Bioreactor production of rVSV-based vectors in Vero cell suspension cultures.
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ABSTRACT:
SUBMITTER: Kiesslich S
PROVIDER: S-EPMC8252067 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.
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Project description:The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms-technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.
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Project description:Vero cells, as approved by the World Health Organization, have been the most commonly used continuous cell line for viral vaccine production over the last 25 years, but their adherent phenotype continues to limit productivity. Adapting to a suspension culture would overcome this restriction and reduce production costs. First, a Vero suspension isolate was obtained and metabolically characterized. Second, RNA sequencing analysis was used to identify differentially expressed genes between adherent and suspension cells, which revealed complete downregulation of adhesion and matrix-associated genes. Additionally, signaling pathways involving Wnt and other tyrosine kinase receptors were identified as potential leads for growth optimization. In particular, supplementation with fibroblast growth factor 2 allowed for a 20% increase in cell density. Finally, a comparative viral productivity assay revealed a 30% increase in poliovirus production in suspension Vero cells compared to adherent cells depending on the serotype, as well as a 140% increase in respiratory syncytial virus production and a 150% increase in yellow fever virus production.This work establishes the potential of the suspension Vero cell line as a new cell platform for viral vaccine production.
2024-06-18 | GSE250417 | GEO
Project description:The Vero cell line is considered the most used continuous cell line for the production of viral vectors and vaccines. Historically, it is the first cell line that was approved by the WHO for the production of human vaccines. Comprehensive experimental data on the production of many viruses using the Vero cell line can be found in the literature. However, the vast majority of these processes is relying on the microcarrier technology. While this system is established for the large-scale manufacturing of viral vaccine, it is still quite complex and labor intensive. Moreover, scale-up remains difficult and is limited by the surface area given by the carriers. To overcome these and other drawbacks and to establish more efficient manufacturing processes, it is a priority to further develop the Vero cell platform by applying novel bioprocess technologies. Especially in times like the current COVID-19 pandemic, advanced and scalable platform technologies could provide more efficient and cost-effective solutions to meet the global vaccine demand. Herein, we review the prevailing literature on Vero cell bioprocess development for the production of viral vectors and vaccines with the aim to assess the recent advances in bioprocess development. We critically underline the need for further research activities and describe bottlenecks to improve the Vero cell platform by taking advantage of recent developments in the cell culture engineering field.
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Project description:Glucosinolates, a group of sulfur-containing specialized metabolites of the Brassicales, have attracted a lot of interest in nutrition, medicine and agriculture due to their positive health effects and their involvement in plant defense. Their biological activities and the extensive knowledge of their biosynthesis have inspired research into development of crops with enhanced glucosinolate contents as well as their biotechnological production in homologous and heterologous systems. Here, we provide proof-of-concept for transgenic suspension cultures of carrot (Daucus carota, Apiacae) as a scalable production platform for plant specialized metabolites using benzylglucosinolate as a model. Two T-DNAs carrying in total six genes of the benzylglucosinolate biosynthesis pathway from Arabidopsis thaliana as well as NPTII and BAR as selectable markers were transferred to carrot cells by Agrobacterium tumefaciens-mediated transformation. Putative transformants selected based on their kanamycin and BASTA resistances were subjected to HPLC-MS analysis. Of 79 putative transformants, 17 produced benzylglucosinolate. T-DNA-integration was confirmed for the five best producers. Callus from these transformants was used to establish suspension cultures for quantitative analysis. When grown in 60-ml-cultures, the best transformants produced roughly 2.5 nmol (g fw)-1 benzylglucosinolate, together with up to 10 nmol (g fw)-1 desulfobenzylglucosinolate. Only one transformant produced more benzylglucosinolate than desulfobenzylglucosinolate. The concentration of sulfate in the medium was not a major limiting factor. High production seemed to be associated with poor growth and vice versa. Therefore, future research should try to optimize medium and cultivation process and to separate growth and production phase by using an inducible promoter.
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Project description:The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.
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