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Functional articular cartilage regeneration remains an unmet medical challenge, increasing the interest for innovative biomaterial-based tissue engineering (TE) strategies. Hydrogels, 3D macromolecular networks with hydrophilic groups, present articular cartilage-like features such as high water content and load-bearing capacity. In this study, 3D porous polyethylene glycol diacrylate (PEGDA) hydrogels were fabricated combining the gas foaming technique and a UV-based crosslinking strategy. The 3D porous PEGDA hydrogels were characterized in terms of their physical, structural and mechanical properties. Our results showed that the size of the hydrogel pores can be modulated by varying the initiator concentration. In vitro cytotoxicity tests showed that 3D porous PEGDA hydrogels presented high biocompatibility both with human chondrocytes and osteoblast-like cells. Importantly, the 3D porous PEGDA hydrogels supported the viability and chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cell (hBM-MSC)-based spheroids as demonstrated by the positive staining of typical cartilage extracellular matrix (ECM) (glycosaminoglycans (GAGs)) and upregulation of chondrogenesis marker genes. Overall, the produced 3D porous PEGDA hydrogels presented cartilage-like mechanical properties and supported MSC spheroid chondrogenesis, highlighting their potential as suitable scaffolds for cartilage TE or disease modelling strategies.
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Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.
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Técnicas de Cultivo de Célula , Incontinencia Fecal , Humanos , Técnicas de Cultivo de Célula/métodos , Calidad de Vida , Reactores Biológicos , MúsculosRESUMEN
Over recent years, the field of cell and gene therapy has witnessed rapid growth due to the demonstrated benefits of using living cells as therapeutic agents in a broad range of clinical studies and trials. Bioprocess economic models (BEMs) are fundamental tools for guiding decision-making in bioprocess design, being capable of supporting process optimization and helping to reduce production costs. These tools are particularly important when it comes to guiding manufacturing decisions and increasing the likelihood of market acceptance of cell-based therapies, which are often cost-prohibitive because of high resource and quality control costs. Not only this, but the inherent biological variability of their underlying bioprocesses makes them particularly susceptible to unforeseen costs arising from failed or delayed production batches. The present work reviews important concepts concerning the development of bioprocesses for stem cell therapy products and highlights the valuable role which BEMs can play in this endeavor. Additionally, some theoretical concepts relevant to the building and structuring of BEMs are explored. Finally, a comprehensive review of the existent BEMs so far reported in the scientific literature for stem cell-related bioprocesses is provided to showcase their potential usefulness.
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In this work, the oxygen transport and hydrodynamic flow of the PBS Vertical-Wheel MINI™ 0.1 bioreactor were characterized using experimental data and computational fluid dynamics simulations. Data acquired from spectroscopy-based oxygenation measurements was compared with data obtained from 3D simulations with a rigid-lid approximation and LES-WALE turbulence modeling, using the open-source software OpenFOAM-8. The mass transfer coefficients were determined for a range of stirring speeds between 10 and 100 rpm and for working volumes between 60 and 100 mL. Additionally, boundary condition, mesh refinement, and temperature variation studies were performed. Lastly, cell size, energy dissipation rate, and shear stress fields were calculated to determine optimal hydrodynamic conditions for culture. The experimental results demonstrate that the kL can be predicted using Sh=1.68Re0.551Sc13G1.18, with a mean absolute error of 2.08%. Using the simulations and a correction factor of 0.473, the expression can be correlated to provide equally valid results. To directly obtain them from simulations, a partial slip boundary condition can be tuned, ensuring better near-surface velocity profiles or, alternatively, by deeply refining the mesh. Temperature variation studies support the use of this correlation for temperatures up to 37 °C by using a Schmidt exponent of 1/3. Finally, the flow was characterized as transitional with diverse mixing mechanisms that ensure homogeneity and suspension quality, and the results obtained are in agreement with previous studies that employed RANS models. Overall, this work provides new data regarding oxygen mass transfer and hydrodynamics in the Vertical-Wheel bioreactor, as well as new insights for air-water mass transfer modeling in systems with low interface deformation, and a computational model that can be used for further studies.
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Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.
