Search Results (85)

Bioreactors enable scalable cell cultivation by providing controlled environments for temperature, oxygen, and nutrient regulation, maintaining viability and enhancing expansion efficiency. Automated systems improve reproducibility and minimize contamination risks, making them ideal for high-density cultures. While fed-batch bioreactors dominate biologics production, continuous systems like perfusion cultures offer superior resource efficiency and productivity. The Quantum hollow-fiber perfusion bioreactor supports cell expansion via semi-permeable capillary membranes and a closed modular design, allowing continuous media exchange while retaining key molecules. We developed a multiple-harvest protocol for suspension cells in the Quantum system, yielding 2.5 × 10¹⁰ MEL-745A cells within 29 days, with peak densities of 4 × 10⁷ cells/mL—a 15-fold increase over static cultures. Viability averaged 91.3%, with biweekly harvests yielding 3.1 × 10⁹ viable cells per harvest. Continuous media exchange required more basal media to maintain glucose and lactate levels but meaningfully less growth supplement than the 2D culture. Stable transgene expression suggested phenotypic stability. Automated processing reduced hands-on time by one-third, achieving target cell numbers 12 days earlier than 2D culture. Despite higher media use, total costs for the automated were lower compared to the manual process. Quantum enables high-density suspension cell expansion with cost advantages over conventional methods. Full article

Protein-coated ultra-high viscosity (UHV)-alginate hydrogels are essential to mimic the physiological in vivo environment of humans in several in vitro applications. This work presents an optimized bioreactor-integrated freeze-drying process for Matrigel^TM-coated UHV-alginate microcarriers in the context of human induced pluripotent stem cell (hiPSC) expansion. The impact of freeze-drying on the UHV-alginate microcarriers using trehalose 100 mg/mL in 0.9% NaCl as a lyoprotective agent, as well as the stem cell response using hiPSCs, was analyzed using microscopy-based screenings. First observations of the process showed that the integrity of the cake was preserved in the samples with a maximum vapor exchanging rate. Following rehydration, the UHV-alginate microcarriers retained their original morphology. Upon the addition of Poloxamer 188, stickiness and bubble formation were reduced. The expansion of hiPSCs in a suspension bioreactor resulted in a 5–7-fold increase in total cell count, yielding at least 1.3 × 10⁷ cells with viability exceeding 80% after seven days of cultivation. In flow cytometry analysis, the pluripotency factors OCT3/4 and SSEA4 resulted in positive signals in over 98% of cells, while the differentiation factor SSEA1 was positive in fewer than 10% of cells. Supported by preceding in silico predictions of drying time, this study presents, for the first time, basic steps toward a “ready-to-use” bioreactor-integrated freeze-drying process for UHV-alginate microcarriers in the iPSC context. Full article

The production of influenza A virus (IAV) using Madin-Darby Canine Kidney (MDCK) cells is a key strategy for efficient influenza vaccine manufacturing. However, challenges remain in optimizing cell culture processes for higher yield and efficiency. This study aims to evaluate different process intensification strategies on two distinct clonal MDCK suspension cell lines (C59 and C113) for improved IAV production. A semi-perfusion strategy was used to push cells towards high cell density (HCD), achieving up to 17 × 10⁶ C113 cells/mL and 42 × 10⁶ C59 cells/mL, respectively. Next, a Tangential Flow Depth Filtration (TFDF)-based perfusion process with direct harvest during IAV production was established, resulting in high titers and a 10-fold higher space-time yield for C59 and a 4-fold improvement for C113 compared to batch operation. In addition, the suitability of N-1 perfusion was evaluated for batch and intensified fed-batch processes. Cells taken from the N-1 perfusion showed different cell-specific growth rates, but this had no effect on virus titers except for processes started from oxygen-deprived precultures. Finally, comparable virus titers were obtained when the production bioreactor was directly inoculated from an HCD cryovial. Taken together, seed train intensification and TFDF-based perfusion majorly reduced process times and improved IAV production. Full article

Plumbagin is an important naphthoquinone with potent anticancer properties besides multitudinous uses in healthcare. It is produced in a limited number of species and families but mostly in the roots of Plumbaginaceae family members. The biosynthetic pathway and the genes that regulate plumbagin synthesis are not completely known, but details of these are being revealed. Several species, including Plumbago, Drosera, and others, are being uprooted for the extraction of plumbagin by pharmaceutical industries, leading to the destruction of natural habitats. The pharmaceutical industry is therefore facing an acute shortage of plant material. This necessitates enhancing the accumulation of plumbagin using suspensions and hairy roots to meet market demands. Many factors, such as the aggregate size of the inoculum, stability of the culture, and the sequential effects of elicitors, immobilization, and permeabilization, have been demonstrated to act synergistically and markedly augment plumbagin accumulation. Hairy root cultures can be used for the large-scale production, growth, and plumbagin accumulation, and the exploration of their efficacy is now imperative. The secretion of compounds into the spent medium and their in situ adsorption via resin has remarkable potential, but this has not been thoroughly exploited. Improvements in the quality of biomass, selection of cell lines, and production of plumbagin in bioreactors have thus far been sporadic, and these parameters need to be further exploited. In this review, we report the advances made relating to the importance of stable cell line selection for the accumulation of compounds in long-term cultures, hairy root cultures for the accumulation of plumbagin, and its semicontinuous production via total cell recycling in different types of bioreactors. Such advances might pave the way for industrial exploitation. The steps in the biosynthetic pathway that are currently understood might also aid us in isolating the relevant genes in order to examine the effects of their overexpression or heterologous downregulation or to edit the genome using CRISPR-Cas9 technology in order to enhance the accumulation of plumbagin. Its potential as an anticancer molecule and its mode of action have been amply demonstrated, but plumbagin has not been exploited in clinics due to its insolubility in water and its highly lipophilic nature. Plumbagin-loaded nanoemulsions, plumbagin–silver, or albumin nanoparticle formulations can overcome these problems relating to its solubility and are currently being tried to improve its bioavailability and antiproliferative activities, as discussed in the current paper. Full article

