Bioreactor Culture Research Articles

Markerless mutagenesis enables isoleucine biosynthesis solely from threonine in Methanothermobacter marburgensis.

The autotrophic, hydrogenotrophic methanogen Methanothermobacter marburgensis is one of the best-studied model organisms in the field of thermophilic archaea. The fact that M. marburgensis shows robust growth and scalability in bioreactor systems makes it a highly suitable candidate for industrial-scale bioprocesses. Additionally, the reported study provides the tools for genetic engineering that enable sequential genome modification in M. marburgensis. Scalable bioreactor cultivation, the ability to genetically engineer, and the recent discovery of natural amino acid secretion in M. marburgensis set the cornerstone for the generation of the first cell factories in archaeal biotechnology to economically produce carbon dioxide-derived commodity and high-value chemicals at industrial scale.

Hyperhydricity-Induced Physiological Changes and Catechin Accumulation in Blueberry Hybrids (Vaccinium corymbosum × V. angustifolium)

Hyperhydricity is a significant challenge in the tissue culture of blueberry plantlets, affecting their propagation, survival and quality, which results in economic losses for industrial blueberry micropropagation. The in vitro liquid propagation of two half-highbush blueberry hybrids, HB1 and HB2, showed that a Growtek stationary bioreactor culture system containing a liquid medium exhibited a higher hyperhydricity percentage than a Sigma glass culture system with a semi-solid medium. The percentage of hyperhydricity (75.21 ± 1.89%) and water content (72%) of HB2 was more than that of HB1. A scanning electron microscopy study revealed that hyperhydric plantlets from both genotypes developed slowly, had closed stomata, and displayed enlarged intercellular spaces between the palisade and spongy parenchyma layers. Disrupted vascular bundles, underdeveloped sieve elements and a weak connection between phloem and xylem tissue were also observed in hyperhydric plantlets. An analysis of mesophyll and stem tissues highlighted a compressed adaxial epidermis, which led to compact palisade parenchyma, with irregularly shaped mesophyll cells. Hyperhydric plants showed strong nuclear magnetic resonance (NMR) signals in the aliphatic, aromatic, and sugar regions, specifically at peaks of 2.0, 2.5, 4.0, 4.5, 6.0, and 6.7 ppm. These signals were attributed to the presence of catechin (C15H14O6), a flavonoid compound, suggesting its significant role or accumulation in these plants under hyperhydric conditions. Despite the negative effects of hyperhydricity on commercial propagation, hyperhydric plants were found to contain higher levels of valuable untargeted metabolites, such as β-P-arbutin, chlorogenic acid, quercetin-3-O-glucoside, epicatechin, 2-O-caffeoyl arbutin, various fatty acids, β-glucose, linolenic acid, and acetyl than both in vitro and ex vitro conditions. The enrichment of bioactive compounds in blueberry enhances its antioxidant properties, nutritional profile, and potential health benefits, making them significant for plant defense mechanisms and stress adaptation.

Flux Sampling Suggests Metabolic Signatures of High Antibody-Producing CHO Cells.

Chinese hamster ovary (CHO) cells remain the industry standard for producing numerous therapeutic proteins, particularly monoclonal antibodies (mAbs). However, achieving higher recombinant protein titers remains an ongoing challenge and a fundamental understanding of the cellular mechanism driving improved bioprocess performance remains elusive. To directly address these challenges and achieve substantial improvements, a more in-depth understanding of cellular function within a bioprocess environment may be required. Over the past decade, significant advancements have been made in the building of genome-scale metabolic models (GEMs) for CHO cells, bridging the gap between high information content 'omics data and the ability to perform in silico phenotypic predictions. Here, time-course transcriptomics has been employed to constrain culture phase-specific GEMs, representing the early exponential, late exponential, and stationary/death phases of CHO cell fed-batch bioreactor culture. Temporal bioprocess data, including metabolite uptake and secretion rates, as well as growth and productivity, has been used to validate flux sampling results. Additionally, high mAb-producing solutions have been identified and the metabolic signatures associated with improved mAb production have been hypothesized. Finally, constraint-based modeling has been utilized to infer specific amino acids, cysteine, histidine, leucine, isoleucine, asparagine, and serine, which could drive increased mAb production and guide optimal media and feed formulations.

N-1 semi-continuous transient perfusion in shake flask for ultra-high density seeding of CHO cell cultures in benchtop bioreactors.

