Cell Culture Production Research Articles

An innovative hybrid modeling approach for simultaneous prediction of cell culture process dynamics and product quality.

The use of hybrid models is extensively described in the literature to predict the process evolution in cell cultures. These models combine mechanistic and machine learning methods, allowing the prediction of complex process behavior, in the presence of many process variables, without the need to collect a large amount of data. Hybrid models cannot be directly used to predict final product critical quality attributes, or CQAs, because they are usually measured only at the end of the process, and more mechanistic knowledge is needed for many classes of CQAs. The historical models can instead predict the CQAs better; however, they cannot directly relate manipulated process parameters to final CQAs, as they require knowledge of the process evolution. In this work, we propose an innovative modeling approach based on combining a hybrid propagation model with a historical data-driven model, that is, the combined hybrid model, for simultaneous prediction of full process dynamics and CQAs. The performance of the combined hybrid model was evaluated on an industrial dataset and compared to classical black-box models, which directly relate manipulated process parameters to CQAs. The proposed combined hybrid model outperforms the black-box model by 33% on average in predicting the CQAs while requiring only around half of the data for model training to match performance. Thus, in terms of model accuracy and experimental costs, the combined hybrid model in this study provides a promising platform for process optimization applications.

A strategy of novel molecular hydrogen-producing antioxidative auxiliary system improves virus production in cell bioreactor

In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture.

A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2′,7′-dichlorodihydrofluorescein diacetate assay

Exposure to xenobiotics can increase the production of reactive oxygen species (ROS). When detoxification organs such as the intestines and liver cannot neutralise these xenobiotics, it can induce oxidative stress and cause damage to tissues. Therefore, cell-based bioassays that indicate intracellular ROS production are a useful screening tool to evaluate the effect of these chemicals. Although flow cytometry is commonly used to measure ROS in cells, many research laboratories in the Global South do not always have access to such specialised instrumentation. Therefore, we describe a sensitive but low-cost method that can easily be used to determine ROS production in vitro. This method employs the fluorogenic dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), which emits fluorescence after being oxidised to a fluorescent derivative. Since the H2DCF-DA bioassay indicates non-specific ROS production it can be used as a marker of overall oxidative stress. This method was validated by exposing human duodenum epithelial adenocarcinoma (HuTu-80) and rat liver epithelial hepatoma (H4IIE-luc) cells to agricultural soil samples.•Production of ROS can be determined in vitro in intestinal and liver cells.•This method is inexpensive and can be easily performed in standard laboratories.•The method provides a tool for the high-throughput screening of environmental samples.

Features of baseline and lipopolysaccharide-induced cytokine secretion in mononuclear leukocyte cultures from patients with the erythema migrans form of acute lyme borreliosis based on clinical parameters

Introduction. Features of cytokine production in mononuclear cell cultures from Lyme borreliosis patients based on clinical data remained poorly studied. The study aim was to estimate the patterns of baseline and lipopolysaccharide-induced cytokine-secretory activity of peripheral blood mononuclear leukocytes from patients with erythema migrans form of acute Lyme borreliosis based on clinical parameters. Materials and methods. Groups of 22 and 12 patients with the diagnoses of mild or moderate severity of monoinfection and co-infection with tick-borne encephalitis of Lyme borreliosis with erythema migrans were examined twice: on week 1 after disease onset and day 14. The control group included 17 healthy donors. Basal and lipopolysaccharide-induced IL-6, IL-10, and TNFα secretion levels were assessed in mononuclear leukocyte culture supernatants applying enzyme immunoassay. Statistical analysis was performed by using the Mann–Whitney U-test, Wilcoxon test, and Spearman’s rank correlation. Results. The group of moderate severity patients was clinically distinguished by severer fever and intoxication manifestations. At the disease onset, the basal TNFα, IL-6 and IL-10 secretion levels in the moderate severity patient cultures were significantly higher than in those of the other groups. After antibiotics treatment, the baseline TNFα and IL-10 levels tended to decrease. At the onset, lipopolysaccharide-induced cultures from the moderate severity patients showed significantly suppressed TNFα production and increased IL-10 secretion as compared to the other groups. Lipopolysaccharide-induced IL-6 secretion in the moderate vs. mild severity group supernatants was significantly lower. In dynamics, the induced TNFα levels in the moderate severity patients were increased to the magnitude exceeding that in the controls. Positive correlations between the IL-6 and TNFα basal levels and maximum body temperature or the C-reactive protein serum concentrations were revealed in the patients. Induced TNFα levels showed negative correlations with fever levels or with IL-10 secretion. Conclusions. It was demonstrated that basal TNFα, IL-6 and IL-10 secretion levels in the mononuclear cell cultures of acute Lyme borreliosis patients increased with the increasing disease severity. Suppression of lipopolysaccharide-induced TNFα production in the moderate severity patient cultures was presumably associated with the regulatory cytokine IL-10 effects.

