High Cell Density Cultivation Research Articles

A holistic approach for process intensification of nicotinamide mononucleotide production via high cell density cultivation under exponential feeding strategy

Nicotinamide mononucleotide (NMN) subsists in all living organisms and has drawn tremendous attention as a nutraceutical and pharmaceutical product for several diseases such as Alzheimer’s, cancer, aging, and vascular dysfunction. Here, NMN was produced intracellularly in a high cell density bioreactor using an engineered Escherichiacoli strain via exponential feeding of co-substrates. Fed-batch culture via exponential feeding of co-substrate (glucose) and continuous feeding of substrate (nicotinamide) were performed using different cumulative nicotinamide concentrations. The highest concentration of 19.3 g/L NMN with a dry cell weight of 117 g/L was acquired from a cumulative nicotinamide concentration of 7.2 g/L with a conversion of 98 % from nicotinamide in 28 h. Further, liquid chromatography-mass spectrometry analysis validated the NMN production. This approach will be beneficial in achieving simultaneously low cost and ensuring high quality and quantity of NMN production.

Bioprocess development to produce a hyperthermostable S-methyl-5'-thioadenosine phosphorylase in Escherichia coli.

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5'-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1-90.1 g L-1 were observed together with an overproduction of ApMTAP in a 1.9%-3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg-1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch

Enhanced production of nicotinamide mononucleotide by high cell density culture of engineered Escherichia coli

Nicotinamide mononucleotide (NMN), an intermediate product of NAD+ biosynthesis, is commonly found intracellularly in living species. Recently, NMN has attracted great interest for the treatment of different diseases. Here, we developed a high cell density fed-batch culture system to produce NMN at higher concentrations using an engineered E. coil strain. Fed-batch culture using linear feeding strategy exhibited 9.38 ± 0.8, 14.8 ± 0.56, and 5.04 ± 0.21 g/L NMN concentrations, and 59.5 ± 0.44, 68.8 ± 0.49, and 36.2 ± 0.32 g/L dry cell weights, using different concentrations of nicotinamide (NAM) of 3, 5, and 10 g/L, respectively. A maximum NMN concentration of 17.2 ± 1.3 g/L with a productivity of 0.956 g/L/h was achieved using two intermittent additions of 5 g/L NAM after 6 h and 17 h. NMN obtained from the fermentation broth was purified using size exclusion chromatography, generating a yield of 53.9 % NMN. Liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis was additionally used to validate the purity of NMN after purification. The fed-batch fermentation process applied in this study, using cheaper feed-stocks, demonstrated a remarkable enhancement in the production of therapeutically important NMN, making this a promising strategy for industrial-scale production of NMN.

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia.

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O2, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O2 permeability. High flow rates of 520μLmin-1 prevented biofilm sloughing in 1m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O2 concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8gm-2 and, thus, a successful CBR scale-up by a factor of 25. Practical application: Cyanobacteria use light energy to upgrade CO2, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O2 accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.

Continuous preparation of low-phenylalanine formulations by treatment of edible protein with immobilized phenylalanine ammonia lyase

Dietary restriction of phenylalanine is the predominant treatment for phenylketonuria. However, the currently available medicated formulations depend on an arduous and unsustainable chemical extraction procedure, which inflates the product’s price. Here, we describe a method that uses immobilized phenylalanine ammonia lyase (PAL) and offers a sustainable solution to generate a low-phenylalanine protein supplement for phenylketonuria patients. This enzyme deaminates phenylalanine to trans-cinnamic acid. Here, we optimized the high cell density cultivation of a recombinant Escherichia coli strain harboring Anabaena variabilis PAL (AvPAL) and achieved protein titers greater than 400 mg/l. This AvPAL protein was immobilized on multiwall carbon nanotubes, and the bioconjugate retained > 50 % activity after ten successive cycles with significantly improved thermostability. Batch deamination with the bioconjugate showed an 80 % reduction in the total phenylalanine content of soya protein hydrolysate. In a continuous packed bed column, > 98 % of the phenylalanine content was depleted at a feed flow rate of 0.1 ml/min, resulting in a space-time yield of cinnamate of 6.65 g/l/h. In a 72-h continuous run, the bioconjugate lost only 2 % of its activity with an operational inactivation constant of 3.9 × 10−5 min−1. This simple covalent immobilization method offers the potential to generate low-phenylalanine diet for phenylketonuria patients.

