XENO-Free production and recovery of human pluripotent stem cells using synthetic dissolvable microcarriers

The implementation of scalable culture platforms for the large-scale production of human pluripotent stem cells (hPSC) and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of these cells for cell therapies and other in vitro biomedical applications, such as drug screening, toxicology assays and disease modeling. Recent progress includes the development of chemically-defined culture conditions for manufacturing of hPSC and their derivatives, namely the development of xeno-free microcarrier platforms to meet good manufacturing practice (GMP) quality requirements [1]. One challenge that remains to be addressed is the establishment of a robust, scalable, and cost-effective downstream processing for cell recovery and removal of the microcarriers. Since hPSC have the tendency to create multilayers of cells on the microcarriers, often forming very large cell-microcarrier aggregates, the process of cell recovery can be technically challenging and time consuming. In this work, we developed a robust and efficient platform for large-scale production of hPSC using synthetic dissolvable microcarriers, which can be quickly dissolved by a non-proteolytic enzyme. This allows an easy cell recovery without the need of the microcarrier separation step, facilitating the downstream processing. Moreover, these synthetic microcarriers are sterile and ready-to-use, and are functionalized with the Synthemax® surface, based on a peptide-acrylate matrix designed for long-term support of hESC self-renewal [2]. hPSC were able to attach and grow on the dissolvable microcarriers and the expansion process was evaluated in a scalable stirred culture system. The cells growth performance on these microcarriers was comparable with the ones obtained when culturing hPSCs in non-dissolvable microcarriers (backbone of polystyrene coated with different ECM molecules), being possible to obtain 1.3x106 cells/mL during 5 days. Importantly, hPSCs cultured on these novel microcarriers were efficiently recovered without the need of the filtration step to separate the microcarriers from the cells and maintained their typical colony morphology and pluripotency-associated marker-expression after re-plating on tissue culture plates. Moreover, their potential for spontaneous differentiation into cells of the three embryonic germ layers was demonstrated through formation of embryoid bodies containing cells expressing typical markers of endoderm, ectoderm and mesoderm. These novel synthetic dissolvable microcarriers allow an easy and efficient downstream processing for hPSCs recovery after expansion/differentiation, without compromising the quality of the cells (viability, potency and functionality), which are a major process breakthrough for stem cell manufacturing. [1] Badenes SM, et al., “Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems”, PlosOne (2016), 11(3):e0151264. [2] Melkoumian Z, et al, “Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells”, Nat Biotechnol (2010), 28(6): 606-10. Acknowledgements: We acknowledge CORNING Incorporated for supplying the dissolvable microcarriers

Magnetic stimulation of the angiogenic potential of mesenchymal stromal cells in vascular tissue engineering

The authors acknowledge the financial support from Fundação para a Ciência e a Tecnologia (FCT-MEC), Portugal, through the dedicated project [PTDC/EDM-EDM/30828/2017] (BeLive) and PhD grant [SFRH/BD/114043/2015] and  through the project [EXPL/CTM-POL/1117/1135/2012] Moreover, the authors thanks POR Lisboa 2020 for the research project [PRECISE, Project N. 16394]. We acknowledge Dr. Marta Teixeira and the IPATIMUP facilities for the development of the ex vivo CAM experiments. The authors acknowledge Prof. Reyes Mallada (University of Zaragoza, Spain) for the use of the vibrating sample magnetometer (VSM) equipment and Dr. Pavel Strichovanec (University of Zaragoza, Spain) for the technical assistance provided during the experiments. We also acknowledge the Instituto de Medicina Molecular (IMM, Lisboa) for the services provided concerning the use of the Confocal Scanning Microscopy (Zeiss LSM 710). Publisher Copyright: © 2021 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.The growing prevalence of vascular diseases worldwide has emphasized the need for novel tissue-engineered options concerning the development of vascularized 3D constructs. This study reports, for the first time, the use of external magnetic fields to stimulate mesenchymal stromal cells (MSCs) to increase the production of vascular endothelial growth factor-A (VEGF-A). Polyvinylalcohol and gelatin-based scaffolds, containing iron oxide nanoparticles, were designed for optimal cell magnetic stimulation. While the application of static magnetic fields over 24 h did not impact on MSCs proliferation, viability and phenotypic identity, it significantly increased the production of VEGF-A and guided MSCs morphology and alignment. The ability to enhance MSCs angiogenic potential was demonstrated by the increase in the number of new vessels formed in the presence of MSCs conditioned media through in vitro and in vivo models. Ultimately, this study uncovers the potential to manipulate cellular processes through short-term magnetic stimulation.publishersversionpublishe

A Novel Engineering Systems Approach for Bioengineering Education: the MIT-Portugal Collaboration

This paper discusses the importance of an engineering systems approach to international bioengineering education and how a new educational research program, the MIT-Portugal Program Bioengineering Systems focus area, aims to develop future global bioengineering leaders. The program, comprising both post-graduate advanced studies and doctoral programs, commences in September 2007. Several other international-collaborative educational and research programs—such as the Cambridge-MIT Institute, the Singapore MIT Alliance, and the Socrates/Erasmus “Erasmus Programme”—offer lessons learned in international collaboration. The MPP Bioengineering Systems program differs from these programs in several respects. The unique collaboration in MPP offers an engineering systems approach, a joint degree offered by three Portuguese universities, and collaborative teaching and research efforts between MIT and Portuguese faculty and students

A Novel Engineering Systems Approach for Bioengineering Education: the MIT-Portugal Collaboration