Cell culture product quality attribute prediction using convolutional neural networks and Raman spectroscopy.

Advanced process control in the biopharmaceutical industry often lacks real-time measurements due to resource constraints. Raman spectroscopy and Partial Least Squares (PLS) models are often used to monitor bioprocess cultures in real-time. In spite of the ease of training, the accuracy of the PLS model is impacted if it is not used to predict quality attributes for the cell lines it is trained on. To address this issue, a deep convolutional neural network (CNN) is proposed for offline modeling of metabolites using Raman spectroscopy. By utilizing asymmetric least squares smoothing to adjust Raman spectra baselines, a generic training data set is created by amalgamating spectra from various cell lines and operating conditions. This data set, combined with their derivatives, forms a two-dimensional model input. The CNN model is developed and validated for predicting different quality variables against measurements from various continuous and fed-batch experimental runs. Validation results confirm that the deep CNN model is an accurate generic model of the process to predict real-time quality attributes, even in experimental runs not included in the training data. This model is robust and versatile, requiring no recalibration when deployed at different sites to monitor various cell lines and experimental runs.

Endothelial lipid droplets suppress eNOS to link high fat consumption to blood pressure elevation.

Metabolic syndrome, today affecting more than 20% of the US population, is a group of 5 conditions that often coexist and that strongly predispose to cardiovascular disease. How these conditions are linked mechanistically remains unclear, especially two of these: obesity and elevated blood pressure. Here, we show that high fat consumption in mice leads to the accumulation of lipid droplets in endothelial cells throughout the organism and that lipid droplet accumulation in endothelium suppresses endothelial nitric oxide synthase (eNOS), reduces NO production, elevates blood pressure, and accelerates atherosclerosis. Mechanistically, the accumulation of lipid droplets destabilizes eNOS mRNA and activates an endothelial inflammatory signaling cascade that suppresses eNOS and NO production. Pharmacological prevention of lipid droplet formation reverses the suppression of NO production in cell culture and in vivo and blunts blood pressure elevation in response to a high-fat diet. These results highlight lipid droplets as a critical and unappreciated component of endothelial cell biology, explain how lipids increase blood pressure acutely, and provide a mechanistic account for the epidemiological link between obesity and elevated blood pressure.

Intensified Influenza Virus Production in Suspension HEK293SF Cell Cultures Operated in Fed-Batch or Perfusion with Continuous Harvest.

Major efforts in the intensification of cell culture-based viral vaccine manufacturing focus on the development of high-cell-density (HCD) processes, often operated in perfusion. While perfusion operations allow for higher viable cell densities and volumetric productivities, the high perfusion rates (PR) normally adopted-typically between 2 and 4 vessel volumes per day (VVD)-dramatically increase media consumption, resulting in a higher burden on the cell retention device and raising challenges for the handling and disposal of high volumes of media. In this study, we explore high inoculum fed-batch (HIFB) and low-PR perfusion operations to intensify a cell culture-based process for influenza virus production while minimizing media consumption. To reduce product retention time in the bioreactor, produced viral particles were continuously harvested using a tangential flow depth filtration (TFDF) system as a cell retention device and harvest unit. The feeding strategies developed-a hybrid fed-batch with continuous harvest and a low-PR perfusion-allowed for infections in the range of 8-10 × 106 cells/mL while maintaining cell-specific productivity comparable to the batch control, resulting in a global increase in the process productivity. Overall, our work demonstrates that feeding strategies that minimize media consumption are suitable for large-scale influenza vaccine production.