Targeted gene manipulation of Leloir pathway genes for the constitutive expression of β-galactosidase and its transgalactosylation product galacto-oligosaccharides from Kluyveromyces lactis GG799 and knockout strains

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical industry. At present, production of GOS involves the enzymatic transformation of lactose by transgalactosylation using β-galactosidase. The yeast Kluyveromyces lactis can utilize lactose as its carbon and energy source. In this species lactose is hydrolyzed by an intracellular β-galactosidase (EC 3.2.1.23) which is induced by its substrate and related compounds like galactose. The molecular details of gene regulation in kluyveromyces lactis, we have used multiple knockout approaches to study the constitutive expression by which galactose induces β-galactosidase. The present study involved carrying out to a method of enhancing the constitutive expression of β-galactosidase through galactose induction and its trans-galactosylation reaction for the production of galacto-oligosaccharides (GOS) in Kluyveromyces lactis (K. Lactis) by applying a knockout based approach on Leloir pathway genes based on fusion-overlap extension polymerase chain reaction and transformation into its genome. The k.lactis strain subjected to Leloir pathway genes knockout, resulted in the accumulation of galactose intracellularly and this internal galactose acts as an inducer of galactose regulon for constitutive expression of β-galactosidase at early stationary phase was due to the positive regulatory function of mutant gal1p, gal7p and both. These resulted strains used for trans-galactosylation of lactose by β – galactosidase is characterized for the production of galacto-oligosaccharides. Galactose-induced constitutive expression of β-galactosidase during the early stationary phase of knockout strains was analysed qualitatively & quantitatively. The activity of β-galactosidase of wild type, gal1z, gal7k and gal1z & gal7k strains were 7, 8, 9 and 11 U/ml respectively using high cell density cultivation medium. Based on these expression differences in β-galactosidase, the trans-galactosylation reaction for GOS production and percentage yield of GOS were compared at 25% w/v of lactose. The percentage yield of GOS production of wild type, Δgal1z Lac4+, Δgal7k Lac4++ and Δgal1z Δgal7k Lac4+++mutants strains were 6.3, 13, 17 and 22 U/ml, respectively. Therefore, we propose that the availability of galactose can be used for constitutive over expression of β – galactosidase in Leloir pathway engineering applications and also for GOS production. Further, increased expression of β – galactosidases can be used in dairy industry by-products like whey to produce added value products such as galacto-oligosaccharides.

A quantitative meta-analysis comparing cell models in perfused organ on a chip with static cell cultures

As many consider organ on a chip for better in vitro models, it is timely to extract quantitative data from the literature to compare responses of cells under flow in chips to corresponding static incubations. Of 2828 screened articles, 464 articles described flow for cell culture and 146 contained correct controls and quantified data. Analysis of 1718 ratios between biomarkers measured in cells under flow and static cultures showed that the in all cell types, many biomarkers were unregulated by flow and only some specific biomarkers responded strongly to flow. Biomarkers in cells from the blood vessels walls, the intestine, tumours, pancreatic island, and the liver reacted most strongly to flow. Only 26 biomarkers were analysed in at least two different articles for a given cell type. Of these, the CYP3A4 activity in CaCo2 cells and PXR mRNA levels in hepatocytes were induced more than two-fold by flow. Furthermore, the reproducibility between articles was low as 52 of 95 articles did not show the same response to flow for a given biomarker. Flow showed overall very little improvements in 2D cultures but a slight improvement in 3D cultures suggesting that high density cell culture may benefit from flow. In conclusion, the gains of perfusion are relatively modest, larger gains are linked to specific biomarkers in certain cell types.

