Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. This work describes the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. Since initial cell density and aggregate size have an important impact in the expansion and commitment of these cells into a particular lineage, a combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 µm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was then performed in 50 mL spinner flasks, and process optimization using a factorial design approach was developed involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. Furthermore, after scalable differentiation, hiPSC-derived neural progenitors still retained their multipotent potential, being able to give rise to neuronal and glial cells. The results presented in this work should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using totally controlled, automated and reproducible large-scale bioreactor culture systems

Magnetic field dynamic strategies for the improved control of the angiogenic effect of mesenchymal stromal cells

project PTDC/EDM-EDM/30828/2017 SFRH/BD/114043/2015 co-financed by the ERDF under the PT2020 Partnership Agreement (POVI-01-0145-FEDER-007265), as well as from POR Lisboa 2020 grant PRECISE (Project N. 16394). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.This work shows the ability to remotely control the paracrine performance of mesenchymal stromal cells (MSCs) in producing an angiogenesis key molecule, vascular endothelial growth factor (VEGF-A), by modulation of an external magnetic field. This work compares for the first time the application of static and dynamic magnetic fields in angiogenesis in vitro model, exploring the effect of magnetic field intensity and dynamic regimes on the VEGF-A secretion potential of MSCs. Tissue scaffolds of gelatin doped with iron oxide nanoparticles (MNPs) were used as a platform for MSC proliferation. Dynamic magnetic field regimes were imposed by cyclic variation of the magnetic field intensity in different frequencies. The effect of the magnetic field intensity on cell behavior showed that higher intensity of 0.45 T was associated with increased cell death and a poor angiogenic effect. It was observed that static and dynamic magnetic stimulation with higher frequencies led to improved angiogenic performance on endothelial cells in comparison with a lower frequency regime. This work showed the possibility to control VEGF-A secretion by MSCs through modulation of the magnetic field, offering attractive perspectives of a non-invasive therapeutic option for several diseases by revascularizing damaged tissues or inhibiting metastasis formation during cancer progression.publishersversionpublishe

Effects of culture media and suspension expansion technologies in mesenchymal stem cell manufacturing - A computational bioprocess and bioeconomics study

Mesenchymal stem cell (MSC) based therapies are promising for a large spectrum of unmet medical needs. Despite this promise, the scaling-up of production of clinical grade MSCs is hindered by the use of planar technologies that require intensive labor and are not enough to meet market demands, as well as due to high product and process variability introduced by the use of xenogeneic materials. This work presents a new bioprocess and bioeconomics model of stem cell expansion to support informed decisions for stem cells process scaling up at reduced annual costs. The intrinsic equations and parameters that capture the cell biological features, according with their source and media used, are embedded in the model. A target number of cells per dose of 140 million and a GMP facility of 400 sq mt with 4 BSCs and 8 incubators will be used as the baseline for expansion of both bone marrow MSCs (BM-MSCs) and adipose stem cells (ASCs) using planar expansion technologies. The current standard medium for MSC culture containing fetal bovine serum (FBS) will be compared with the xeno-free alternative of human platelet lysate (hPL). The use of hPL for both cell sources results in an increase of the number of doses produced and a decrease of the cost of goods (CoG) per dose (Table 1). In order to improve the production capacity, 8 bioreactors with capacity up to 50L were input in the model, using xeno-free plastic microcarriers for cell adhesion and hPL as the culture medium. The model results indicate that the investment in the use of suspension cultures is valuable due to a considerable increase in the production and a decrease of CoG/dose. As the number of doses produced per year increases, the reagent costs dominate relatively to the facility costs (Fig. 1). Sensitivity analysis was performed by varying 11 model variables by +/- 33%. The main factors that influence annual capacity and CoGs are related to harvesting density and yield, growth rates and microcarrier area and concentration (Table 2). These findings may be used to improve the design of expansion methods with fully xeno-free materials and highlight the relevance of the optimization of harvesting and downstream processing protocols. Please click Additional Files below to see the full abstract