Plant cell suspension cultures offer a sustainable method for producing valuable secondary metabolites, such as bioactive pentacyclic triterpenes. This study established a high-triterpene-yielding cell suspension culture from the apple cultivar “Cox Orange Pippin”. Through transcriptomic analysis and triterpene profiling across growth phases, we uncovered complex regulatory networks that govern biomass production and triterpene biosynthesis. Key biological processes, including cell cycle regulation, cell wall biosynthesis, lipid metabolism, and stress response mechanisms, play pivotal roles in culture dynamics. Differential gene expression linked to these processes revealed how the culture adapts to growth conditions and nutrient availability at each growth phase. Methyl jasmonate elicitation enhanced phenylpropanoid and flavonoid biosynthesis, along with specific triterpene production pathways, highlighting its potential for optimizing secondary metabolite production. Key enzymes, such as oxidosqualene cyclase 4 and a putative C-2α hydroxylase, were identified as promising targets for future metabolic engineering efforts. This study represents the first in-depth report on the molecular mechanisms underlying plant cell growth in bioreactors, specially focusing on a cell suspension culture derived from a semi-russeted apple cultivar. The findings reveal key regulatory pathways in biomass accumulation and triterpene production, offering valuable insights for optimizing bioreactor cultures for industrial applications. Full article

Solanum betaceum Cav. (tamarillo) has a strong biotechnological potential given the ease of obtaining cell lines from it that can be genetically transformed. However, genetic transformation of tamarillo cell suspension cultures has not yet been described. This study presents a simple method for Agrobacterium-mediated transformation of these cells and demonstrates the successful insertion of the β-glucuronidase gene (gusA) and the yellow fluorescent protein gene (eyfp) in their genome. For the success of this protocol, the selection of actively growing sub-cultured callus as explant and isolation of bacterial colonies with a cell density OD₆₀₀ of 0.6–0.8 were key steps. Also, the inoculation of the callus in a bacteria liquid culture, the use of sonication, and the addition of antioxidants were essential. The transient expression of the gusA gene in tamarillo callus was confirmed and quantified, and no significant differences were observed between using LBA4404 or EHA105 strains. Finally, the insertion of the eyfp gene in the tamarillo genome enabled the in vivo confirmation of the transformation success. The present study showed that tamarillo cell suspension cultures can be genetically modified, opening the way for metabolite production in transformed cells and future scaling-up in bioreactors. Full article

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

The prediction of the cell yield in large-scale bioreactor culture is an important factor for various cell therapy bioprocess operations to ensure consistency in cell quality and efficient use of resources. However, the shear sensitivity of cells used in cell therapy manufacturing can make such predictions difficult, particularly in large-scale suspension cultures that have significant stresses without representative scale down models. The PBS Vertical-Wheel (VW) bioreactors have been demonstrated to provide a homogeneous hydrodynamic environment with low shear for cell culture at various scales (0.1–80 L) and is thereby employed for various shear-sensitive cells. In this study, the oxygen transfer rate for surface aeration for three large-scale VW bioreactors was measured along with the specific oxygen uptake rate (sOUR) of iPSCs cultured in the bioreactors. The oxygen mass transfer coefficient was measured in PBS-3/15/80 L bioreactors at different agitation rates, headspace gas flowrates, and working volumes using the static gassing-out method. The sOUR of iPSCs was measured using the dynamic method in the PBS-0.1 L Mini with a custom DO probe configuration. The results from both experiments were combined to calculate the theoretical maximum cell density before oxygen limitation across VW bioreactors at 2 L/3 L/10 L/15 L/50 L/80 L working volumes at a different agitation speed and aeration rate. Full article