Design and characterization of a novel perfusion reactor for biopharmaceuticals production

The demand for commercially valuable biopharmaceuticals able to treat different immunopathological diseases has accelerated in the last few decades. In recent years the industry has shifted the preferred mode of operation towards continuous strategies and has favoured the adoption of perfusion approaches for the cell culture step. In this mode successful perfusion process examples have reached up to 10-fold higher cell densities and product titres than fed-batch, while maintaining product quality at reduced costs. Perfusion processes are operated at high cell densities where cells are retained, while product and waste are continuously removed. This leads to different requirements in flow and mixing in comparison to lower cell density operations, potentially influencing the cellular production performance and therefore product quality. As the pharmaceutical industry is highly regulated, ensuring homogeneity in the bulk throughout the process is critical, while oxygen and mixing requirements of high cell density cultures must be continuously met. This paper presents the design and characterization of a novel 250 mL stirred tank reactor (STR) developed to work in perfusion mode. The results presented include the experimental measurement of the power consumption, the mixing analysis of the flow within the bioreactor which informs design consideration and scaling efforts and the biological data of the perfusion stage with an industrially relevant cell line producing an IgG monoclonal antibody. The characterization allows the operating window of the reactor to be established, resulting in increased productivity of intensified cultures.

Cultivation of the polyextremophile Cyanidioschyzon merolae 10D during summer conditions on the coast of the Red Sea and its adaptation to hypersaline sea water.

The west coast of the Arabian Peninsula borders the Red Sea, a water body which maintains high average temperatures and increased salinity compared to other seas or oceans. This geography has many resources which could be used to support algal biotechnology efforts in bio-resource circularity. However, summer conditions in this region may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiophyceae are a class of polyextremophilic red algae that natively inhabit acidic hot springs. C. merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at elevated temperatures and acidic pH. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, β-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme coastal desert summer environments.

Increasing gene dosage and chaperones co-expression facilitate the efficient dextranase expression in Pichia pastoris

Dextranase (DEX) is an important hydrolase in the food industry, which can degrade the dextran into low-molecular-weight polysaccharides. However, its commercial applications are limited by ineffective heterologous recombination system. Here, we build an efficient Pichia pastoris system for DEX following two different engineering strategies. First, through increasing the gene dosage, the DEX activity increased from 40.13 U/mL to 121.02 U/mL. The up-regulation of the endoplasmic reticulum stress gene ERO1 was detected by the qPCR. Since chaperones are involved in the protein folding and modification, co-expression of chaperones was applied to relieve the endoplasmic reticulum stress and facilitated further increase in DEX activity to 164.78 U/mL. Then, high cell density cultivation of P. pastoris was performed in a 5 L fermenter (cell dry weight reached 105.37 g/L), resulting in the DEX activity to 619.78 U/mL. Finally, under the conditions of 25 °C, pH 5.0, 5% sorbitol as co-substrates, and a feed rate of 7.2 mL/h, a DEX yield of 3257.23 U/mL was obtained through 5-L fed-batch fermentation. DCFH-DA staining showed that the reactive oxygen species level decreased by 68.83% due to sorbitol co-feeding. Our study thus represents an important step for building a robust P. pastoris expression system for recombinant DEX production.

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures.

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%-30%) and productivity (~30%). A threshold PF-68 concentration of 5g/L for high-density cell culture (up to 100 × 106 cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.

High Cell-Density Cultivation of Methylococcus capsulatus Bath for Efficient Methane-Derived Mevalonate Production.

The engineered Methylococcus capsulatus Bath presents a promising approach for converting methane, a potent greenhouse gas, into valuable chemicals. High cell-density culture (HCDC) is necessary for high-titer growth-associated bioproducts, but it often requires time-consuming and labor-intensive optimization processes. In this study, we aimed to achieve efficient HCDC of M. capsulatus Bath by measuring the residual nutrient levels during bioreactor operations and analyzing the specific uptake of each medium component. By controlling the concentrations of nutrients, particularly calcium and phosphorus via intermittent feeding, we achieved a high cell density of 28.2 g DCW/L and a significantly elevated production of mevalonate at a concentration of 1.8 g/L from methane. Our findings demonstrate that the methanotroph HCDC approach presented herein offers a promising strategy for promoting sustainable development, with an exceptional g-scale production titer for value-added synthetic biochemicals.