Influence of heat treatments on the functionality of soy protein hydrolysates in animal cell cultures.

Soy protein hydrolysates enhance integral viable cell density (IVCD) and recombinant protein production (Immunoglobulin, IgG) in cell cultures, but their functionality varies from batch-to-batch. This is undesirable since it affects both quantity and characteristics of the recombinant proteins. It is hypothesized that the variability of hydrolysates is due to variations in meal and hydrolysate processing treatments. To study this, hydrolysates were produced from meals heated at 121°C/0-120min. The heating decreased free amino acid and reducing monosaccharide contents in meals (0.72-0.27% and 3.3-2.6%) and hydrolysates (14.7-7.1% and 16.9-7.9%). Dry heating introduced large variation in the IVCD ((115-316%), but additional heating in suspension reduced it (131-159%). The decrease in IVCD variation corresponded with decreased variation in carboxymethyl-lysine (CML) and lysinoalanine (LAL) contents. Thus, meal and hydrolysate processing induced substantial variation in hydrolysate functionality. It is therefore critical to establish strict process controls for meal and hydrolysate production to ensure consistency.

Toxicological evaluation of ginsenoside-rich cell culture biomass of Panax japonicus produced in a large-scale bioreactor system

An environmentally friendly, resource-efficient substitute for the production of vegetative biomass and the bioactive components of medicinal plants is provided by novel biotechnology-based suspension cells cultured in bioreactors. Despite the cultured biomass is produced in the controlled conditions, it requires detailed chemical analysis and toxicological evaluation before being approved for human consumption. This study reports the results of biochemical, toxicological, and elemental composition analysis of the suspension cell culture of a medicinal species Panax japonicus (T. Nees) C.A. Mey. cultivated in a large-scale bioreactor system. For the scaling up the process of cultivation, the suspension culture was grown sequentially in a series of laboratory (20-L), pilot (75-L), and industrial (630-L) bioreactors. The biomass productivity of cell culture was comparable or higher than of wild plants or in vitro cultured hairy roots reported in the literature. The resulting cell biomass was analyzed for ginsenoside contents using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC–ESI–MS). For the elemental composition of cells, inductively coupled argon plasma mass spectrometry (ICP–MS) was used. In addition, the biomass was evaluated for acute toxicity and skin sensibilization effects on in vivo animal models. The analyses confirmed that suspension cells cultured in bioreactors accumulate a broad spectrum of ginsenosides and their malonylated derivatives at a total concentration of 7.54 % (w/w) based on dry weight and contain essential macro- (K, Ca, Mg, Na) and micro- (Zn, Mn, Fe, B, Al, Cu) elements in dietary-safe concentrations. Acute toxicity tests using the administered doses of 2000 and 5000 mg dry biomass per kg animal weight resulted in no changes in animal organ weights and no or minor changes in hematological and biochemical parameters of blood. No skin irritation or sensitizing effects were observed. Therefore, bioreactor cultivation of P. japonicus cell suspension is highly productive and provides a ginsenoside-rich and non-toxic cell biomass which is a potentially valuable component of functional foods, food additives, and natural health products.