This paper discusses the importance of an engineering systems approach to international bioengineering education and how a new educational research program, the MIT-Portugal Program Bioengineering Systems focus area, aims to develop future global bioengineering leaders. The program, comprising both post-graduate advanced studies and doctoral programs, commences in September 2007. Several other international-collaborative educational and research programs—such as the Cambridge-MIT Institute, the Singapore MIT Alliance, and the Socrates/Erasmus “Erasmus Programme”—offer lessons learned in international collaboration. The MPP Bioengineering Systems program differs from these programs in several respects. The unique collaboration in MPP offers an engineering systems approach, a joint degree offered by three Portuguese universities, and collaborative teaching and research efforts between MIT and Portuguese faculty and students

A cell rolling cytometer reveals the correlation between mesenchymal stem cell dynamic adhesion and differentiation state

This communication presents quantitative studies of the dynamic adhesion behavior of mesenchymal stem cells (MSCs) enabled by the combination of cell-surface receptor–ligand interactions and three-dimensional hydrodynamic control by microtopography.National Institutes of Health (U.S.) (Grant HL-095722)National Institutes of Health (U.S.) (Grant HL-097172)National Science Foundation (U.S.) (CAREER Award 0952493)Korea (South). Ministry of Science, ICT and Future Planning (National Research Foundation of Korea. Pioneer Research Center Program 2013M3C1A3064777)National Research Foundation of Korea (Framework of International Cooperation Program 2013K2A1A2053078

Cutinase structure, function and biocatalytic applications

This review analyses the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase from Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three dimensional structure of this cutinase is also analysed and its function as a lipase discussed and compared with other lipases. The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media where the different types of enzymatic reactions have been performed, namely hydrolysis, esterification, transesterification and resolution of racemic mixtures. The stability of cutinase preparations is discussed, particularly in anionic reversed micelles considering the role of hexanol as substrate, co-surfactant and stabilizer. Process development based on the operation of cutinase reactors is also reviewed

Effects of glycosaminoglycan supplementation in the chondrogenic differentiation of bone marrow- and synovial- derived mesenchymal stem/stromal cells on 3D-extruded poly (ε-caprolactone) scaffolds

The lack of effective and long-term treatments for articular cartilage defects has increased the interest for innovative tissue engineering strategies. Such approaches, combining cells, biomaterial matrices and external biochemical/physical cues, hold promise for generating fully functional cartilage tissue. Herein, this study aims at exploring the use of the major cartilage glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronic acid (HA), as external biochemical cues to promote the chondrogenic differentiation of human bone marrow- and synovium-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) on custom-made 3 D porous poly (ε-caprolactone) (PCL) scaffolds. The culture conditions, namely the chondrogenic medium and hypoxic environment (5% O2 tension), were firstly optimized by culturing hBMSCs on PCL scaffolds without GAG supplementation. For both MSC sources, GAG supplemented media, particularly with HA, promoted significantly cartilage-like extracellular matrix (ECM) production (higher sulfated GAG amounts) and chondrogenic gene expression. Remarkably, in contrast to tissues generated using hBMSCs, the hSMSC-based constructs showed decreased expression of hypertrophic marker COL X. Histological, immunohistochemical and transmission electron microscopy (TEM) analysis confirmed the presence of typical articular cartilage ECM components (GAGs, aggrecan, collagen fibers) in all the tissue constructs produced. Overall, our results highlight the potential of integrating GAG supplementation, hSMSCs and customizable 3 D scaffolds toward the fabrication of bioengineered cartilage tissue substitutes with reduced hypertrophy.info:eu-repo/semantics/publishedVersio

Evaluating the impact of culture conditions on human mesenchymal stem/stromal cell-derived exosomes through FTIR spectroscopy

In the last decade, the therapeutic effects of mesenchymal stem/stromal cells (MSCs) have been attributed to a paracrine activity exerted by extracellular vesicles secreted by MSCs, as exosomes. Their properties as intercellular communication vehicles have led to an increase interest in their use for cell-free therapeutic applications. The present work aimed to evaluate how different culture conditions, as culture medium (xenogeneic -free (XF) vs serum-containing medium), conditioning time (1, 2 and 3 days) and different MSC donors (n=6), affect the chemical characteristics of exosomes. For that, purified MSC-derived exosomes were characterized by Fourier-Transform InfraRed (FTIR) spectroscopy, a highly sensitive, fast and high throughput technique. The principal component analysis (PCA) of pre-processed FTIR spectra of purified exosomes was conducted, enabling the evaluation of the replica variance of the exosomes chemical fingerprint in a reduced dimensionality space. For that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. It was observed that the chemical fingerprint of exosomes is more dependent of the medium used for MSCs cultivation than the MSC donor and conditioning days. Exosomes secreted by MSCs cultured with serum-containing medium presented a more homogenous chemical fingerprint than exosomes obtained with XF medium. Moreover, for a given medium (XF or serum-containing medium), the exosomes chemical fingerprint depends more of the MSC donor than of the conditioning days. The regression vector of the PCA enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. Ratios between these spectral bands were determined, since these attenuate artifacts due to cell quantity and baseline distortions underneath each band. Statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. It was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of exosomes chemical fingerprint in function of the medium used for MSC grown and the MSC donor. This work is therefore a step forward into understanding how different culture conditions and MSC donors affect MSC exosomes characteristics

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 [2,20 - m-(phenylene)-5,50 -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, 40 ,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.info:eu-repo/semantics/publishedVersio