One strategy to enhance the production of biological therapeutics is using transient perfusion in the preculture (N-1 stage) to seed the production culture (N stage) at ultra-high cell densities (>10 x 106 viable cells/mL). This very high seeding density improves cell culture performance by shortening the timeline and/or achieving higher final product concentrations. Typically, an N-1 seed train employs bioreactors with alternating tangential flow filtration (ATF) or tangential flow filtration (TFF) perfusion systems or Wave cell bag bioreactor with integrated filtration membrane, which have costs and technical complexity. Here, we propose an alternative method using semi-continuous transient perfusion through media exchange in shake flasks, which is suitable for benchtop-scale intensification process development. Daily media exchange was necessary to prevent nutrient limitations. The observed limitation of maximum viable cell densities (VCD) in various flask sizes was demonstrated to be due to oxygen limitations through the measurements of maximum oxygen transfer rates (OTR) using the sulfite system. By increasing agitation frequency from 200 to 300 RPM, maximum OTR in 500-mL shake flasks was increased by 62.3%, allowing an increase in maximum VCD of 29.6%. However, in 1000-mL shake flasks, an increase in agitation rate resulted in early cell death. After demonstrating that media exchange in shake flasks by centrifugation had no significant impact on cell growth rates, metabolism, and productivity, a benchtop bioreactor was seeded from semi-continuous transient perfusion cell expansion. The ultra-high cell density seeding resulted in a 49.3% increase in space-time-yield (STY) when compared to a standard low seeding density culture.

Screening of Non-Conventional Yeasts on Low-Cost Carbon Sources and Valorization of Mizithra Secondary Cheese Whey for Metabolite Production

The production of microbial metabolites such as (exo)polysaccharides, lipids, or mannitol through the cultivation of microorganisms on sustainable, low-cost carbon sources is of high interest within the framework of a circular economy. In the current study, two non-extensively studied, non-conventional yeast strains, namely, Cutaneotrichosporon curvatus NRRL YB-775 and Papiliotrema laurentii NRRL Y-3594, were evaluated for their capability to grow on semi-defined lactose-, glycerol-, or glucose-based substrates and produce value-added metabolites. Three different nitrogen-to-carbon ratios (i.e., 20, 80, 160 mol/mol) were tested in shake-flask batch experiments. Pretreated secondary cheese whey (SCW) was used for fed-batch bioreactor cultivation of P. laurentii NRRL Y-3594, under nitrogen limitation. Based on the screening results, both strains can grow on low-cost substrates, yielding high concentrations of microbial biomass (>20 g/L) under nitrogen-excess conditions, with polysaccharides comprising the predominant component (>40%, w/w, of dry biomass). Glucose- and glycerol-based cultures of C. curvatus promote the secretion of mannitol (13.0 g/L in the case of glucose, under nitrogen-limited conditions). The lipids (maximum 2.2 g/L) produced by both strains were rich in oleic acid (≥40%, w/w) and could potentially be utilized to produce second-generation biodiesel. SCW was nutritionally sufficient to grow P. laurentii strain, resulting in exopolysaccharides secretion (25.6 g/L), along with dry biomass (37.9 g/L) and lipid (4.6 g/L) production.

Harnessing Apple Cell Suspension Cultures in Bioreactors for Triterpene Production: Transcriptomic Insights into Biomass and Triterpene Biosynthesis.

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.

Model-Based Characterization of the Metabolism of Recombinant Adeno-Associated Virus (rAAV) Production via Human Embryonic Kidney (HEK293) Cells

In this paper, we present a kinetic–metabolic model describing adeno-associated virus (AAV) production via HEK293 cells that encompasses the main metabolic pathways, namely, glycolysis, tricarboxylic acid cycle (TCA), pyruvate fates, the pentose phosphate pathway, anaplerotic reaction, amino acid metabolism, nucleotides synthesis, biomass synthesis, and the metabolic pathways of protein synthesis of the AAV (capsid and Rep proteins). For the modeling, Michaelis–Menten kinetics was assumed to define the metabolic model. A dataset from bioreactor cultures containing metabolite profiles of adeno-associated virus 6 (AAV6) production via triple transient transfection in a low-cell-density culture, including the concentration profiles of glutamine, glutamic acid, glucose, lactate, and ammonium, was utilized for fitting and computing the model parameters. The model that resulted from the adjusted parameters defined the experimental data well. Subsequently, a Sobol-based global sensitivity analysis procedure was applied to determine the most sensitive parameters in the final model.

