Search Results (22)

This study examines the differences in performance between orbitally shaken bioreactors (OSBs) and stirred tank bioreactors (STBs) in Chinese Hamster Ovary (CHO) cell perfusion culture in response to the growing market demand for monoclonal antibodies (mAbs). Although OSBs demonstrated higher cell densities, a notable reduction in specific antibody production rates was observed during the mid-to-late phases of the culture compared with STBs. To elucidate the underlying mechanisms, the rheological behaviour of high-density cell suspensions in both reactor types was initially characterised, confirming their adherence to the Sisko fluid model. Computational Fluid Dynamics (CFD) analysis revealed the influence of these rheological properties on the shear stress distribution and mass transfer. This analysis identified the key limiting factors for achieving higher cell densities: mass transfer efficiency in OSBs and shear stress in STBs. Using an Euler–Lagrangian cell-tracking methodology to analyse cellular “lifelines”, it was determined that OSBs exhibited approximately twice the number and frequency of shear stress peak occurrences compared to STBs. This persistent mechanical stimulation likely contributes to the reduced specific antibody production rates observed. This comprehensive investigation not only clarifies the comparative advantages and limitations of different bioreactor types in perfusion culture but also provides a robust theoretical basis and technical guidance for informed reactor selection, optimisation, and scale-up in industrial production environments. Full article

Plant biotechnology creates opportunities for the cultivation of plants regardless of their natural habitats, which are often protected or difficult to access. Maintaining suspension cell cultures in bioreactors is an advanced part of biotechnological research that provides possibilities for obtaining plant tissue on a large scale. In this study, the suspension culture cultivation of a Chinese endemic plant, Schisandra henryi, in a stirred tank bioreactor was elaborated for the first time. The phytochemical profile of the tissue extracts was determined with UHPLC-MS/MS for the lignans (fifteen dibenzocyclooctadiene lignans, one aryltetralin lignan, and two neolignans) and UHPLC-DAD-ESI-MS³ for the phenolic compounds (procyanidins and their derivatives and catechin). The maximum total lignan content of 1289 µg/100 g DW was detected for the extracts from suspensions cultured in a bioreactor for over 10 days. For the phenolic compounds, catechin was the dominant compound (390.44 mg/100 g DW). The biological activity of the extracts was tested too. To determine antioxidant potential we used DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Molybdenum reduction, and β-carotene bleaching tests. The inhibition activity of the S. henryi extract on the enzymes responsible for skin aging, hyaluronidase and tyrosinase, was assessed with spectrophotometry. The cytotoxic activity of the extracts was estimated on human ovarian SKOV-3, cervical HeLa, and gastric AGS cancer cells and non-cancer, normal fibroblasts by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results showed the great potential of the obtained cell biomass extracts. The results of the antioxidant tests indicated their strong ability to reduce the level of free radicals, similarly to that of ascorbic acid, as well as the weak capacity to protect lipids from oxidation. Moreover, anticancer potential, particularly on the cervical and gastric cancer cells, was confirmed too. Full article

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEK_Dyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands. Full article

Interrupted blood flow in the brain due to ischemic injuries such as ischemic stroke or traumatic brain injury results in irreversible brain damage, leading to cognitive impairment associated with inflammation, disruption of the blood–brain barrier (BBB), and cell death. Since the BBB only allows entry to a small class of drugs, many drugs used to treat ischemia in other tissues have failed in brain-related disorders. The administration of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has shown promise in improving the functional recovery of the brain following cerebral ischemia by inducing blood vessel formation. To facilitate such a treatment approach, it is necessary to develop bioprocesses that can produce therapeutically relevant MSC-EVs in a reproducible and scalable manner. This study evaluated the feasibility of using stirred suspension bioreactors (SSBs) to scale-up the serum-free production of pro-angiogenic MSC-EVs under clinically relevant physioxic conditions. It was found that MSCs grown in SSBs generated EVs that stimulated angiogenesis in cerebral microvascular endothelial cells, supporting the use of SSBs to produce MSC-EVs for application in cerebral ischemia. These properties were impaired at higher cell confluency, outlining the importance of considering the time of harvest when developing bioprocesses to manufacture EV populations. Full article

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 10⁹ TCID50/mL) in the culture supernatant, the final formulations were prepared at 10¹⁰ TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented. Full article

