Mammalian Cell Culture Media Research Articles

Production of Staphylococcal Complement Inhibitor (SCIN) and Other Immune Modulators during the Early Stages of Staphylococcus aureus Biofilm Formation in a Mammalian Cell Culture Medium.

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth of Staphylococcus aureus Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how does S. aureus protect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined during in vitro biofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmed scn (SCIN) and, to a lesser extent, chp (CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating that S. aureus is able to modulate the innate immune system already during the early stages of biofilm formation in vitro These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilms in vivo and present opportunities to develop preventive strategies.

Combination of nutrients in a mammalian cell culture medium kills cryptococci.

We found that a large inoculum of Cryptococcus gattii cells, when plated on Dulbecco's modified eagle's medium (DMEM) incorporated into agar, died within a few hours provided that DMEM agar plates had been stored in darkness for approximately 3 days after preparation. Standard conditions were developed for quantification of killing. The medium lost its fungicidal activity when exposed to visible light of wave length ∼400nm. The amount of energy required was estimated at 5.8 × 104 joules @ 550nm. Liquid DMEM conditioned by incubation over DMEM agar plates stored in darkness was fungicidal. We found that fungicidal activity was heat-stable (100°C). Dialysis tubing with MWC0 < 100 Daltons retained fungicidal activity. Neutral pH was required. Strains of Cryptococcus were uniformly sensitive, but some Candida species were resistant. Components of DMEM required for killing were pyridoxal and cystine. Micromolar amounts of iron shortened the time required for DMEM agar plates to become fungicidal when stored in the dark. Organic and inorganic compounds bearing reduced sulfur atoms at millimolar concentrations inhibited fungicidal activity. Our results point to a light-sensitive antifungal compound formed by reaction of pyridoxal with cystine possibly by Schiff base formation.

A Novel Method for Studying Zinc Deficiency in vitro and its Application

Zinc deficiency is correlated with many pathologies, but the molecular connection between disease states and insufficient zinc is still poorly understood. To investigate the connection between zinc deficiency and organismal phenotypes, we developed a technique to specifically diminish zinc levels in mammalian cell culture media without perturbing the concentrations of other metal ions. Growth of cells in such depleted medium evokes some of the transcriptional responses previously noted in organismal models of zinc deficiency, which can be restored upon addition of zinc to the original levels. RNASeq and miRNA data similarly support a zinc‐specific response to the depletion. We also report how the zinc transcriptome remodels in the context of zinc limiting growth. This robust and transportable methodology offers investigators a unique tool for studying zinc deficiency in the context of mammalian cell culture.Support or Funding InformationThe work was supported by the US National Institutes of Health and the Mallinckrodt Faculty Scholar Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low glucose concentrations within typical industrial operating conditions have minimal effect on the transcriptome of recombinant CHO cells.

Typically, mammalian cell culture medium contains high glucose concentrations that are analogous to diabetic levels in humans, suggesting that mammalian cells are cultivated in excessive glucose. Using RNA-Seq, this study characterized the Chinese hamster ovary (CHO) cell transcriptome under two glucose concentrations to assess the genetic effects associated with metabolic pathways, in addition to other global responses. The initial extracellular glucose concentrations used represented high (30 mM) and low (10 mM) glucose conditions, where at the time the transcriptomes were compared, the glucose concentrations were approximately 24 and 4.4 mM for the mid-exponential cultures, where 4.4 mM represents a common target concentration in the biopharmaceutical industry for controlled fed-batch cultures. A recombinant CHO cell line producing a monoclonal antibody was used, such that the impact on glycosylation genes could be evaluated. Relatively few genes were identified as being significantly different (FDR ≤ 0.01) between the high and low glucose conditions, for example, only 575 genes, and only 40 of these genes had 2-fold or greater differences. Gene expression differences for glycolysis, TCA cycle, and glycosylation-related reactions were minimal and unlikely to have biological significance. This transcriptome study indicates that low glucose concentrations in the culture medium are unlikely to cause any biologically significant or detrimental changes to CHO cells at the transcriptome level. Furthermore, it is well-known that maintaining low glucose concentrations in fed-batch cultures can reduce lactate production, which in turn improves process outcomes. Taken together, the transcriptome data supports the continued development of low glucose-based processes to control lactate. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:771-785, 2017.

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture.