Tendon Tissue Regeneration With Cell Orientation Using an Injectable Alginate-Cell Cross-linked Gel.

Tendons have a limited blood supply and form inferior scar tissue during repair, which increases the risk of reruptures, causes complications, and limits regenerative capacity. Current methods to repair injured tendon tissue use solid scaffolds, which carry the risk of contamination (infections) and require open surgery for transplantation. Alginate-cell cross-linked gels, which can be applied by a percutaneous injection and transmit mechanical stress to cells via direct cell interaction, could induce tendon tissue regeneration. Controlled laboratory study. A cross-linked gel was prepared to suspend azide-modified mesenchymal stromal cells (MSCs) in a dibenzocyclooctyne-modified branched alginic acid solution. The cross-linked gel was cultured in a bioreactor. In vivo, the Achilles tendon defects of 104 Lewis rats were injected with saline (control group), alginate gel alone (alginate group), alginate gel with MSCs (MSC group), and cross-linked gel (cross-link group). At 2 and 4 weeks postoperatively, histological and biochemical evaluations were performed. The biomechanical properties of repaired tissue were assessed at 4 weeks. In the bioreactor culture, the cell orientation in the cross-linked gel was parallel to the direction of tension. Histological analysis of the cross-link group showed significantly more repaired tendon tissue and improved collagen fiber orientation compared with the alginate group or MSC group. The biomechanical properties of the cross-link group included higher stiffness. The cross-linked gel was injectable at the injury site and was able to induce tissue regeneration with cell-oriented adaptability to the mechanical environment of tissue defects. Intercellular cross-linking technology holds the potential for clinical application as a minimally invasive therapeutic approach that can contribute to the qualitative improvement of tendon tissue regeneration.

Measurement of Oxygen Transfer Rate and Specific Oxygen Uptake Rate of h-iPSC Aggregates in Vertical Wheel Bioreactors to Predict Maximum Cell Density Before Oxygen Limitation

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.

Characterizing the effect of impeller design in plant cell fermentations using CFD modeling

Cultivation of plant cell cultures in conventional bioreactors designed for microbial cells often results in decrease of biomass productivity as compared to that in shake flasks, presumably due to the imbalance between the mass transfer requirements and compromise with cell viability. Hit and trial methods for bioreactor design are generally performed to achieve high biomass productivity in the bioreactor. In this study, a rational approach has been adopted to choose a suitable impeller for Viola odorata cell suspension culture using computational fluid dynamics (CFD). A two-phase CFD model was employed to characterize the non-Newtonian fluid dynamics of the plant cell suspension in a stirred tank reactor using different impeller designs, a setric, Rushton and marine impeller. The simulations were performed adopting Euler-Euler approach for the two-phase flow and dispersed turbulence model. The numerical model was validated with good agreement with experimental determination of volumetric mass transfer coefficient. The impact of impeller design was then investigated on critical process parameters like mixing, oxygen mass transfer and shear. The developed CFD model demonstrated that setric impeller is a suitable choice for V. odorata cell cultivation among the three impellers offering low-shear environment at equivalent velocity magnitudes at reactor bottom with higher cell-lift capabilities which is preferable in high cell-density plant cell cultivations.

Production of Protease Inhibitor With Penicillium sp. - Optimization of the Medium for Growth in Pellet Form and Cytotoxicity Testing.

Penicillium sp. (IBWF 040-09) produces a protease inhibitor that can potentially be used against the main protease of human African trypanosomiasis. Since the target substance is formed intracellularly (under nutrient limitation), the fungal pellet is preferred compared to the free mycelia in bioreactor cultivation. The optimization of the production of protease inhibitor became the main focus of this study. The effects of the concentrations of spores, calcium chloride, and Pluronic F68 were investigated with regard to fungal growth, pellet morphology, and the production of protease inhibitor. The combination of adjusting the spore concentration and adding Pluronic F68 and calcium chloride increased the probability of achieving the desired morphology. This ensured better reproducibility of the production of the target substance by Penicillium sp. (IBWF 040-09) with the bioreactor system used. In addition, the protease inhibitor was tested in a resazurin assay and showed no noticeable cytotoxic effects on peripheral blood mononuclear cells isolated from whole blood cells.