Zinc aspartate induces proliferation of resting and antigen-stimulated human PBMC under high-density cell culture condition

BackgroundZinc, one of the most important essential trace elements in the human body, regulates a wide range of cellular functions of immune cells, such as proliferation, differentiation and survival. Zinc deficiency affects both the innate and adaptive immune system. Zinc supplementation was discussed as possible therapy for infectious diseases and T cell-mediated autoimmune diseases. However, the influence of commercial zinc preparations on proliferation and cytokine production of resting and antigen-stimulated peripheral blood mononuclear cells (PBMC) has not yet been completely investigated. MethodsHere, we examined whether zinc aspartate (Unizink®), an approved drug to treat zinc deficiency in patients, induces proliferation, cytokine production, and induction of apoptosis/caspase 3/7 activity of resting PBMC under high-density cell culture condition. In addition, we performed antigen-specific proliferation experiments, where PBMCs of healthy donors vaccinated against Influenza A (H1N1) and/or SARS-CoV-2 were stimulated with Influenza A (H1N1) peptides or SARS-CoV-2 peptides as well as the Mixed Lymphocyte Culture (MLC) in the presence of increasing concentrations of zinc aspartate. ResultsWe observed a dose-dependent enhancement of proliferation and induction of cytokine production (IFN-γ, IL-5, GM-CSF and CXCL10) of resting PBMC in presence of zinc aspartate. The number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis steadily decreased in presence of zinc aspartate. Moreover, zinc aspartate was capable of stimulating antigen-specific PBMC proliferation using MLC or influenza A (H1N1) and SARS-CoV-2 peptides in both a dose-dependent and a donor-specific manner. In the absence of zinc aspartate, we clearly could discriminate two groups of responders: low and high responders to antigenic stimulation. The addition of increasing concentration of zinc aspartate significantly stimulated the proliferation of PBMC from low responders, but not from high responders. ConclusionTaken together, our results suggest that zinc aspartate induces the proliferation of resting and antigen-stimulated PBMCs under high-density cell culture conditions. Thus, zinc might represent a supportive treatment in patients suffering from infectious diseases.

Cellular Contact Guidance on Liquid Crystalline Networks with Anisotropic Roughness.

Cell contact guidance is widely employed to manipulate cell alignment and differentiation in vitro. The use of nano- or micro-patterned substrates allows efficient control of cell organization, thus opening up to biological models that cannot be reproduced spontaneously on standard culture dishes. In this paper, we explore the concept of cell contact guidance by Liquid Crystalline Networks (LCNs) presenting different surface topographies obtained by self-assembly of the monomeric mixture. The materials are prepared by photopolymerization of a low amount of diacrylate monomer dissolved in a liquid crystalline solvent, not participating in the reaction. The alignment of the liquid crystals, obtained before polymerization, determines the scaffold morphology, characterized by a nanometric structure. Such materials are able to drive the organization of different cell lines, e.g., fibroblasts and myoblasts, allowing for the alignment of single cells or high-density cell cultures. These results demonstrate the capabilities of rough surfaces prepared from the spontaneous assembly of liquid crystals to control biological models without the need of lithographic patterning or complex fabrication procedures. Interestingly, during myoblast differentiation, also myotube structuring in linear arrays is observed along the LCN fiber orientation. The implementation of this technology will open up to the formation of muscular tissue with well-aligned fibers in vitro mimicking the structure of native tissues.

High cell density culture of Neochloris oleoabundans in novel horizontal thin-layer algal reactor: Effects of localized aeration, nitrate concentration and mixing frequency

A horizontal thin-layer algal reactor was designed, constructed and tested for high cell density culture of an oil-rich green alga, Neochloris oleoabundans, at a thickness of 6 mm. A hollow fiber membrane sparger was used for localized aeration with CO2-enriched air, which allowed control of culture pH at a narrow range of pH 7.0–7.5. The optimal nitrate concentration under the investigated conditions was 345 mg-N/L with a cell density of ∼6.6 × 108 cells/mL and biomass concentration of ∼8 g/L. The volumetric and areal biomass productivities were 1.81 g/L/day and 10.85 g m− 2/day, respectively, and the maximum volumetric and areal productivities 3.57 g/L/day and 21.42 g m− 2/day, respectively. Intermittent mixing at a frequency of once every two hours (five min mixing each time) produced a cell growth profile similar to that of continuous mixing. Such intermittent mixing resulted in 98.38 % saving on mixing energy and 33.9 % reduction in water evaporation. It was observed that metabolic stress such as high pH and high nitrate concentration could inhibit cell division while causing cell size to enlarge abnormally. The results indicate that it is technically feasible to have high cell density culture of photoautotrophic microalgae with intermittent mixing and no culture circulation, opening the door towards the development of open algal farming systems of low capital, operational, and maintenance costs.