Background: The callus cultures from the fronds of the lycophyte Phlegmariurus taxifolius produce the huperzine A (HupA) alkaloid, which is used in Alzheimer’s disease treatment. This study aimed to establish the growth kinetics and HupA production by the newly HupS21 cell line grown in 250 mL flasks and in a 2 L airlift bioreactor. Methods: Batch-type kinetics were carried out for 60 days in 250 mL flasks and for 20 days in a 2 L airlift bioreactor. Measurements of dry weight (DW), specific growth rate (μ), doubling time (dt), pH, carbohydrate consumption, and HupA quantification were performed. The acetylcholinesterase (AChE) inhibitory assay of the HupS21 alkaloidal extract was determined. Results: The 250 mL flasks kinetic reached a maximum cell growth of 8.17 g/L DW, with a μ of 0.045 day⁻¹ and a dt of 15.40 days. The maximum HupA production was of 2.03 μg/g DW at day 45. In the 2 L airlift reactor, a maximum growth of 16.70 g/L DW, a μ of 0.062 day⁻¹, a dt of 11.20 days, and HupA production of 2.48 μg/g DW at day 15 were obtained. The alkaloidal extract from the HupS21 cell line at 100 μg/mL showed an AChE inhibitory activity of 85.6 ± 1.27%. Conclusions: The airlift reactor outperformed the flask cultures in maximum cell growth, specific growth rate, doubling time, and HupA production. To our knowledge, this research is the first report on the establishment of suspension cell cultures of P. taxifolius in shaken flasks and in an airlift bioreactor, providing a foundation for scaling up HupA production for pharmaceutical use. 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

Microbioreactors increase information output in biopharmaceutical screening applications because they can be operated in parallel without consuming large quantities of the pharmaceutical formulations being tested. A capillary wave microbioreactor (cwMBR) has recently been reported, allowing cost-efficient parallelization in an array that can be activated for mixing as a whole. Although impedance spectroscopy can directly distinguish between dead and viable cells, the monitoring of cells in suspension within bioreactors is challenging because the signal is influenced by the potentially varying properties of the culture medium. In order to address this challenge, an impedance sensor consisting of two sets of microelectrodes in a cwMBR is presented. Only one set of electrodes was covered by a two-photon cross-linked hydrogel to become insensitive to the influence of cells while remaining sensitive to the culture medium. With this impedance sensor, the biomass of Saccharomyces cerevisiae could be measured in a range from 1 to 20 g L⁻¹. In addition, the sensor can compensate for a change in the conductivity of the suspension of 5 to 15 mS cm⁻¹. Moreover, the two-photon cross-linking of hydroxyethyl starch methacrylate hydrogel, which has been studied in detail, recommends itself for even much broader sensing applications in miniaturized bioreactors and biosensors. 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

Anaerobic wastewater treatment is, in many cases, a justified alternative to typical activated sludge processes, from a technological, economic, and ecological point of view. The optimisation of fermentation reactors is primarily concerned with increasing the biodegradation of organic compounds and biogas production, as well as improving efficiency in the removal of nitrogen and phosphorus compounds. The aim of the research was to determine the impact of using low-cost recycled filling on the efficiency of treating real confectionery wastewater in a vertical anaerobic labyrinth flow bioreactor. The experiments focused on selecting the organic loading rate that would allow for the effective biodegradation and removal of pollutants, as well as the efficient production of biomethane. It was found that the tested reactor can operate efficiently at a maximum organic loading rate (OLR) of 7.0–8.0 g of chemical oxygen demand (COD)/L·d. In this OLR range, high efficiency was guaranteed for both wastewater treatment and biogas production. However, increasing the OLR value to 8.0 g COD/L·d had a significant negative effect on the methane (CH₄) content in the biogas. The most efficient variants achieved a biodegradation efficiency of around 90% of the organic compounds, a CH4 content of over 70% in the biogas, and a biogas yield of over 400 L/kg of COD removed. A significant influence of the applied OLR on the ratio of free organic acids (FOS) to total alkaline capacity (TAC) and pH was observed, as well as a strong correlation of these indicators with the specific biogas yield and CH4 content. The application of a solution based on the use of a hybrid system of anaerobic granulated sludge and an anaerobic filter resulted in an efficient treatment process and an almost complete elimination of suspensions from the wastewater. 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

A self-forming dynamic membrane bioreactor (SFD MBR) is a cost-effective alternative to conventional MBR, in which the synthetic membrane is replaced by a “cake layer,” an accumulation of the biological suspension over a surface of inert, low-cost support originated by filtration itself. Under optimized conditions, the cake layer is easy to remove and quick to form again, resulting a “dynamic membrane.” The permeate of the SFD MBR has chemo-physical characteristics comparable to those of conventional ultrafiltration-based MBR. In this paper, two nylon meshes with pore sizes of 20 and 50 µm, respectively, were tested in a bench-scale SFD MBR in which an air mass load (AML) was periodically supplied tangentially to the filtration surface to maintain filtration effectiveness. The SFD MBR equipped with 20 µm nylon mesh coupled with 5 min of AML every 4 h showed the best performance, ensuring both a permeate with turbidity values always below 3 NTU and revealing no increases in transmembrane pressure (TMP) with manual maintenance needs. A benchmark test with the only difference of a suction break (relaxation) instead of AML was conducted under identical operating conditions for validation with an already known maintenance strategy. This latter test produced a permeate of very good quality, but it needed frequent TMP increases and consequent manual cleanings, showing that a periodic AML coupled with the use of a 20 µm mesh can be an optimal strategy for long-term operation of SFD MBR. Full article