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The importance of Mg in the human body, its key role in bone tissue development, in addition to its application to improve and modify physical, thermal, and mechanical properties of bioactive silicate glasses, make Mg a very interesting element as a component of bioactive glasses for medical applications. Although Mg is a typical element in the composition of numerous developed bioactive glasses, the analysis of the literature reveals that further research is required to gain comprehensive understanding about the effects of MgO on bioactive glass structure and properties. This article presents a comprehensive overview of the field of Mg‐containing bioactive glasses discussing available compositions and summarizing existing knowledge on effects of MgO on glass properties. The biomedical applications of several developed glasses are discussed highlighting the distinct effects of Mg in relevant areas, such as bioactivity and cell response, and focusing on the most common applications for these glasses, such as bone cements, bioactive coatings, and scaffolds for bone tissue engineering. The effect of Mg on bone development is discussed and avenues for future research in the field are proposed, emphasizing the need to investigate unstudied properties of (already developed) Mg‐containing glass compositions, such as angiogenesis‐stimulating action and the effect on osteoclast functions, to develop new Mg‐containing bioactive glasses as well as to make products of different shape designs such as nanoparticles, fibers, and complex porous structures.

With an increasing demand for efficacious, safe, and affordable vaccines for human and animal use, process intensification in cell culture-based viral vaccine production demands advanced process strategies to overcome the limitations of conventional batch cultivations. However, the use of fed-batch, perfusion, or continuous modes to drive processes at high cell density (HCD) and overextended operating times has so far been little explored in large-scale viral vaccine manufacturing. Also, possible reductions in cell-specific virus yields for HCD cultivations have been reported frequently. Taking into account that vaccine production is one of the most heavily regulated industries in the pharmaceutical sector with tough margins to meet, it is understandable that process intensification is being considered by both academia and industry as a next step toward more efficient viral vaccine production processes only recently. Compared to conventional batch processes, fed-batch and perfusion strategies could result in ten to a hundred times higher product yields. Both cultivation strategies can be implemented to achieve cell concentrations exceeding 107 cells/mL or even 108 cells/mL, while keeping low levels of metabolites that potentially inhibit cell growth and virus replication. The trend towards HCD processes is supported by development of GMP-compliant cultivation platforms, i.e., acoustic settlers, hollow fiber bioreactors, and hollow fiber-based perfusion systems including tangential flow filtration (TFF) or alternating tangential flow (ATF) technologies. In this review, these process modes are discussed in detail and compared with conventional batch processes based on productivity indicators such as space-time yield, cell concentration, and product titers. In addition, options for the production of viral vaccines in continuous multi-stage bioreactors such as two- and three-stage systems are addressed. While such systems have shown similar virus titers compared to batch cultivations, keeping high yields for extended production times is still a challenge. Overall, we demonstrate that process intensification of cell culture-based viral vaccine production can be realized by the consequent application of fed-batch, perfusion, and continuous systems with a significant increase in productivity. The potential for even further improvements is high, considering recent developments in establishment of new (designer) cell lines, better characterization of host cell metabolism, advances in media design, and the use of mathematical models as a tool for process optimization and control.

Cardiac fibroblast differentiation to myofibroblast is a crucial process in the development of cardiac fibrosis and is tightly dependent on transforming growth factor beta-1 (TGF-β1). The transcription factor forkhead box O1 (FoxO1) regulates many cell functions, including cell death by apoptosis, proliferation, and differentiation. However, several aspects of this process remain unclear, including the role of FoxO1 in cardiac fibroblast differentiation and the regulation of FoxO1 by TGF-β1. Here, we report that TGF-β1 stimulates FoxO1 expression, promoting its dephosphorylation, nuclear localization and transcriptional activity in cultured cardiac fibroblasts. TGF-β1 also increases differentiation markers such as α-smooth muscle actin, connective tissue growth factor, and pro-collagen I, whereas it decreases cardiac fibroblast proliferation triggered by fetal bovine serum. TGF-β1 also increases levels of p21waf/cip-cycle inhibiting factor protein, a cytostatic factor promoting cell cycle arrest and cardiac fibroblast differentiation. In addition, TGF-β1 increases cardiac fibroblast contractile capacity as assessed by collagen gel contraction assay. The effect of TGF-β1 on cardiac fibroblast differentiation was prevented by FoxO1 down-regulation and enhanced by FoxO1 overexpression. Thus, our findings reveal that FoxO1 is regulated by TGF-β1 and plays a critical role in cardiac fibroblast differentiation. We propose that FoxO1 is an attractive new target for anti-fibrotic therapy.

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Defective interfering particles (DIPs) are a natural byproduct of influenza A virus (IAV) replication. DIPs interfere with the propagation and spread of infectious standard virus (STV), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. These properties open exciting possibilities for the development of DIP-based antivirals. Exploring options for cell culture-based DIP production, we have established a fully continuous cultivation process, where one bioreactor is used to grow cells that are fed to two bioreactors operated in parallel for virus production. This system allows head-to-head comparisons of STV and DIP replication dynamics over extended time periods. Cultivations were performed at two residence times (RT, 22 and 36 h) using MDCK suspension cells grown in a fully defined medium. For infection, we used a virus seed generated by reverse genetics containing STVs and a known DIP carrying a deletion in segment 1 (delS1(1)). Four days post infection, DIPs achieved maximum concentrations of 7.0·109 virions/mL and 8.4·109 virions/mL for RTs of 22 and 36 h, respectively. Furthermore, oscillations in virus titers with two to three maxima were found for DIP accumulation at 36 and 22 h RT, respectively. To complement the study, a basic mathematical model using simple kinetics and a reasonable number of parameters to describe DIP-propagation in continuous cultures was established. Upon fitting the model individually to each of the two data sets, oscillations in the viral dynamics and the cell population dynamics were described well. Modeling suggests that both STV inactivation and virus degradation have to be taken into account to achieve good agreement of simulations and experimental data for longer RTs. Together, the high DIP titers obtained, and the successful simulation of the experimental data showed that the combination of continuous bioreactors and mathematical models can enable studies regarding DIP dynamics over extended time periods and allow large scale manufacturing of DIP-based antivirals.

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers. Because this vector is unable to replicate in human recipients, a high amount of virions per dose is required for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA‐CR19 is an option to obtain very high MVA yields. Here we compared different options for cell retention in perfusion mode using conventional stirred‐tank bioreactors including an alternating tangential flow filtration system, an acoustic settler and an inclined settler. The last two options allowed continuous MVA virus harvesting. Furthermore, we studied hollow‐fiber based bioreactors and an orbital‐shaken bioreactor in perfusion mode, both available for single‐use. Productivity for the virus strain MVA‐CR19 was compared to results from batch and continuous production reported in literature. Our results demonstrate that MVA virus is highly stable at 37°C in cell culture so that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred‐tank bioreactor, a perfusion strategy during the whole run with working volume expansion after virus infection resulted in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5  ×  10 9 virions/mL were achieved in these intensified processes. Taken together, the study shows a novel perspective on high‐yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single‐use perfusion platforms. This article is protected by copyright. All rights reserved