A high-density 3-dimensional culture model of human glioblastoma for rapid screening of therapeutic resistance

Glioblastoma is among the most lethal cancers, with no known cure. A multitude of therapeutics are being developed or in clinical trials, but currently there are no ways to predict which patient may benefit the most from which drug. Assays that allow prediction of the tumor’s response to anti-cancer drugs may improve clinical decision-making. Here, we present a high-density 3D primary cell culture model for short-term testing from resected glioblastoma tissue that is set up on the day of surgery, established within 7 days and viable for at least 3 weeks. High-density 3D cultures contain tumor and host cells, including microglia, and retain key histopathological characteristics of their parent tumors, including proliferative activity, expression of the marker GFAP, and presence of giant cells. This provides a proof-of-concept that 3D primary cultures may be useful to model tumor heterogeneity. Importantly, we show that high-density 3D cultures can be used to test chemotherapy response within a 2–3-week timeframe and are predictive of patient response to Temozolomide therapy. Thus, primary high-density 3D cultures could be a useful tool for brain cancer research and prediction of therapeutic resistance.

High cell density cultivation of Bacillus subtilis NCIM 2063: Modeling, optimization and a scale-up procedure

Bacillus subtilis is a non-pathogenic, sporulating, Gram-positive bacteria with pronounced antimicrobial and metabolic activity and great potential for wide application in various fields. The aim of this paper was to determine the optimum B. subtilis NCIM 2063 growth conditions and to scale up biomass production from shake flasks to a bioreactor level. The critical growth para-meters and their interaction effects were studied using Box?Behnken experimental design and response surface methodology. Developed model equations were statistically significant with good prediction capability. It was found that during shake flask cultivation glucose should be added in concentration up to 5 g L-1 in DSM medium, OTR at 10 mol m-3 h-1 and temperature of 33?C, to achieve the maximum number of viable cells and spores. To scale up the process from shake flasks to the bioreactor level kLa was used as a main criterion. Scale up effect was evaluated by comparing the growth kinetics in the shake flasks and in a laboratory bioreactor. The total number of cells obtained in the bioreactor was 4.57x109 CFU mL-1 which is 1.41 times higher than the number of cells in the shake flasks (3.24x109 CFU mL-1), proving that the scale-up procedure was conducted successfully.

Unbiased Transcriptomic Analysis of Chondrocyte Differentiation in a High-Density Cell Culture Model

In the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.

Promoting soluble expression of hybrid mussel foot proteins by SUMO-TrxA tags for production of mussel glue

Mussel foot proteins (Mfps) display application potential with strong adhesion, enabling mussels to adhere firmly to various surfaces. Mytilus galloprovincialis foot protein 3B (Mgfp-3B) exhibits this characteristic remarkably. However, it remains a challenge for further research due to the low soluble expression of heterologous production. In this study, a small ubiquitin-related modifier (SUMO) and thioredoxin A (TrxA), which catalyzed the proper folding of disulfide bridges, were selected to increase the soluble expression of mfps. An additional ribosome binding site was introduced between the molecular chaperones and Mgfp-3B (fp-3) to form a bicistronic translation-coupled expression vector for co-expression. The results revealed that the combination of SUMO-TrxA increased the soluble expression of fp-3 by 18.07 %. Furthermore, the SUMO-TrxA also boosted the soluble expression of hybrid mfps Mgfp-3B-Mfp-1 (fp-3-1) by 11.29 %, Mgfp-3B-Mgfp-3B (fp-3-3) by 19.91 %, and Mgfp-3B-Mgfp-5 (fp-3-5) by 14.03 %. Ultimately, by high cell density cultivation in a 5 L bioreactor, the yields of fp-3, fp-3-3, and fp-3-5 co-expressed with SUMO-TrxA reached 217.75 mg/L, 127.2 mg/L, and 97.28 mg/L, respectively. Consequently, soluble production of mfps holds great potential for the sustainable supply of protein adhesive materials.