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The culture microenvironment has been demonstrated to regulate stem cell fate and to be a crucial aspect for quality-controlled stem cell maintenance and differentiation to a specific lineage. In this context, extracellular matrix (ECM) proteins are particularly important to mediate the interactions between the cells and the culture substrate. Human induced pluripotent stem cells (hiPSCs) are usually cultured as anchorage-dependent cells and require adhesion to an ECM substrate to support their survival and proliferation in vitro. Matrigel, a common substrate for hiPSC culture is a complex and undefined mixture of ECM proteins which are expensive and not well suited to clinical application. Decellularized cell-derived ECM has been shown to be a promising alternative to the common protein coatings used in stem cell culture. However, very few studies have used this approach as a niche for neural differentiation of hiPSCs. Here, we developed a new stem cell culture system based on decellularized cell-derived ECM from neural progenitor cells (NPCs) for expansion and neural differentiation of hiPSCs, as an alternative to Matrigel and poly-l-ornithine/laminin-coated well plates. Interestingly, hiPSCs were able to grow and maintain their pluripotency when cultured on decellularized ECM from NPCs (NPC ECM). Furthermore, NPC ECM enhanced the neural differentiation of hiPSCs compared to poly-l-ornithine/laminin-coated wells, which are used in most neural differentiation protocols, presenting a statistically significant enhancement of neural gene expression markers, such as ßIII-Tubulin and MAP2. Taken together, our results demonstrate that NPC ECM provides a functional microenvironment, mimicking the neural niche, which may have interesting future applications for the development of new strategies in neural stem cell research.
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The generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) represents a valuable tool for a myriad of in vitro applications, including drug screening, disease modeling and regenerative medicine. However, the success of these applications is dependent on the establishment of reliable, efficient, simple, and cost-effective differentiation methods. In this chapter, we describe an efficient and robust 3D platform for the generation of hPSC-CMs based on the use of a microwell culture system, which can be applied in any laboratory environment. Additionally, we will also describe protocols for the structural and functional characterization of the obtained CMs for further quality control upon differentiation.
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Miocitos Cardíacos , Células Madre Pluripotentes , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , HumanosRESUMEN
Bioelectricity drives several processes in the human body. The development of new materials that can deliver electrical stimuli is gaining increasing attention in the field of tissue engineering. In this work, novel, highly electrically conductive nanofibers made of poly [2,2'-m-(phenylene)-5,5'-bibenzimidazole] (PBI) have been manufactured by electrospinning and then coated with cross-linked poly (3,4-ethylenedioxythiophene) doped with poly (styrene sulfonic acid) (PEDOT:PSS) by spin coating or dip coating. These scaffolds have been characterized by scanning electron microscopy (SEM) imaging and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The electrical conductivity was measured by the four-probe method at values of 28.3 S·m-1 for spin coated fibers and 147 S·m-1 for dip coated samples, which correspond, respectively, to an increase of about 105 and 106 times in relation to the electrical conductivity of PBI fibers. Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) cultured on the produced scaffolds for one week showed high viability, typical morphology and proliferative capacity, as demonstrated by calcein fluorescence staining, 4',6-diamidino-2-phenylindole (DAPI)/Phalloidin staining and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay. Therefore, all fiber samples demonstrated biocompatibility. Overall, our findings highlight the great potential of PEDOT:PSS-coated PBI electrospun scaffolds for a wide variety of biomedical applications, including their use as reliable in vitro models to study pathologies and the development of strategies for the regeneration of electroactive tissues or in the design of new electrodes for in vivo electrical stimulation protocols.
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Neural tissue engineering strategies are paramount to create fully mature neurons, necessary for new therapeutic strategies for neurological diseases or the creation of reliable in vitro models. Scaffolds can provide physical support for these neurons and enable cues for enhancing neural cell differentiation, such as electrical current. Coaxial electrospinning fibers, designed to fulfill neural cell needs, bring together an electroconductive shell layer (PCL-PANI), able to mediate electrical stimulation of cells cultivated on fibers mesh surface, and a soft core layer (PGS), used to finetune fiber diameter (951 ± 465 nm) and mechanical properties (1.3 ± 0.2 MPa). Those dual functional coaxial fibers are electroconductive (0.063 ± 0.029 S cm-1, stable over 21 days) and biodegradable (72% weigh loss in 12 hours upon human lipase accelerated assay). For the first time, the long-term effects of electrical stimulation on induced neural progenitor cells were studied using such fibers. The results show increase in neural maturation (upregulation of MAP2, NEF-H and SYP), up-regulation of glutamatergic marker genes (VGLUT1 - 15-fold) and voltage-sensitive channels (SCN1α - 12-fold, CACNA1C - 32-fold), and a down-regulation of GABAergic marker (GAD67 - 0.09-fold), as detected by qRT-PCR. Therefore, this study suggest a shift from an inhibitory to an excitatory neural cell profile. This work shows that the PGS/PCL-PANI coaxial fibers here developed have potential applications in neural tissue engineering.