Yeast extract (YE), as a non-animal source additive for mammalian cell culture medium, has been widely used for manufacturing of therapeutic proteins. In the present study, one particular YE was found to have significantly improved the specific productivity (q p) of Fc-fusion protein in recombinant Chinese hamster ovary (rCHO) cell culture. In order to elucidate the intracellular effects of YE on protein productivity, steps of the target protein synthesis process were investigated to unveil their variations caused by YE addition. Stepwise analysis on Fc-fusion protein synthesis process showed that YE enhanced Fc-fusion protein gene transcription with cell cycle arrest at G1 phase; mammalian target of rapamycin (mTOR) signaling pathway was activated to enhance the translation of Fc-fusion protein, and the block in post-translational steps of Fc-fusion protein was alleviated by YE addition as well. Our results revealed the responses of multiple protein production steps to the addition of YE and provided a practical guidance for the separation and application of active compounds from hydrolysates.

Development of protein-free medium for therapeutic protein production in mammalian cells: recent advances and perspectives

Mammalian cell culture media used for the manufacture of therapeutic protein have advanced systematically from serum-containing into animal-free, protein-free and chemically defined formulations over the past decades. Initially driven by patient safety concerns associated with the use of animal-derived medium components, and later by inconsistent cell culture performance due to variability in plant-derived raw material lots, many biologics manufacturers redirect their focus on the development of proprietary media formulations and implementation of well-controlled chemically defined raw materials in all cell culture media and feeds for production processes. This article will provide an overview of current trends and objectives of industrial medium development efforts for therapeutic protein production.

Cell culture media supplementation of bioflavonoids for the targeted reduction of acidic species charge variants on recombinant therapeutic proteins.

Charge variants in recombinant proteins are an important series of protein modifications, whose potential role on protein stability, activity, immunogenicity, and pharmacokinetics continues to be studied. Monoclonal antibodies in particular have been shown to have a wide range of acidic species variants, including those associated with the addition of covalent modifications as well as the chemical degradation at specific peptide regions on the antibody. These variants play a significant role toward the overall heterogeneity of recombinant therapeutic proteins and are typically monitored during manufacturing to ensure they lie within proven acceptable ranges. In this work, it has been found that the supplementation of members of the bioflavonoid chemical family into mammalian cell culture media was effective toward the reduction of acidic species charge variants on recombinant monoclonal antibodies and dual variable domain immunoglobulins. The demonstrated reduction in acidic species through the use of bioflavonoids facilitates the manufacturing of a less heterogeneous product with potential improvements in antibody structure and function.

Effect of Biofield Treatment on Physical, Thermal, and Spectral Properties of SFRE 199-1 Mammalian Cell Culture Medium

SFRE 199-1 medium (SFRE-M) is important mammalian cell culture medium, used for the culture of primary cells of mammals such as baboon kidney cells. The present study was attempted to evaluate the impact of biofield energy treatment on the physical, thermal and spectral properties of SFRE-M. The study was accomplished in two groups; one was set as control while another was subjected to Mr. Trivedi’s biofield energy treatment and coded as treated group. Subsequently, the control and treated samples were analyzed using various analytical techniques. The CHNO analysis showed about 2.16, 4.87, and 5.89% decrease in percent contents of carbon, hydrogen, and oxygen, respectively; while 9.49% increase in nitrogen contents of treated sample as compared to the control. X-ray diffraction (XRD) analysis showed 7.23% decrease in crystallite size of treated sample as compared to the control. The thermogravimetric analysis (TGA) analysis showed the increase in onset temperature of thermal degradation by 19.61% in treated sample with respect to the control. The control sample showed the 48.63% weight loss during the thermal degradation temperature (Tmax) while the treated sample showed only 13.62% weight loss during the Tmax. The differential scanning calorimetry (DSC) analysis showed the 62.58% increase in the latent heat of fusion of treated sample with respect to the control sample. The Fourier transform infrared spectroscopy (FT-IR) spectrum of treated SFRE-M showed the alteration in the wavenumber of C-O, C-N and C-H vibrations in the treated sample as compared to the control. Altogether, the XRD, TGA-DTG, DSC, and FT-IR analysis suggest that Mr. Trivedi’s biofield energy treatment has the impact on physical, thermal and spectral properties of SFRE-M. The treated SFRE-M was more thermal stable than the control SFRE-M and can be used as the better culture media for mammalian cell culture.

The optimization of the nitric oxide quantitative analysis for its determination in the cultural medium of mammalian cell culture

The protocol for the quantitative analysis of nitric oxide as nitrite-ion suitable for determination of its production by a mammalian cell culture was developed. The optimal results were obtained using microvolume-adjusted Griess method after the preliminary reduction of NO3- to NO2- with non-activated cadmium. The protocol was verified on a rat glioma C6 cell culture. The developed method may be used for the nitric oxide determination in 96-well and 48-well microplates; the detection limit is 2.1 ± 0.1 μM for NO2- and 2.9 ± 0.1 μM for NO3-.

Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

Proteomics analysis of altered cellular metabolism induced by insufficient copper level

Insufficient copper level in the mammalian cell culture medium resulted in lactate accumulation while maintaining similar growth and culture viability profiles. Label-free, LC-MS/MS-based shotgun proteomics method was applied to compare the protein expression profiles obtained from the cultures exposed to suboptimal copper level to those provided with sufficient amount of copper. Under copper deficient condition, a substantial reduction of the protein levels of the multiple subunits of Complex IV, also known as cytochrome c oxidase, of the mitochondrial electron transport chain was observed for all three different Chinese Hamster Ovary (CHO) cell lines expressing therapeutic monoclonal antibodies tested. Additional proteins affected by suboptimal copper level included peroxiredoxin (PRDX) and hepatocyte-derived growth factor (HDGF), which were affected during early phase of the fed-batch production, several days prior to initiation of lactate accumulation. In contrast, proteins such as syntenin (SDCBP) and integral membrane 2C (ITM2C) showed altered expression patterns toward the end of culture duration, after lactate divergence had occurred. For all conditions tested, time was the most predominant factor facilitating the direction of global protein expression trend, with substantial number of proteins subjected to time-dependent changes in expression, independent of copper.

Activation of aryl hydrocarbon receptor signaling by extracts of teak and other wood dusts.

Wood dusts, as a group, are categorized as known human carcinogens, but the risks of exposure to specific types of wood dusts and the carcinogenic chemicals they contain are not well studied. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is linked to the carcinogenic action of specific classes of chemicals. Here we examined whether chemicals in various wood dusts had the potential to activate AhR signaling as a potential toxic mechanism of action. We found that methanol extracts of teak, walnut, mahogany, and poplar dusts contained a wide range of AhR ligand activity, whereas extracts of oak, pine, and other softwoods did not contain appreciable activity. Teak dust extract, being particularly potent, was subjected to chemical analysis. The 2-methylanthraquinone (2-MAQ) accounted for the AhR ligand activity and was present at an average concentration of 0.27 parts per hundred in teak dust. Pure 2-MAQ potently induced AhR signaling (EC50 115 nM), confirming that this was the active ligand. Aqueous extracts of teak dust made using yeast or mammalian cell culture medium also contained robust AhR activity, suggesting the 2-MAQ ligand is soluble at bioactive concentrations in physiologically relevant fluids. The high concentration and potency of 2-MAQ in teak wood suggest it may mediate toxic effects through activation of AhR signaling in exposed wood workers.

How to assess chemically defined media and feeds from 9 suppliers on CHO cells producing mAb

Mammalian cell culture medium development has widely evolved in recent years. The use of hydrolysates as serum replacement has led to process variability due to lot-to-lot variations. The undefined composition of these media could also increase the process optimization timelines, sometimes with limited impact on process performances. With the reduction of process development activities for preclinical and Phase I studies, medium and feed platforms raised. The objective of the media was to ensure cell growth only in order to go as fast as possible to production bioreactors while the feeds were responsible for productivity and production length. Either companies spent several months if not years to develop their own generic medium and feed platforms or they used commercial ones, sometimes under licenses. The medium and feed platform assessment also started earlier in the product development process. Clone screening was performed more and more in fed-batch conditions rather than batch ones. Thus screening tools, scale-down models of bioreactors, with lower and lower working volumes were designed. Another cell culture process evolution was the development of new expression systems without any selection agents. In order to assess our screening scale-down model, between 20 to 35 chemically defined platforms from 9 suppliers were screened with 3 CHO host cell lines/expression systems.

The optimization of a rapid low-cost alternative of large-scale medium sterilization

Background One of the most important unit operations in upstream animal cell bioprocesses at scales over 100 L is the preparation and sterilization of the medium. This complex, sensitive, and expensive process requires a considerable investment in both material and time [1]. Traditionally, large-scale medium sterilization is performed with costly single-use dead-end filters. To optimize and reduce the cost of this unit operation, we investigated the sterilization of mammalian cell culture medium at volumes larger than 100 L.

