Influence Of Medium Composition Research Articles

Nutritionally physiological cell culture medium and 3D culture influence breast tumour proteomics and anti-cancer drug effectiveness

Many drugs have been discontinued during phase II/III breast cancer clinical trials due to lack of clinical efficacy, indicating shortcomings in predictive value of preclinical data. Nutrient availability in the tumour cell microenvironment and the dimensionality of in vitro tumour cells likely impact on drug responsiveness. Global proteomics experiments were conducted to assess the impact of nutrient availability and dimensionality of culture. Protein set enrichment analyses identified “pathways in cancer”, “focal adhesion” and “ECM receptor in interaction” related to cell culture dimensionality in MDA-MB-231 cells. In MCF-7 cells, 4 pathways were influenced by medium composition, and 2 pathways were influenced by cell culture dimensionality (2D vs. 3D). These pathways were also identified using KEGG analyses. Eight drugs were selected for investigation according to the differential expression of their putative or known target proteins. The influence of medium composition on drug effectiveness was explored using the “Melbourne Medium” (MM), developed to have nutritionally physiological levels of metabolites as compared with conventional (hyper-nutritional) cell culture medium (CM). The influence of dimensionality on drug effectiveness was also explored, using an innovative 3D viability assessment combining automated confocal microscopy and image analysis. Dimensionality of culture appeared to have a greater influence on the proteome and drug effects than variation in nutrient levels. The number of differentially expressed proteins in the different media was greater in 2D than 3D. We conclude that the risk of qualifying inactive compounds in preclinical assessment may be mitigated using additional models incorporating physiological media and 3-dimensionality.

Using Static Culture Method to Increase the Production of Acetobacter Xylinum Bacterial Cellulose

ABSTRACT Bacterial cellulose is a natural polymer, usually produced through synthetic (chemically defined) or natural media. However, the current methods of bacterial cellulose production are not ideal because of their low productivity and large number of byproducts. In order to improve the yield of bacterial cellulose, the influence of medium composition, culture time, culture temperature and culture pH on the production of bacterial cellulose by Acetobacter xylinum ATCC 23,767 was studied by single factor method. The optimal medium composition for Acetobacter xylinum can be analyzed by Thermal field emission scanning electron microscopy (TF-SEM) as glucose (20 g/L), yeast extract (5 g/L), peptone (5 g/L), citric acid (2 g/L). Using the logarithmic calculation method, we can determine that the optimal culture conditions are controlled by initial pH of the culture medium of 6 and constant static culture at 28°C for 9 days. Using TF-SEM, Fourier transform infrared (FT-IR) spectroscopy and water contact angle test, the best post-treatment solution can be selected as sodium hydroxide (1%). The results showed that the yield of bacterial cellulose could reach up to 8.5 g/L under the above conditions, and the obtained product exhibited a dense three-dimensional network structure.

Features of Hemolysin Biosynthesis by Morganella morganii.

We studied the influence of medium composition and aeration on the hemolytic activity of uropathogenic Morganella morganii strain MM 190. The maximum level of hemolysis was observed in LB (59%), DMEM supplemented with fetal bovine serum (62%), and urine (53%) under aeration conditions during the exponential growth phase. The presence of 2% urea in the medium suppressed hemolysin synthesis. Moreover, addition of bacterial culture fluid containing hemolysin to a monolayer of T-24 bladder carcinoma and OKP-GS kidney carcinoma cells led to 25 and 42% cell death, respectively. We found that the maximum expression of the hemolysin gene hlyA was observed in 2-h culture in LB medium, which correlated with the hemolytic activity of the bacteria in this medium and indicated the predominance of the short hlyCA transcript in the cells.

The Influence of Medium Composition on EUCAST and Etest Antifungal Susceptibility Testing.

There is an ongoing effort to optimize and revise antifungal susceptibility testing (AFST) methods due to the rising number of fungal infections and drug-resistant fungi. The rising antifungal resistance within Candida and Aspergillus species, which are common contributors to invasive fungal infections (IFIs), is a cause for concern, prompting an expanding integration of in vitro AFST to guide clinical decisions. To improve the relevance of in vitro AFST results to therapy outcomes, influential factors should be taken into account. The tested medium is one of several factors that could affect the results of AFST. The present study evaluated the effect of two complex media (Sabouraud dextrose and Columbia) versus the standard defined medium (RPMI 1640) on the AFST results of amphotericin B, posaconazole, and voriconazole against Candida spp. and Aspergillus spp. representatives, utilizing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest methods. Overall, Candida species exhibited higher variability in minimum inhibitory concentration (MIC) across different media (more than three log2 dilutions) comparing to Aspergillus spp., while quality control isolates showed consistency regardless of tested media, antifungals, and methods. When comparing tested methods, MIC variation was mostly detected using EUCAST than it was using Etest.