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Nanofibras , Estimulación Eléctrica , Humanos , Poliésteres , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Research on human stem cells, such as pluripotent stem cells and mesenchymal stromal cells, has shown much promise in their use for regenerative medicine approaches. However, their use in patients requires large-scale expansion systems while maintaining the quality of the cells. Due to their characteristics, bioreactors have been regarded as ideal platforms to harbour stem cell biomanufacturing at a large scale. Specifically, single-use bioreactors have been recommended by regulatory agencies due to reducing the risk of product contamination, and many different systems have already been developed. This review describes single-use bioreactor platforms which have been used for human stem cell expansion and differentiation, along with their comparison with reusable systems in the development of a stem cell bioprocess for clinical applications.
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Human-induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented a reproducible and scalable production of cerebellar organoids by using the novel single-use Vertical-Wheel bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic conditions, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests, and studying pathological pathways involved in cerebellar degeneration.
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Cerebelo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , RNA-Seq , Cerebelo/citología , Matriz Extracelular/metabolismo , Humanos , Hidrogeles/química , Células Madre Pluripotentes Inducidas/citología , Organoides/citologíaRESUMEN
The ability to culture and differentiate neural stem cells (NSCs) to generate functional neural populations is attracting increasing attention due to its potential to enable cell-therapies to treat neurodegenerative diseases. Recent studies have shown that electrical stimulation improves neuronal differentiation of stem cells populations, highlighting the importance of the development of electroconductive biocompatible materials for NSC culture and differentiation for tissue engineering and regenerative medicine. Here, we report the use of the conjugated polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS CLEVIOS P AI 4083) for the manufacture of conductive substrates. Two different protocols, using different cross-linkers (3-glycidyloxypropyl)trimethoxysilane (GOPS) and divinyl sulfone (DVS) were tested to enhance their stability in aqueous environments. Both cross-linking treatments influence PEDOT:PSS properties, namely conductivity and contact angle. However, only GOPS-cross-linked films demonstrated to maintain conductivity and thickness during their incubation in water for 15 days. GOPS-cross-linked films were used to culture ReNcell-VM under different electrical stimulation conditions (AC, DC, and pulsed DC electrical fields). The polymeric substrate exhibits adequate physicochemical properties to promote cell adhesion and growth, as assessed by Alamar Blue® assay, both with and without the application of electric fields. NSCs differentiation was studied by immunofluorescence and quantitative real-time polymerase chain reaction. This study demonstrates that the pulsed DC stimulation (1 V/cm for 12 days), is the most efficient at enhancing the differentiation of NSCs into neurons.
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Replenishing neurons in patients with neurodegenerative diseases is one of the ultimate therapies for these progressive, debilitating and fatal diseases. Electrical stimulation can improve neuron stem cell differentiation but requires a reliable nanopatterned electroconductive substrate. Potential candidate substrates are polycaprolactone (PCL) - polyaniline:camphorsulfonic acid (PANI:CSA) nanofibers, but their nanobiophysical properties need to be finetuned. The present study investigates the use of the pseudo-doping effect on the optimization of the electroconductivity of these polyaniline-based electrospun nanofibers. This was performed by developing a new solvent system that comprises a mixture of hexafluoropropanol (HFP) and trifluoroethanol (TFE). For the first time, an electroconductivity so high as 0.2 S cm-1 was obtained for, obtained from a TFE:HFP 50/50 vol% solution, while maintaining fiber biocompatibility. The physicochemical mechanisms behind these changes were studied. The results suggest HFP promotes changes on PANI chains conformations through pseudo-doping, leading to the observed enhancement in electroconductivity. The consequences of such change in the nanofabrication of PCL-PANI fibers include an increase in fiber diameter (373 ± 172 nm), a decrease in contact angle (42 ± 3°) and a decrease in Young modulus (1.6 ± 0.5 MPa), making these fibers interesting candidates for neural tissue engineering. Electrical stimulation of differentiating neural stem cells was performed using AC electrical current. Positive effects on cell alignment and gene expression (DCX, MAP2) are observed. The novel optimized platform shows promising applications for (1) building in vitro platforms for drug screening, (2) interfaces for deep-brain electrodes; and (3) fully grown and functional neurons transplantation.
