Continuous collection of human mesenchymal-stromal-cell-derived extracellular vesicles from a stirred tank reactor operated under xenogeneic-free conditions for therapeutic applications.

Background: Mesenchymal-stromal-cell-derived extracellular vesicles (MSC-EVs) play a key role in the paracrine effects of MSC and have demonstrated therapeutic potential in various preclinical models. However, clinical translation is hindered by manufacturing practices relying on planar culture systems, fetal bovine serum (FBS)-supplemented media, and non-scalable, low-purity EV isolation methods that fail to meet dose and safety requirements, underscoring the need for innovative approaches. In this study, we developed a scalable platform to manufacture human MSC-EVs at clinically relevant numbers, integrating continuous collection of EV-enriched conditioned media (CM) using a stirred-tank reactor (STR) under xenogeneic-free conditions and a scalable downstream process.

Fabrication and Characterization of Porous PEGDA Hydrogels for Articular Cartilage Regeneration.

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.

Unsupervised analysis of whole transcriptome data from human pluripotent stem cells cardiac differentiation.

The main objective of the present work was to highlight differences and similarities in gene expression patterns between different pluripotent stem cell cardiac differentiation protocols, using a workflow based on unsupervised machine learning algorithms to analyse the transcriptome of cells cultured as a 2D monolayer or as 3D aggregates. This unsupervised approach effectively allowed to portray the transcriptomic changes that occurred throughout the differentiation processes, with a visual representation of the entire transcriptome. The results allowed to corroborate previously reported data and also to unveil new gene expression patterns.

Developing a Cell-Microcarrier Tissue-Engineered Product for Muscle Repair Using a Bioreactor System.

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.

Neural progenitor cell-derived extracellular matrix as a new platform for neural differentiation of human induced pluripotent stem cells.

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 .

Bioprocess Economic Modeling: Decision Support Tools for the Development of Stem Cell Therapy Products.

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.

Characterization of the Aeration and Hydrodynamics in Vertical-Wheel Bioreactors.

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.

Cell Culture Process Scale-Up Challenges for Commercial-Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

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.

PEDOT:PSS-Coated Polybenzimidazole Electroconductive Nanofibers for Biomedical Applications.

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.

Electrical stimulation of neural-differentiating iPSCs on novel coaxial electroconductive nanofibers.

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.

Single-Use Bioreactors for Human Pluripotent and Adult Stem Cells: Towards Regenerative Medicine Applications.

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.

Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals faster commitment under dynamic conditions.

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.

Polyaniline-polycaprolactone fibers for neural applications: Electroconductivity enhanced by pseudo-doping.

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 effect of electrospun scaffolds on the glycosaminoglycan profile of differentiating neural stem cells.

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.

Suspension Culture of Human Induced Pluripotent Stem Cells in Single-Use Vertical-Wheel™ Bioreactors Using Aggregate and Microcarrier Culture Systems.

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.

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

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.

Functionalization of Electrospun Nanofibers and Fiber Alignment Enhance Neural Stem Cell Proliferation and Neuronal Differentiation.

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.

3D Microwell Platform for Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.

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.

POLYBENZIMIDAZOLE NANOFIBERS FOR NEURAL STEM CELL CULTURE.

Neurodegenerative diseases compromise the quality of life of increasing numbers of the world's aging population. While diagnosis is possible no effective treatments are available. Strong efforts are needed to develop new therapeutic approaches, namely in the areas of tissue engineering and (DBS).

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.

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.

Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

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 the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs.

Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture.

The cerebellum plays a critical role in all vertebrates, and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models, cerebellar neurons differentiated from pluripotent stem cells have been used.