Characterization of AT-02, a pan-amyloid-binding peptide fusion immunoglobulin with high binding potency, complement activation, and immune cell stimulation

Abstract Background Cardiac amyloidosis is an ominous manifestation of systemic amyloidosis, notably in AL and ATTR-associated types, resulting in a restrictive hypertrophic cardiomyopathy with electrical conduction and atrioventricular strain dysfunction (1). Amyloid is not naturally cleared by the immune system due to the presence of extracellular matrix components in the deposits (2). However, recruiting and stimulating the innate immune system, notably phagocytic macrophages, is a promising strategy to effect amyloid removal. To facilitate this, we have generated AT-02, a humanized IgG1 incorporating the pan-amyloid-binding peptide, p5R (3), at the light chain C-terminus. This reagent binds diverse types of amyloid via the peptide interactions and retains immunomodulatory capacity. Purpose The goal of these studies was to characterize the bioactivity of AT-02 that underlies its potential therapeutic activity. Methods AT-02 was produced from a CHO pool using a perfusion cell culture production process, purified by Protein A, anion exchange, and cation exchange chromatographies, and characterized by mass spectrometry and SDS-capillary electrophoresis. AT-02 was biotinylated and used for immunohistochemical detection of both AL and ATTR amyloidosis in formalin fixed tissues (Figure 1A). Binding to heparin (as a surrogate for amyloid-associated hypersulfated heparan sulfate proteoglycans) and synthetic AL fibrils was studied using both ELISA and surface plasmon resonance assays (Figure 1B). Activation of complement following binding to amyloid substrates was determined using a C5b9 liberation assay in the presence of human plasma. The effect of AT-02 on amyloid fibril growth was determined using a fluorescence fibril elongation assay. In vivo clearance of amyloid and cell activation was demonstrated using a murine model of AL amyloidoma. Results AT-02 bound human cardiac amyloid in tissue sections with high specificity and intensity. The potency for heparin and synthetic amyloid fibrils, as well as human cardiac amyloid extracts, was sub-nanomolar in the ELISA assay with kinetics consistent with a high affinity antibody. In the presence of plasma, complement activation of amyloid-bound AT-02 was significantly higher as compared to controls, and the reagent inhibited synthetic amyloid fibril growth by ∼40%. Treatment of human amyloid with AT-02 expedited phagocytosis by murine macrophages in vivo and enhanced the intensity of immune response to the implanted amyloid. Conclusion Clearance of cardiac amyloid is a significant clinical unmet need for patients with systemic amyloidosis. The humanized IgG1-peptide fusion, AT-02, potently binds cardiac amyloid and may serve as an opsonin and induce macrophage-mediated phagocytosis and clearance of cardiac amyloid deposits.

Enabling mRNA Therapeutics: Current Landscape and Challenges in Manufacturing.

Recent advances and discoveries in the structure and role of mRNA as well as novel lipid-based delivery modalities have enabled the advancement of mRNA therapeutics into the clinical trial space. The manufacturing of these products is relatively simple and eliminates many of the challenges associated with cell culture production of viral delivery systems for gene and cell therapy applications, allowing rapid production of mRNA for personalized treatments, cancer therapies, protein replacement and gene editing. The success of mRNA vaccines during the COVID-19 pandemic highlighted the immense potential of this technology as a vaccination platform, but there are still particular challenges to establish mRNA as a widespread therapeutic tool. Immunostimulatory byproducts can pose a barrier for chronic treatments and different production scales may need to be considered for these applications. Moreover, long-term storage of mRNA products is notoriously difficult. This review provides a detailed overview of the manufacturing steps for mRNA therapeutics, including sequence design, DNA template preparation, mRNA production and formulation, while identifying the challenges remaining in the dose requirements, long-term storage and immunotolerance of the product.

The Influenza B Virus Victoria and Yamagata Lineages Display Distinct Cell Tropism and Infection-Induced Host Gene Expression in Human Nasal Epithelial Cell Cultures.

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus-Yamagata and Victoria-circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses.

Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in Camptotheca.

Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

Unprecedented Neoverrucosane and Cyathane Diterpenoids with Anti-Neuroinflammatory Activity from Cultures of the Culinary-Medicinal Mushroom Hericium erinaceus.