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Doping en los Deportes , Nanofibras , Compuestos de Anilina , Humanos , Poliésteres , Ingeniería de TejidosRESUMEN
The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.
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Diferenciación Celular , Sulfatos de Condroitina/biosíntesis , Heparitina Sulfato/biosíntesis , Células-Madre Neurales/metabolismo , Andamios del Tejido/química , Línea Celular Transformada , Humanos , Células-Madre Neurales/citologíaRESUMEN
The induced pluripotent stem cells (iPSCs) are derived from somatic cells by using reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc (OSKM) or Oct4, Sox2, Nanog and Lin28 (OSNL). They resemble embryonic stem cells (ESCs) and have the ability to differentiate into cell lineage of all three germ-layer, including cardiomyocytes (CMs). The CMs can be generated from iPSCs by inducing embryoid bodies (EBs) formation and treatment with activin A, bone morphogenic protein 4 (BMP4), and inhibitors of Wnt signaling. However, these iPSC-derived CMs are a heterogeneous population of cells and require purification and maturation to mimic the in vivo CMs. The matured CMs can be used for various therapeutic purposes in regenerative medicine by cardiomyoplasty or through the development of tissue-engineered cardiac patches. In recent years, significant advancements have been made in the isolation of iPSC and their differentiation, purification, and maturation into clinically usable CMs. Newer small molecules have also been identified to substitute the reprogramming factors for iPSC generation as well as for direct differentiation of somatic cells into CMs without an intermediary pluripotent state. This review provides a concise update on the generation of iPSC-derived CMs and their application in personalized cardiac regenerative medicine. It also discusses the current limitations and challenges in the application of iPSC-derived CMs. Graphical abstract.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular/genética , Células Madre Embrionarias , Miocitos Cardíacos , Medicina RegenerativaRESUMEN
Human induced pluripotent stem cells (hiPSCs) have the potential to be used in a variety of biomedical applications, including drug discovery and Regenerative Medicine. The success of these approaches is, however, limited by the difficulty of generating the large quantities of cells required in a reproducible and controlled system. Bioreactors, widely used for industrial manufacture of biological products, constitute a viable strategy for large-scale production of stem cell derivatives. In this chapter, we describe the expansion of hiPSCs using the Vertical-Wheel™ bioreactor, a novel bioreactor configuration specifically designed for the culture of shear-sensitive cells. We provide protocols for the expansion of hiPSCs in suspension, both as floating aggregates and using microcarriers for cell adhesion. These methods may be important for the establishment of a scalable culture of hiPSCs, allowing the manufacturing of industrial- or clinical-scale cell numbers.
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Tecnología Biomédica/métodos , Reactores Biológicos/normas , Células Madre Pluripotentes Inducidas/citología , Cultivo Primario de Células/métodos , Tecnología Biomédica/instrumentación , Tecnología Biomédica/normas , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Guías de Práctica Clínica como Asunto , Cultivo Primario de Células/instrumentación , Cultivo Primario de Células/normasRESUMEN
Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and -19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.
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Neural stem cells (NSCs) have the potential to generate the cells of the nervous system and, when cultured on nanofiber scaffolds, constitute a promising approach for neural tissue engineering. In this work, the impact of combining nanofiber alignment with functionalization of the electrospun poly-ε-caprolactone (PCL) nanofibers with biological adhesion motifs on the culture of an NSC line (CGR8-NS) is evaluated. A five-rank scale for fiber density was introduced, and a 4.5 level, corresponding to 70-80% fiber density, was selected for NSC in vitro culture. Aligned nanofibers directed NSC distribution and, especially in the presence of laminin (PCL-LN) and the RGD-containing peptide GRGDSP (PCL-RGD), promoted higher cell elongation, quantified by the eccentricity and axis ratio. In situ differentiation resulted in relatively higher percentage of cells expressing Tuj1 in PCL-LN, as well as significantly longer neurite development (41.1 ± 1.0 µm) than PCL-RGD (32.0 ± 1.0 µm), pristine PCL (25.1 ± 1.2 µm), or PCL-RGD randomly oriented fibers (26.5 ± 1.4 µm), suggesting that the presence of LN enhances neuronal differentiation. This study demonstrates that aligned nanofibers, functionalized with RGD, perform as well as PCL-LN fibers in terms of cell adhesion and proliferation. The presence of the full LN protein improves neuronal differentiation outcomes, which may be important for the use of this system in tissue engineering applications.
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The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.