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein. Full article

HEK293 is a widely used cell line in the fields of research and industry. It is assumed that these cells are sensitive to hydrodynamic stress. The aim of this research was to use particle image velocimetry validated computational fluid dynamics (CFD) to determine the hydrodynamic stress in both shake flasks, with and without baffles, and in stirred Minifors 2 bioreactors to evaluate its effect on the growth and aggregate size distribution of HEK293 suspension cells. The HEK FreeStyle^TM 293-F cell line was cultivated in batch mode at different specific power inputs (from 63 $\mathrm{W} {\mathrm{m}}^{-3}$ to 451 $\mathrm{W} {\mathrm{m}}^{-3}$), whereby $\approx 60 \mathrm{W} {\mathrm{m}}^{-3}$ corresponds to the upper limit, which is what has been typically described in published experiments. In addition to the specific growth rate and maximum viable cell density VCD${}_{\max }$, the cell size distribution over time and cluster size distribution were investigated. The VCD${}_{\max }$ of $(5.77\pm 0.02)·{10}^6$$\mathrm{cells}$$\mathrm{m}$${\mathrm{L}}^{-1}$ was reached at a specific power input of 233 $\mathrm{W} {\mathrm{m}}^{-3}$ and was $23.8\%$ higher than the value obtained at 63 $\mathrm{W} {\mathrm{m}}^{-3}$ and $7.2\%$ higher than the value obtained at 451 $\mathrm{W} {\mathrm{m}}^{-3}$. No significant change in the cell size distribution could be measured in the investigated range. It was shown that the cell cluster size distribution follows a strict geometric distribution whose free parameter p is linearly dependent on the mean Kolmogorov length scale. Based on the performed experiments, it has been shown that by using CFD-characterised bioreactors, the VCD${}_{\max }$ can be increased and the cell aggregate rate can be precisely controlled. Full article

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries. Full article

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. The mass transfer coefficients were determined for a range of stirring speeds between 10 and 100 rpm and for working volumes between 60 and 100 mL. Additionally, boundary condition, mesh refinement, and temperature variation studies were performed. Lastly, cell size, energy dissipation rate, and shear stress fields were calculated to determine optimal hydrodynamic conditions for culture. The experimental results demonstrate that the $k_L$ can be predicted using $Sh=1.68R{e}^{0.551}S{c}^{\frac{1}{3}}{G}^{1.18}$, with a mean absolute error of 2.08%. Using the simulations and a correction factor of 0.473, the expression can be correlated to provide equally valid results. To directly obtain them from simulations, a partial slip boundary condition can be tuned, ensuring better near-surface velocity profiles or, alternatively, by deeply refining the mesh. Temperature variation studies support the use of this correlation for temperatures up to 37 °C by using a Schmidt exponent of 1/3. Finally, the flow was characterized as transitional with diverse mixing mechanisms that ensure homogeneity and suspension quality, and the results obtained are in agreement with previous studies that employed RANS models. Overall, this work provides new data regarding oxygen mass transfer and hydrodynamics in the Vertical-Wheel bioreactor, as well as new insights for air-water mass transfer modeling in systems with low interface deformation, and a computational model that can be used for further studies. Full article

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) hold great potential to generate novel and curative cell therapy products. However, the current large-scale cultivation of hUCMSCs is based on empirical geometry-dependent methods, limiting the generation of high-quantity and high-quality hUCMSCs for clinical therapy. Herein, we develop a novel scale-up strategy based on computational fluid dynamics (CFD) to effectively expand the hUCMSCs in a 3D tank bioreactor. Using a standardized hUCMSCs line on microcarriers, we successfully translated and expanded the hUCMSCs from a 200 mL spinner flask to a 1.5 L computer-controlled bioreactor by matching the shear environment and suspending the microcarrier. Experimental results revealed that the batch-cultured hUCMSCs in bioreactors with an agitation speed of 40 rpm shared a more favorable growth and physiological state, similar to that run at 45 rpm in a 200 mL spinner flask, showing comparability in both culture systems. Notably, the maximum cell density reached up to 27.3 × 10⁵ cells/mL in fed-batch culture, 2.9 folds of that of batch culture and 20.2 times of seeding cells. As such, efficient process optimization and scale-up expansion of hUCMSCs were achieved in the microcarrier-based bioreactor system by the developed CFD simulation strategy, which provided an alternative toolbox to generate massive and standardized curative cell therapy products. Full article