Formation of Zinc-Containing Nanoparticles from Zn2+ Ions in Cell Culture Media: Implications for the Nanotoxicology of ZnO

Zinc ions generate a range of poorly soluble Zn-containing nanoparticles when added to commonly used mammalian cell culture media. The formation of these nanoparticles confounds the use of soluble Zn salts as positive controls during cytotoxicity testing of other Zn-containing nanoparticles, such as ZnO. These nanoprecipitates can either be crystalline or amorphous and vary in composition depending upon the concentration of Zn(II) within the medium. The cytotoxicity and immune system response of these nanoparticles in situ are similar to those of 30 nm ZnO nanoparticles. The low residual level of truly soluble Zn species (taken as species passing through a 2 kDa membrane) in cell culture media with serum is insufficient to elicit any appreciable cytotoxicity. These observations highlight the importance of employing appropriate controls when studying ZnO nanoparticle toxicity and suggest a re-evaluation of the conclusions drawn in some previous cytotoxicity studies.

Comparison of the Virulence Potential of Acinetobacter Strains from Clinical and Environmental Sources

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 102 and 104 bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1β, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use.

Toxicity of pamam-coated gold nanoparticles in different unicellular models

Polyamidoamine (PAMAM) dendrimers are used for many pharmaceutical and biomedical applications. However, the toxicological risks of several PAMAM-based compounds are still not fully evaluated, despite evidences of PAMAM deleterious effects on biological membranes, leading to toxicity. In this report, we investigated the toxicity of generation 0 PAMAM-coated gold nanoparticles (AuG0 NPs) in four different models to determine how different cellular systems are affected by PAMAM-coated NPs. Toxicity was evaluated in two mammalian cell lines, Neuro 2A and Vero, in the green alga Chlamydomonas reinhardtii and the bacteria Vibrio fischeri. AuG0 NP treatments reduced cell metabolic activity in algal and bacterial cells, measured by esterase enzymatic activity (C. reinhardtii) and luminescence emission (V. fischeri). EC50 value after 30 min of treatment was similar in both organisms, with 0.114 and 0.167 mg mL(-1) for C. reinhardtii and V. fischeri, respectively. On the other hand, AuG0 NPs induced no change of mitochondrial activity in mammalian cells after 24 h of treatment to up to 0.4 mg mL(-1) AuG0 NPs. Change in the absorption spectra of AuG0 NP in the mammalian cell culture media may indicate an alteration of NP properties that contributed to the low toxicity of AuG0 NPs in mammalian cells. For a safe development of PAMAM-based nanomaterials, the difference of sensitivity between mammalian and microbial cells, as well as the modulation of NPs toxicity by medium properties, should be taken into account when designing PAMAM NPs for applications that may lead to their introduction in the environment.

Potential of Mid-Infrared Spectroscopy for On-Line Monitoring of Mammalian Cell Culture Medium Components

This study proposed a methodology to evaluate the potential of midinfrared (MIR) spectroscopy as a process analytical technology (PAT) tool for in situ (in-line) monitoring of cell culture media constituents, paving the way for on-line bioprocess monitoring and control of mammalian cell cultures. The methodology included a limit of detection (LOD) analysis and external influence investigation in addition to the calibration model development. The LOD analysis in the initial step provided a detailed procedure by which to evaluate the monitoring potential of the instrument of choice, for the application in question. The external influence study highlighted the potential difficulties when applying this technique to a typical mammalian cell culture. A comparative investigation between a fixed conduit immersion probe and flexible fiber-optic immersion probe was also carried out. Limitations associated with the use of MIR spectroscopy in the cell culture environment were also examined. A preliminary investigation, on components typically found in mammalian cell cultures, involving spectral characterization and limit of detection analysis was completed. It was evident at this initial stage that glutamine, could not be accurately detected at levels typically found in a mammalian cell culture medium. Results for glucose and ammonia, however, proved promising. A seven-concentration-level experimental design was used, and partial least squares regression employed, to develop calibration models. Optimized model results echoed the results of the preliminary analysis with the percentage error of prediction for glucose as low as 6.03% with the fixed conduit probe and glutamine having a higher error of 63.06% for the same probe. Comparison of the model results obtained from both probes supported the fixed conduit as the more accurate of the two probes for this experimental setup. The effect of external influences on the MIR spectra and hence the concentrations predicted by the model were also examined. These were subjected to statistical analysis to determine the significance of the effect. This study demonstrates that MIR spectroscopy as a PAT tool has limited potential for mammalian cell culture monitoring due to low concentrations of analytes present and outlines a method to allow the system to be evaluated.

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host-pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscove's modified Dulbecco's medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a (15)N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.

A Kinetic Aggregation Assay Allowing Selective and Sensitive Amyloid-β Quantification in Cells and Tissues

The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for quantifying Aβ fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated Aβ fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ(1-40) reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half-time (t(50)) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.