Engineering Halomonas bluephagenesis for pilot production of terpolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyvalerate from glucose.

Engineering Halomonas bluephagenesis for pilot production of terpolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyvalerate from glucose.

Long-term bioreactor cultivation affects dioscin content and the ratio of 25(S)- and 25(R)-protodioscin isomers in the suspension cell culture of Dioscorea deltoidea Wall.

Long-term bioreactor cultivation affects dioscin content and the ratio of 25(S)- and 25(R)-protodioscin isomers in the suspension cell culture of Dioscorea deltoidea Wall.

Advances and Challenges in Modeling Autosomal Dominant Polycystic Kidney Disease: A Focus on Kidney Organoids.

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent hereditary disorder characterized by distinct phenotypic variability that has posed challenges for advancing in-depth research. Recent advancements in kidney organoid construction technologies have enabled researchers to simulate kidney development and create simplified in vitro experimental environments, allowing for more direct observation of how genetic mutations drive pathological phenotypes and disrupt physiological functions. Emerging technologies, such as microfluidic bioreactor culture systems and single-cell transcriptomics, have further supported the development of complex ADPKD organoids, offering robust models for exploring disease mechanisms and facilitating drug discovery. Nevertheless, significant challenges remain in constructing more accurate ADPKD disease models. This review will summarize recent advances in ADPKD organoid construction, focusing on the limitations of the current techniques and the critical issues that need to be addressed for future breakthroughs. New and Noteworthy: This review presents recent advancements in ADPKD organoid construction, particularly iPSC-derived models, offering new insights into disease mechanisms and drug discovery. It focuses on challenges such as limited vascularization and maturity, proposing potential solutions through emerging technologies. The ongoing optimization of ADPKD organoid models is expected to enhance understanding of the disease and drive breakthroughs in disease mechanisms and targeted therapy development.

Neural progenitor cell-derived exosomes in ischemia/reperfusion injury in cardiomyoblasts.

The physiologic relationship between the brain and heart is emerging as a novel therapeutic target for clinical intervention for acute myocardial infarction. In the adult human brain, vestigial neuronal progenitor stem cells contribute to neuronal repair and recovery following cerebral ischemic injury, an effect modulated by secreted exosomes. Ischemia conditioned neuronal cell derived supernatant and experimental stroke has been shown to be injurious to the heart. However, whether unconditioned neuronal progenitor cell derived-exosomes can instead protect myocardium represents a profound research gap. We investigated the effects of unconditioned neural stem cell derived exosomes as post-injury treatment for cardiomyoblasts from three neuronal culture conditions; adherent cultures, neurosphere cultures and bioreactor cultures. Small extracellular vesicles were enriched with serial ultracentrifugation, validated via nanoparticle tracking analysis, transmission electron microscopy and Western blot analysis prior to utilization as post-injury treatment for H9c2 cardiomyoblasts following oxygen and glucose deprivation. LDH assay was used to assess viability and Seahorse XF high-resolution respirometry analyzer to investigate post-injury cardiomyocyte bioenergetics. We found no evidence that unconditioned neural stem cell derived exosomes are cardiotoxic nor cardioprotective to H9c2 cardiomyoblasts following ischemia-reperfusion injury. Based on our findings, utilizing unconditioned neural stem cell derived exosomes as post-injury treatment for other organs should not have adverse effects to the damaged cardiac cells.

Microalgae cultivation in semi-transparent photovoltaic bioreactor for sustainable power generation, wastewater treatment and biodiesel production

Microalgae cultivation in semi-transparent photovoltaic bioreactor for sustainable power generation, wastewater treatment and biodiesel production

Assessment of different nitrogen sources and bioreactor cultivation strategies during growth of Aurantiochytrium limacinum on spruce sugars

Assessment of different nitrogen sources and bioreactor cultivation strategies during growth of Aurantiochytrium limacinum on spruce sugars

Model-based fed-batch cultivation of Viola odorata plant cells exhibiting antimalarial and anticancer activity.