The culinary medicinal mushroom Hericium erinaceus holds significant global esteem and has garnered heightened interest within increasingly ageing societies due to its pronounced neuroprotective and anti-neuroinflammatory properties. Within this study, two novel diterpenes, 16-carboxy-13-epi-neoverrucosane (1) and Erinacine L (2); three known xylosyl cyathane diterpenoids, Erinacine A (3), Erinacine C (4), and Erinacine F (5); and four lanostane-type triterpenoids, and three cyclic dipeptides (10-12), in addition to orcinol (13), were isolated from the rice-based cultivation medium of H. erinaceus. Their structures were determined by NMR, HR-ESI-MS, ECD, and calculated NMR. Compound 1 marks a pioneering discovery as the first verrucosane diterpene originating from basidiomycetes, amplifying the scope of fungal natural product chemistry, and the intricate stereochemistry of Compound 5 has been comprehensively assessed for the first time. Compounds 2-5 not only showed encouraging neurotrophic activity in rat adrenal pheochromocytoma PC-12 cells, but also significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia cell cultures with IC50 values as low as 5.82 ± 0.18 μM. To elucidate the mechanistic underpinnings of these bioactivities, molecular docking simulation was used to analyze and support the interaction of 1 and 2 with inducible NO synthase (iNOS), respectively. In particular, compound 2, a cyathane-xyloside containing an unconventional hemiacetal moiety, is a compelling candidate for the prevention of neurodegenerative diseases. In summation, this investigation contributes substantively to the panorama of fungal diterpene structural diversity, concurrently furnishing additional empirical substantiation for the role of cyathane diterpenes in the amelioration of neurodegenerative afflictions.

Regulation of serotonin production by specific microbes from piglet gut

BackgroundSerotonin is an important signaling molecule that regulates secretory and sensory functions in the gut. Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models. However, how gut microbes regulate intestinal serotonin production in piglets remains vague. To investigate the relationship between microbiota and serotonin specifically in the colon, microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion. Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investigate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells.ResultsAntibiotic infusion increased quantities of Lactobacillus amylovorus (LA) that positively correlated with increased serotonin concentrations in the colon, while no effects observed for Limosilactobacillus reuteri (LR). To understand how microbes regulate serotonin, representative strains of LA, LR, and Streptococcus alactolyticus (SA, enriched in feces from prior observation) were selected for cell culture studies. Compared to the control group, LA, LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1 (TPH1) expression and promoted serotonin production in IPEC-J2 cells, while in RIN-14B cells only LA exerted similar action. To investigate potential mechanisms mediated by microbe-derived molecules, microbial metabolites including lactate, acetate, glutamine, and γ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacterial supernatant. Among these metabolites, acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1. Similar effects were also recapitulated when treating the cells with AR420626, an agonist targeting free fatty acid receptor 3.ConclusionsOverall, these results suggest that Lactobacillus amylovorus showed a positive correlation with serotonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures. These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host, which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis.

Tuning the Porosity of Dextran Microgels with Supramacromolecular Nanogels as Soft Sacrificial Templates.

Hydrogels, as well as colloidal hydrogels (microgels), are important materials for a large variety of applications in the biomedical field. Microgels with a controlled pore size (meso- and macropores) are required for efficient nutrient support, modulation of cell adhesion, removal of metabolic products in cell cultures, and probiotic loading. Common microgel fabrication techniques do not provide sufficient control over pore sizes and geometry. In this work, the natural polysaccharide dextran modified with methacrylate groups is used to synthesize highly monodisperse meso- and macroporous microgels in a size range of100-150µm via photo cross-linking in microfluidic droplets. The size of mesopores is varied by the concentration of dextran methacrylate chains in the droplets (50-200gL-1 ) and the size of macropores is regulated by the integration of pH-degradable supramacromolecular nanogels with diameters of 300 and 700nm as sacrificial templates. Using permeability assays combined with confocal laser scanning microscopy, it is demonstrated that functional dextran-based microgels with uniform and defined pores could be obtained.

Proapoptotic effects of halogenated bis-phenylthiourea derivatives in cancer cells.