Optimization of Mustard Pakcoy Growth: The Influence of Media Composition and Coconut Water Concentration

This research aimed to investigate the effect of different media compositions and varying coconut water concentrations on the growth and yield of mustard pakcoy (Brassica rapa var. parachinensis). The study was conducted in Modong Village, Sidoarjo Regency, with further laboratory analysis at the Faculty of Science and Technology, Universitas Muhammadiyah Sidoarjo. The experimental design followed a Randomized Block Design (RAK) in a factorial arrangement with two primary factors (media composition and coconut water concentration) and four replications. While no significant interaction was observed between the media composition and the coconut water concentration, independent effects of each factor were notable. The media composition significantly influenced the leaf count at 7, 28, and 35 days after planting (DAP) and the wet weight at 35 DAP. The optimum media composition included 2 kg of topsoil, 90 g of goat manure, and 45 g of rice husk charcoal per polybag, which resulted in the highest leaf count and wet weight. Additionally, coconut water concentration significantly affected the stem diameter at 35 DAP, with a concentration of 100 ml per liter of water yielding the maximum stem diameter. The findings imply potential growth optimization strategies for mustard pakcoy, enhancing yields via targeted alterations in growing media and the use of coconut water, which could be of international interest to agronomists, horticultural scientists, and sustainable agriculture practitioners.

Influence of Media Composition on the Level of Bovine Satellite Cell Proliferation.

It is predicted that already in 2040, 35% of requirements for meat will be provided by in vitro production. Recreating the course of myogenesis in vitro, and thus resembling a structure of muscle tissue, is the basis for research focusing on obtaining cultured meat and requires providing relevant factors supporting the proliferation of satellite cells-being precursors of skeletal muscles. The present work aimed to develop the composition of the medium that would most effectively stimulate the proliferation of bovine satellite cells (BSCs). The modeling and optimization methods included the measurements of the synergistic, co-stimulatory effect of three medium components: the amount of glucose, the type of serum (bovine or horse), and the amount of mitogenic factor-bFGF. Additionally, the qPCR analyses determined the expression of genes involved in myogenesis, such as Pax7 and Myogenic Regulatory Factors, depending on the level of the tested factor. The results showed significant positive effects of serum type (bovine serum) and mitogenic factor (addition of 10 ng/mL bFGF) on the proliferation rate. In turn, qPCR analysis displayed no significant differences in the relative expression level of Pax7 genes and MRF factors for both factors. However, a statistically higher Pax7 and Myf5 gene expression level was revealed when a low glucose medium was used (p < 0.05). In conclusion, the components of the medium, such as bovine serum and the addition of a mitogenic factor at the level of 10 ng/mL, ensure a higher proliferation rate of BSCs and lower glucose content ensured the expression of crucial genes in the self-renewal of the satellite cell population.

Isolation of lysozyme producing Bacillus subtilis Strains, identification of the new strain Bacillus subtilis BSN314 with the highest enzyme production capacity and optimization of culture conditions for maximum lysozyme production

• Screening, isolation and characterization of lysozyme-producing Bacillus subtilis strains. • Bacillus subtilis BSN314 was found to have highest biomass production capacity. • Analysis of results was based on two techniques; single factor and orthogonal method. • By single factor, influence of medium composition and growth conditions was determined. Lysozyme is a naturally occurring enzyme that is ubiquitous and plays an important role in innate immunity through its potent bacteriolytic activity. The main objective of this study was to isolate, screen and identify lysozyme-producing Bacillus subtilis strains by 16S rDNA sequencing. Of the four Bacillus subtilis strains isolated (B18, B66, B70, and B124), only one (B66) was selected for further study because it exhibited the comparatively highest enzymatic activity. The 16S rDNA sequence of strain B66 was submitted to the Genbank database with accession number OM327451 and was assigned the name Bacillus subtilis BSN314. Bacillus subtilis BSN314 was selected for further study to maximize biomass production. To obtain the maximum biomass product of Bacillus subtilis BSN314, two statistical methods were applied, one related to the nutritional conditions affecting the growth medium (chemical components), called the single factor optimization method, and the other showing their effects on the cultivation conditions (orthogonal method). The results showed that the composition of the culture medium (carbon, nitrogen, inorganic salts and surfactants) and the culture conditions (temperature, pH, shaking speed and percentage of inoculum) have a specific influence on the biomass production of Bacillus subtilis BSN314. The culture medium containing the combination of A2B3C1D2 with 1.5% glucose, 2.5% soybean peptone, 0.5% K 2 HPO 4 , and 0.1% Tween-20 was determined to be the optimal medium for biomass production whereas, the order of culture media based on the effects of the selected conditions having lysozyme activity is as follows; glucose % > soyapeptone % > Tween-20 % > K 2 HPO 4 %. The results showed that the A2B3C1D2 ratio had the highest enzyme activity of 55 U/mL.