Viola odorata is a medicinal plant used in the indigenous systems of medicine in India, to treat respiratory tract disorders. V. odorata natural plant source is limited in availability. Bioprocess principles can be applied to develop sustainable methods for the commercial production of high-quality V. odorata plant biomass. To this effect, the in vitro culture conditions of V. odorata were rationally optimized to increase the biomass production up to 21.7 ± 0.8g DW L-1 in 12days in shake flasks. In the current study, a modified stirred tank reactor and a balloon-type bubble column reactor were used to improve the biomass production at the batch reactor level. Sufficient nutrient feeding strategies were developed using first principle-based mathematical modelling to overcome substrate inhibition and achieve higher cell density in the reactor. In addition, bioreactor-cultivated biomass extracts (aqueous/alcoholic) were tested for various bioactivities like hemolytic, cytotoxic, anti-inflammatory, and antiplasmodial. Experimental validation of the fed-batch model-predicted strategy resulted in a two-fold enhancement in biomass production (32.2g DW L-1) at the bioreactor level. Biomass extracts showed no hemolytic activity up to 4mgmL-1 concentrations. Further, the stirred tank cultivated biomass extract displayed cytotoxicity against Caco2 - colon carcinoma cell lines, exhibiting an IC50 of 1.5 ± 0.1mgmL-1. In vitro experiments also indicated the anti-inflammatory property in the bioreactor cultivated plant biomass extracts. As a new application, the biomass extracts also demonstrated up to 80% inhibition of malarial parasite growth in vitro. Additionally, when administered alongside artesunate (1.8mgkg-1d-1), the plant extracts (400mgkg-1d-1) effectively controlled parasite growth in vivo. It is to be noted that a first report on fed-batch cultivation of V. odorata cell suspension culture in lab-scale bioreactors and on the antiplasmodial activity of the V. odorata plant extracts. Overall, the bioactive potential of the in vitro-generated plant biomass extracts is similar to that in the natural plant biomass extracts.

Harnessing cell aggregates for enhanced adeno-associated virus manufacturing: Cultivation strategies and scale-up considerations.

The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated. It was, however, found that the spent medium of the HCDP inhibited the transfection of HEK293T cell aggregates, which was confirmed when testing with single-cell suspension culture. The formation of aggregates in shaken multi-well plates was also investigated to develop a screening system using the average power input as a scale-down criterion, which revealed that cell aggregates could be generated in 12-well plates, however with a larger size than in a STR. Taking into account the reported higher rAAV production of adherent cells in comparison with single cells for triple-plasmid transfection, HEK293T cell aggregates can possibly surpass single-cell suspension in space-time rAAV yield. The formation of HEK293T cell aggregates in a STR system offers a promising approach for scaling up and intensifying rAAV production by triple-plasmid transfection, in comparison with traditional 2D scale-up methods.

Production of Recombinant Adeno-Associated Virus Through High-Cell-Density Transfection of HEK293 Cells Based on Fed-Perfusion Culture.

Adeno-associated virus (AAV)-associated gene therapy has been increasingly promising, in light of the drugs progressed to clinical trials or approved for medications internationally. Therefore, scalable and efficient production of recombinant AAV is pivotal for advancing gene therapy. Traditional methods, such as the triple-plasmid transfection of human embryonic kidney 293 cells in suspension culture, have been widely employed but often hampered by low unit yield. In this study, we optimized the cell culture process with high cell density up to 2 × 107 cells/mL by employing a perfusion culture system with centrifugation and medium exchange in shake flasks and perfusion device in bioreactor. Furthermore, we utilized a design of experiments strategy to systematically modulate a series of transfection-related variables including the quantity of plasmid DNA, the DNA-to-polyethylenimine ratio, incubation duration, and the impact of post-transfection feeding strategies on the yield of recombinant AAV (rAAV). Our comprehensive analysis and subsequent optimizations actualized a remarkable unit yield reaching nearly 2 × 1012 vector genomes (vg)/mL. Importantly, the resulting single-cell yield and biological activity were found to be comparable with those obtained from fed-batch cultures, underscoring the efficacy of our approach. Based on these findings, we investigated rAAV yield via high-density suspend culture in bioreactor, particularly focusing on cell aggregation and the use of perfusion technology. Intriguingly, we attempted to elevate the yield of an oversized recombinant coagulation factor VIII AAV843 vector by 3.5-fold, reaching a yield of 1 × 1012 vg/mL. Concurrently, the medium usage rate was only double that of batch feeding, thereby significantly shrinking the upstream cost of rAAV manufacture. In summary, this strategy significantly benefits large-scale AAV production for both commercial and clinical applications.