New halogenated thiourea derivatives were synthesized via the reaction of substituted phenylisothiocyanates with aromatic amines. Their cytotoxic activity was examined in in vitro studies against solid tumors (SW480, SW620, PC3), a hematological malignance (K-562), and normal keratinocytes (HaCaT). Most of the compounds were more effective against SW480 (1a, 3a, 3b, 5j), K-562 (2b, 3a, 4a), or PC3 (5d) cells than cisplatin, with favorable selectivity. Their anticancer mechanisms were studied by Annexin V-fluorescein-5-isothiocyanate apoptosis, caspase-3/caspase-7 assessment, cell cycle analysis,interleukin-6 (IL-6) release inhibition, and reactive oxygen species(ROS) generation assay. Thioureas 1a, 2b, 3a, and4a were the most potent activators of early apoptosis in K-562 cells, and substances 1a, 3b, 5j triggered late-apoptosis or necrosis in SW480 cells. This proapoptotic effect was proved by the significant increase of caspase-3/caspase-7 activation. Cell cycle analysis revealed that derivatives 1a, 3a, 5j increased the number of SW480 and K-562 cells in the sub-G1 and/or G0/G1 phases, and one evoked cycle arrest at the G2 phase. The most potent thioureas inhibited IL-6 cytokine secretion from PC3 cells and both colon cancer cell lines. Apoptosis-inducing compounds also increased ROS production in all tumor cell cultures, which may enhance their anticancer properties.

WAVE-based intensified perfusion cell culture for fast process development.

This study was to evaluate the feasibility of using a rocking type bioreactor system, specifically the WAVE 25, in an intensified perfusion culture (IPC) mode for monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell line. A disposable perfusion bag with floating membrane was used in the IPC process. An automated filter switching system was employed to continuously clarify the harvested post-membrane culture fluid. The overall cell culture performance, product titer, and quality were compared to those of a typical IPC conducted in a bench-top glass bioreactor. The results showed that the overall trends of cell culture performance, product titer (accumulated harvest volumetric titer) were similar to those of the typical IPC conducted in the glass bioreactor, while the purity related quality were slightly better than the typical run. Furthermore, with the automated filter switching system, the harvested post-membrane culture fluid could be continuously clarified, making it suitable for downstream continuous chromatography. The study demonstrated the feasibility of using the WAVE-based rocking type bioreactor in the N stage IPC process, which increases the flexibility in adopting IPC process. The results suggest that the rocking type bioreactor system could be a viable alternative to traditional stirred tank bioreactors for perfusion culture in the biopharmaceutical industry.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

A fluorescence-based opto-mechatronic screening module (OMSM) for automated 3D cell culture workflows

Cost-effective automated solutions for hydrogel-based 3D cell culture workflows are required to increase throughput, reproducibility and user-independent results. 3D bioprinters can handle viscous biomaterials, however, throughput is low, they are limited to specific biomaterials, and lack post-processing and analysis methods. Conversely, liquid handling robots have higher throughput but miss viscous materials handling capabilities. Furthermore, commercial systems are not only expensive but mostly not ‘open-source’ and hence are not adaptable to specific experimental requirements.The aim of this study was the implementation of an opto-mechatronic screening module (OMSM) for automated 3D cell culture workflows from production to analysis into a previously developed biomanufacturing workstation. An analysis module with a fluorescence wide-field microscope and a motorised XYZ stage was engineered to transport tissue-culture plates from the storage rack of the workstation to the custom-made microscope and to assess fluorescence-based cell parameters. The microscope has two fluorescence channels and a resolution greater than 228 lp/mm. The XYZ-stage achieved an accuracy of 0.8 μm in X-direction (repeatability 2.4 μm) and 1.4 μm in Z-direction (repeatability <0.1 μm). Viability assays and drug-response studies were conducted on 2D and 3D prostate cancer gelatin-methacryloyl hydrogel models demonstrating its capabilities comparable to a conventional fluorescence microscope. Altogether, the integration of an opto-mechatronic screening module into the biomanufacturing workstation enables automated, reproducible, and user-independent production, imaging and analysis of hydrogel-based 3D cell cultures.