Our aim in the experiment was to study the effects of methyl jasmonates (MeJA) on the active compounds of rosemary suspension cells, the metabolites’ change of contents under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 μM MeJA (M100). The results demonstrated that MeJA treatments promoted the accumulation of rosmarinic acid (RA), carnosic acid (CA), flavonoids, jasmonate (JA), gibberellin (GA), and auxin (IAA); but reduced the accumulations of abscisic acid (ABA), salicylic acid (SA), and aspartate (Asp). In addition, 50 and 100 μM MeJA promoted the accumulation of alanine (Ala) and glutamate (Glu), and 50 μM MeJA promoted the accumulation of linoleic acid and alpha-linolenic acid in rosemary suspension cells. Comparative RNA-sequencing analysis of different concentrations of MeJA showed that a total of 30, 61, and 39 miRNAs were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of the target genes of the differentially expressed miRNAs showed that plant hormone signal transduction, linoleic acid, and alpha-linolenic acid metabolism-related genes were significantly enriched. In addition, we found that miR160a-5p target ARF, miR171d_1 and miR171f_3 target DELLA, miR171b-3p target ETR, and miR156a target BRI1, which played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of 12 differentially expressed miRNAs and their target genes showed a high correlation between the RNA-seq and the qRT-PCR result. Amplification culture of rosemary suspension cells in a 5 L stirred bioreactor showed that cell biomass accumulation in the bioreactor was less than that in the shake flask under the same conditions, and the whole cultivation period was extended to 14 d. Taken together, MeJA promoted the synthesis of the active compounds in rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided an overall view of the miRNAs responding to MeJA in rosemary. Full article

Adeno-associated virus vectors (AAV) are reported to have a great potential for gene therapy, however, a major bottleneck for this kind of therapy is the limitation of production capacity. Higher specific AAV vector yield is often reported for adherent cell systems compared to cells in suspension, and a microcarrier-based culture is well established for the culture of anchored cells on a larger scale. The purpose of the present study was to explore how microcarrier cultures could provide a solution for the production of AAV vectors based on the triple plasmid transfection of HEK293T cells in a stirred tank bioreactor. In the present study, cells were grown and expanded in suspension, offering the ease of this type of operation, and were then anchored on microcarriers in order to proceed with transfection of the plasmids for transient AAV vector production. This process was developed in view of a bioreactor application in a 200 mL stirred-tank vessel where shear stress aspects were studied. Furthermore, amenability to a continuous process was studied. The present investigation provided a proof-of-concept of a continuous process based on microcarriers in a stirred-tank bioreactor. Full article

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ β-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling. Full article

Mandarin fish (Siniperca chuatsi) is one of the important cultured fish species in China. Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca Chuatsi rhabdovirus (SCRV) have hindered the development of mandarin fish farming industry. Vaccination is the most effective method for control of viral diseases, however viral vaccine production requires the large-scale culture of cells. Herein, a suspension culture system of Chinese perch brain cell (CPB) was developed on Cytodex 1 microcarrier in a stirred bioreactor. Firstly, CPB cells were cultured using Cytodex 1 microcarrier in 125 mL stirring flasks. With the optimum operational parameters, CPB cells grew well, distributed uniformly, and could fully cover the microcarriers. Then, CPB cells were digested with trypsin and expanded step-by-step with different expansion ratios from the 125 mL stirring bottle to a 500 mL stirring bottle, and finally to a 3-L bioreactor. Results showed that with an expansion ratio of 1:3, we achieved a high cell density level (2.25 × 10⁶ cells/mL) with an efficient use of the microcarriers, which also confirmed the data obtained from the 125 mL stirring flask. Moreover, obvious cytopathic effects (CPE) were observed in the suspended CPB cells post-infection with ISKNV and SCRV. This study provided a large-scale culture system of CPB cells for virus vaccine production. Full article

Stirred single-use bioreactors in combination with microcarriers (MCs) have established themselves as a technology that has the potential to meet the demands of current and future cell therapeutic markets. However, most of the published processes have been performed using fetal bovine serum (FBS) containing cell culture medium and non-biocompatible MCs. This approach has two significant drawbacks: firstly, the inevitable potential risks associated with the use of FBS for clinical applications; secondly, non-biocompatible MCs have to be removed from the cell suspension before implantation, requiring a step that causes loss of viable cells and adds further costs and complications. This study aimed to develop a new platform based on a chemically defined xeno- and serum-free cell culture medium and biodegradable MC that can support the growth of human adipose stem cells (hASCs) while still preserving their undifferentiated status. A specific combination of components and manufacturing parameters resulted in a MC prototype, called “BR44”, which delivered the desired functionality. MC BR44 allows the hASCs to stick to its surface and grow in a chemically defined xeno- and serum-free medium (UrSuppe). Although the cells’ expansion rate was not as high as with a commercial non-biodegradable standard MC, those cultured on BR44 maintained a better undifferentiated status in both static and dynamic conditions than those cultured on traditional 2D surfaces. Full article