Polymeric Microspheres/Cells/Extracellular Matrix Constructs Produced by Auto-Assembly for Bone Modular Tissue Engineering.

Modular tissue engineering (MTE) is a novel “bottom-up” approach to create engineered biological tissues from microscale repeating units. Our aim was to obtain microtissue constructs, based on polymer microspheres (MSs) populated with cells, which can be further assembled into larger tissue blocks and used in bone MTE. Poly(L-lactide-co-glycolide) MS of 165 ± 47 µm in diameter were produced by oil-in-water emulsification and treated with 0.1 M NaOH. To improve cell adhesion, MSs were coated with poly-L-lysine (PLL) or human recombinant collagen type I (COL). The presence of oxygenated functionalities and PLL/COL coating on MS was confirmed by X-ray photoelectron spectroscopy (XPS). To assess the influence of medium composition on adhesion, proliferation, and osteogenic differentiation, preosteoblast MC3T3-E1 cells were cultured on MS in minimal essential medium (MEM) and osteogenic differentiation medium (OSG). Moreover, to assess the potential osteoblast–osteoclast cross-talk phenomenon and the influence of signaling molecules released by osteoclasts on osteoblast cell culture, a medium obtained from osteoclast culture (OSC) was also used. To impel the cells to adhere and grow on the MS, anti-adhesive cell culture plates were utilized. The results show that MS coated with PLL and COL significantly favor the adhesion and growth of MC3T3-E1 cells on days 1 and 7, respectively, in all experimental conditions tested. On day 7, three-dimensional MS/cell/extracellular matrix constructs were created owing to auto-assembly. The cells grown in such constructs exhibited high activity of early osteogenic differentiation marker, namely, alkaline phosphatase. Superior cell growth on PLL- and COL-coated MS on day 14 was observed in the OSG medium. Interestingly, deposition of extracellular matrix and its mineralization was particularly enhanced on COL-coated MS in OSG medium on day 14. In our study, we developed a method of spontaneous formation of organoid-like MS-based cell/ECM constructs with a few millimeters in size. Such constructs may be regarded as building blocks in bone MTE.

Effects of temperature and frequency on the dielectric properties of thiophene compounds and its application in coal microwave-assisted desulfurization

This study aimed to clarify the influence of medium composition, microwave frequency, and temperature on the dielectric properties and desulfurization rates of thiophene components in coal. Thiophene compounds with different structures were selected, and the dielectric parameters at different frequencies and temperatures were investigated. The coal-like substance samples loaded with benzothiophene were prepared and subjected to 915 and 2450 MHz microwave-assisted citric-acid desulfurization at different temperatures. Using X-ray absorption near-edge structure (XANES), changes of organic sulfur components structures and contents in coal-like substances after microwave desulfurization were observed. Results showed that thiophene compounds containing a conjugated structure had poor dielectric properties. The dielectric loss of benzothiophene at 915 MHz was better than that at 2450 MHz. In the temperature range of 20–100 °C, the dielectric loss of benzothiophene increased with the increasing temperature, and both peaked at 90 °C. With the further increase of temperature, the microwave absorbing capacity of benzothiophene decreases. Microwave-assisted citric acid can oxidize part of the benzothiophene in coal-like substances into phenethyl alcohol, sulfoxide, sulfone, sulfonate, sulfite and sulfate. Phenyl alcohol, sulfonate, sulfite and sulfate can be removed by washing with water to remove benzothiophene from coal-like substances. The desulfurization rate was directly related to the dielectric loss of benzothiophene, and the desulfurization rate was highest when the dielectric loss was the largest. When the reaction temperature was 90 °C, 915 MHz frequency microwave radiation citric acid can obtain the best desulfurization effect of benzothiophene in coal-like substances, and the desulfurization rate reached 42.87%.

The influence of medium composition on the microbial secretory production of hydroxyalkanoate oligomers.