Human pluripotent stem cell expansion in vertical-wheel bioreactors

Human induced pluripotent stem cells (hiPSC) have been regarded as an enormous breakthrough for medicine, since they can be derived from patients and be used to generate virtually all types of cells in the human body. One of the great bottlenecks in the usage of these cells for regenerative medicine or drug discovery applications is their expansion to relevant quantities. The Vertical-Wheel Bioreactors (PBS Biotech) present a novel scalable bioreactor configuration, whose agitation mechanism allows for homogeneous mixing conditions inside the single-use vessel, while conveying less shear stress to the cells when compared to traditional alternatives. These characteristics are advantageous for hiPSC expansion and thus, in this work, hiPSC were expanded in the Vertical-Wheel Bioreactor using different strategies, namely culturing the cells 1) on microcarriers and 2) as floating aggregates. In the first approach, cells were cultured under xeno-free conditions, using the Essential 8 medium together with microcarriers and coatings devoid of any animal-derived products [1]. The culture conditions were optimized in terms of initial cell/microcarrier ratio, inoculation method and agitation rate, in the PBS 0.1 vessel (working volume: 80 mL). The cells were successfully expanded, maintaining a normal karyotype, up to a 6.7-fold increase in cell number, after 6 days. These optimized culture conditions were successfully repeated in a larger vessel, the PBS 0.5 (300 mL working volume) demonstrating the scalability of the Vertical-Wheel system. In the second approach, hiPSC were expanded as floating aggregates, a methodology which does not require a separation step at the end of culture, to remove microcarriers, facilitating the downstream processing and Good Manufacturing Practice-compliance of the process. Cells were cultured in the PBS 0.1 (working volume: 60 mL), using mTeSR1, a serum-free medium and were monitored throughout culture regarding growth kinetics, aggregate size distribution and expression of pluripotency markers. The Vertical-Wheel Bioreactors were shown to efficiently keep the cell aggregates in suspension, under lower linear agitation speeds than an equivalent volume spinner flask (7 cm/s vs. 13 cm/s). Following 7 days of culture, cells were expanded up to a 5.2 ± 0.6-fold increase in cell number. The hiPSC aggregates increased in size over time, from an average diameter of 135 ± 61 µm to 397 ± 119 µm after 7 days. Pluripotency was maintained throughout time, as assessed by sustained high (\u3e 80%) expression of pluripotency markers OCT4, SOX2 and TRA-1-60, and low (\u3c 10%) expression of early differentiation marker SSEA-1. The results were validated using a second hiPSC line. This study revealed that the Vertical-Wheel Bioreactor allows hiPSC growth either on microcarriers and as aggregates and suggested it to have advantages versus other configurations. These results make the Vertical-Wheel Bioreactor a promising platform for hiPSC expansion and, prospectively, differentiation approaches, contributing for the generation of bona fide cells for various biomedical applications, namely drug screening, disease modelling, and, ultimately, for Regenerative Medicine. [1] Rodrigues CAV, Silva TP, Nogueira DES, Fernandes TG, Hashimura Y, Wesselschmidt R, Diogo MM, Lee B, Cabral JMS (2018), “Scalable Culture Of Human Induced Pluripotent Cells On Microcarriers Under Xeno‐Free Conditions Using Single‐Use Vertical‐Wheel™ Bioreactors”, Journal of Chemical Technology and Biotechnology, DOI: 10.1002/jctb.573

Development of a scale-down approach to the scalable culture of induced Pluripotent Stem Cells on microcarriers using single-use Vertical-Wheel™ bioreactors under xeno-free conditions

Induced Pluripotent Stem Cells (iPSC) are capable of extensive self-renewal while retaining the ability to differentiate into virtually all cell types of the body. These cells are the subject of much research and development activity aimed at the development of cell-based tools, which may speed drug discovery, and cell-based medical therapies that are being developed to address unmet medical needs. However, development of these therapies is hampered by manufacturing bottlenecks including production scale up to meet the anticipated demand. PBS Biotech, Inc. has developed a single use bioreactor with an innovative Vertical-Wheel™ design that promotes more homogenous and gentle particle suspension, under lower hydrodynamic shear environment than traditional bioreactor vessel design. Vertical-Wheel bioreactors are available from lab-scale vessels (PBS MINI) to larger production units (up to 500L). This study describes the culture of human iPSCs on microcarriers under xeno-free conditions using Vertical-Wheel bioreactors. Human iPSCs were cultured on microcarriers to provide surface for cell attachment using the chemically defined Essential 8 culture medium, a xeno-free, feeder-free culture medium. The culture conditions were optimized in terms of 1) initial cell/microcarrier ratio, 2) inoculation method and 3) agitation rate, in the PBS-0.1 vessel using 80 mL working volume. The cells were successfully expanded, up to a 7-fold increase in cell number, after 6 days in the bioreactor. Glucose consumption and lactate production were analyzed to prevent glucose starvation or excessive lactate accumulation. These optimized culture conditions were successfully repeated in a larger vessel, the PBS-0.5 using 300 mL working volume, demonstrating the scalability of the Vertical-Wheel system. With this PBS-0.5 bioreactor, 3 x 108 cells were produced after 6 days of operation, and the specific growth rate (0.72 day-1) was similar to the one observed with the PBS-0.1 (0.68 day-1). The applications of iPSC cells and their progeny, especially in clinical settings, will require a guarantee of cell quality. After PBS-MINI bioreactor culture, the expression of pluripotency markers, such as Oct4, Nanog, and SSEA4 was assessed by immunocytochemistry and flow cytometry. The directed differentiation into the neural lineage of the expanded cells was performed and the pluripotency of the cells was further tested after embryoid body formation. The robustness of this process method was evaluated by cultivating another iPSC cell line under the same process conditions, resulting in identical growth kinetics in the PBS MINI-0.1. The methodology developed herein, which grows human iPSC on microcarriers in single-use bioreactors using chemically defined xeno-free cultivation reagents provides a foundation upon which further refinement and scale-up of processes can be built for large scale production of iPSCs

Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitro

© 2020 The Authors. S TEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneic-free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4- and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-β levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-β and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.This project received funding from: Fundação para a Ciência e Tecnologia, Portugal, under the Harvard Medical School-Portugal Program project Induction of Immune Tolerance in Human Allogeneic Hematopoietic Stem Cell Transplantation (HMSP-ICT/0001/2011) and UID/BIM/50005/2019, project funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. We also acknowledge the funding received from POR Lisboa 2020 through the project PRECISE – Accelerating progress toward the new era of precision medicine (project no. 16394).info:eu-repo/semantics/publishedVersio

Cutinase activity in supercritical and organic media: water activity, solvation and acid–base effects

We performed a comparative study on the activity of Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY, in n-hexane, acetonitrile, supercritical ethane (sc-ethane) and sc-CO2, at two different water activity (aW) values set by salt hydrate pairs in situ and at acid–base conditions fixed with solid-state buffers of aqueous pKa between 4.3 and 10.6. The reaction studied was the transesterification of vinyl butyrate by (R,S)-2-phenyl-1-propanol. The transesterification activity of cutinase was highest and similar in sc-ethane and in n-hexane,about one order of magnitude lower in acetonitrile and even lower in sc-CO2. Activity coefficients (γ) generated for the two substrates indicated that they were better solvated in acetonitrile and thus less available for binding at the active site than in the other three solvents. γ data also suggested higher reaction rates in sc-ethane than in n-hexane, as observed, and provided evidence for a direct negative effect of sc-CO2 on enzyme activity. Manipulation of the acid–base conditions of the media did not afford any improvement of the initial rates of transesterification relative to the blanks (no added acid–base buffer, only salt hydrate pair), except in the case of cutinase immobilized on zeolite NaA in sc-ethane at aW = 0.7. The poor performance of the blank in this case and the great improvement observed in the presence of a basic buffer suggest a deleterious acidic effect in the medium which, an experiment without additives confirmed, was not due to the known acidic character of the salt hydrate pair used to set aW = 0.7. In acetonitrile, increasing aW was accompanied by a decrease in initial rates of transesterification, unlike in the other solvents. There was considerable hydrolysis in acetonitrile, where initial rates of hydrolysis increased about 20-fold from aW = 0.2 to 0.7. Hydrolysis was less pronounced in sc-ethane and in n-hexane, and only at aW = 0.7, and in sc-CO2 butyric acid was detected only at very long reaction times, in agreement with a generally low catalytic activity. Cutinase enantio-selectivity towards the alcohol substrate was low and unaffected by any manipulation of medium conditions.This work has been supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through the contracts PRAXIS/PBIO/14314/1998 and POCTI/35429/QUI/2000 and the grant PRAXIS XXI/BD/21615/99 (S. Garcia), and by FEDER.We thank Ricardo Baptista for help in the production of cutinase

Scalable generation of cerebellar neurons from pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, our ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of cerebellar neuroepithelium formed from human iPSCs, recapitulating the early developmental events of the cerebellum. Additionally, an efficient maturation of replated cerebellar progenitors into distinct types of functional cerebellar neurons was also achieved under defined and feeder-free conditions. However, developing a scalable protocol that allows to produce large numbers of organoids and high yields of mature neurons in a 3D bioreactor culture systems is still a difficult challenge. In this work, we present a new approach for the reproducible and scalable generation of mid-hindbrain organoids under chemically defined conditions by using the novel PBS 0.1 (100 mL) Vertical-Wheel single-use bioreactor. In this system, an efficient cell aggregation with shape and size-controlled aggregates can be obtained, which is important for homogeneous and efficient differentiation. Moreover, a larger amount of iPSC-derived aggregates can be generated without being excessively labour-intensive, achieving 431 ± 53.6 aggregates/mL at 24 hours after seeding. After differentiation, distinct types of cerebellar neurons were generated, including Purkinje cells (Calbindin+), Granule cells (BARHL1+ and Pax6+), Golgi cells (Neurogranin+ and GAD65+), Deep cerebellar nuclei projection neurons (TBR1+) and Non-Golgi-type interneurons (Parvalbumin+ and Calbindin-). These cells show signs of efficient maturation, staining positive for MAP2, and are able to change intracellular Ca2+ concentration following KCl stimulation. In this system, human iPSC-derived organoids are able to mature into different mature cerebellar neurons and to survive for up to 3 months, without replating and co-culture with feeder layers

Modeling Rett syndrome with human patient-specific forebrain organoids

Copyright © 2020 Gomes, Fernandes, Vaz, Silva, Bekman, Xapelli, Duarte, Ghazvini, Gribnau, Muotri, Trujillo, Sebastião, Cabral and Diogo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.We also acknowledge financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal, through iBB, Institute for Bioengineering and Biosciences (UIDB/04565/2020) and from Programa Operacional Regional de Lisboa 2020 (Project No. 007317). AG was supported by FCT (PD/BD/128373/2017).info:eu-repo/semantics/publishedVersio