6): p Bobis, S., D. Jarocha, and M. Majka, Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol, (4): p Zhu, W., et al., Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells, (2): p Clinical trials. [cited 2012; Available from: 7. Santos, F.D., et al., Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-

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