With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Influence of medium composition on biomineralization and morphology of newgrowths

The research presents rational (from the point of view of processability) types of bacteria, nutrient media, biochemical agents and their quantities for biogenic mineral formation under specified conditions. The principles of the influence of metabolism processes of microorganisms on the change in the alkalinity of medium are established. The dependences of morphological parameters (size, shape) of mineral components formed during biomineralization in simulated media on the type of nutrient in the crystallization medium are established. The authors formed a phenomenological model of the processes occurring as a result of the bacterial activity of various strains, nutrient components and precursors of crystallization initiation as stages of structure formation during the production of building materials.

Influence of media composition and genotype for successfulCannabis sativaL. in vitro introduction

ISHS XXX International Horticultural Congress IHC2018: II International Symposium on Micropropagation and In Vitro Techniques Influence of media composition and genotype for successful Cannabis sativa L. in vitro introduction

Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins.

Effect of various bacteriological media on the plaque morphology of Staphylococcus and Vibrio phages.

The influence of media composition on the life cycle of bacteriophages to exhibit diverse plaque morphology on various bacteriological media was investigated by a double agar overlay method. Both Staphylococcus aureus phage and Vibrio parahaemolyticus phage showed altered plaque morphology from small to large and from clear to turbid, in different culture media used for the double agar overlay method.

Influence of medium composition and physical factors on enhanced production of endoglucanase by locally isolated fungal strain in solid state fermentation

Endoglucanase is one of the most important enzymes of the cellulase group. Endoglucanase are involved in the catalytic hydrolysis of cellulose and plays a pivotal role in different sectors like pharmaceutical, textile, detergent, and food processing as well as paper and pulp industry. With consumers getting more and more aware of environmental issues, industries find enzymes as a better option over other chemical catalysts. In the current research different thermophilic fungal strains were isolated from the different sources. Qualitative screening was carried out on the basis of cellulose hydrolysis zone. The quantitative screening was carried out employing solid state fermentation. The fungal culture, showing highest EG potential was selected identified and assigned the code Aspergillus fumigatus BBT2. Different fermentation media were evaluated and M 2 containing wheat bran gave maximum EG production. The maximal enzyme productivity was recorded in 72 hours, 40°C, pH 5, inoculum size 1.5ml, and moisture content (1:1). Glucose (1%) and peptone (1%) were optimized as best carbon and nitrogen sources, respectively.

Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

Enhanced active extracellular polysaccharide production from Ganoderma formosanum using computational modeling

Extracellular polysaccharide (EPS) is one of the major bioactive ingredients contributing to the health benefits of Ganoderma spp. In this study, response surface methodology was applied to determine the optimal culture conditions for EPS production of Ganoderma formosanum. The optimum medium composition was found to be at initial pH 5.3, 49.2 g/L of glucose, and 4.9 g/L of yeast extract by implementing a three-factor–three-level Box–Behnken design. Under this condition, the predicted yield of EPS was up to 830.2 mg/L, which was 1.4-fold higher than the one from basic medium (604.5 mg/L). Furthermore, validating the experimental value of EPS production depicted a high correlation (100.4%) with the computational prediction response model. In addition, the percentage of β-glucan, a well-recognized bioactive polysaccharide, in EPS was 53±5.5%, which was higher than that from Ganoderma lucidum in a previous study. Moreover, results of monosaccharide composition analysis indicated that glucose was the major component of G. formosanum EPS, supporting a high β-glucan percentage in EPS. Taken together, this is the first study to investigate the influence of medium composition for G. formosanum EPS production as well as its β-glucan composition.

Influence of Medium Composition on Acrylic Acid Solution Polymerization and Process Scale‐Up

SummaryThe main objective of the present study was the production of poly(acrylic acid) through solution polymerizations in a process scale compatible with the commercial manufacture of dental cements. In order to do that, the work was divided into three fundamental steps. In the first step, the effect of system composition on final dental cement performance was analyzed with small reactors (50 mL). Then, the effect of the operation mode (batch or semibatch) on process performance and dental cement properties was also evaluated. Finally, process performance and dental cement properties were evaluated at a larger scale (12 L). It is shown that dental cements with adequate properties can be produced in both batch and semibatch modes with small reactors. However, at larger scales appropriate dental cements and safe process operation can only be obtained with the semibatch operation mode, requiring significant modification of recipes developed with small scale bench reactors.

Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.

Enhancing isobutyric acid production from engineered Acidithiobacillus ferrooxidans cells via media optimization.

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe(2+)) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe(2+)), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe(3+) concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe(3+) to Fe(2+) ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 ± 0.2 mg IBA/L/d which is a 14-fold increase over the previously reported value.