Cell Culture Medium Components Research Articles

Best practices in cell culture: an overview.

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

Best practices for the use and evaluation of animal serum as a component of cell culture medium.

Animal serum is a common additive for cell culture medium and is often required at 5 to 10% (v/v) for the attachment and growth of primary and continuous anchorage-dependent (monolayer) cultures. The use of animal serum in cell culture medium confers several advantages and also some risks. This article discusses the use of animal serum as a component of cell culture medium. The best practices associated with the sourcing, storage, thawing, testing, and mitigation of risk associated with the use of animal sera are among the topics described in this article.

Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l-methionine (met) and l-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759-770, 2017.

Understanding the Interactions of Peptides and Proteins that are Commonly used in Biological Media on Polystyrene Surfaces

Polystyrene(PS) is a commonly used material for designing cell culture surfaces like assay plates, petridishes, T flasks to name a few. These surfaces when used for the culture of adherent cells will modulate cell growth and function depending of the quality of adhesion provided. To enable biological activity on polystyrene, surfaces can be modified by different treatments which result in an increase in the amount of certain surface chemical species like oxygen. To understand the differences between treated and untreated polystyrene surfaces, we examine in this study the interaction between two commonly occurring biological entities in these applications i) Albumin which is a common component of cell culture media and is used to sustain the growth and activity of biological cells and ii) commonly occurring peptide motifs that are a typical part of the ECM proteins which allow the cells to interact with the surface through these binding motifs.

Protein extracts from cultured cells contain nonspecific serum albumin.

Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection.

Cell culture media impact on drug product solution stability.

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016.

Considerations using silver nitrate as a reference for in vitro tests with silver nanoparticles

Most in vitro tests regarding the cellular toxicology of nanoparticulate metals compare particle to associated metal ion exposure. However, it is also a fact, that for example silver ions are reduced by sugars or transformed to silver chloride by chloride salts which are abundant components of cell culture media. These reactions are likely to either complicate or even invalidate comparisons between effects of ions and particles. Here, we present a fast and quantitative method to determine particle formation and numbers in different cell culture media with non-destructive small-angle X-ray scattering (SAXS). Silver nitrate with a concentration of 25μgAgmL−1 was dissolved for up to 24h at 37°C in Dulbeccos Modified Eagle Medium (DMEM) with and without 10% fetal bovine serum (FBS) and a solution of D-glucose (4.5μgmL−1), respectively. Silver nanoparticles were observed in all solutions after 5min. The cell culture media displayed a limited particle-growth. FBS showed an effect on the polydispersity of the generated particles but after 5min the overall particle size was nearly equal in FBS and non FBS supplemented medium. Particles in D-glucose were precipitating after 10min. Particulate silver concentration was between 3 and 4μgmL−1 in both cell culture media (CCM). These results should be taken into account when performing silver ion-toxicity experiments in relevant media.

Identification of the Factors Responsible for the Selective in vitro Cytotoxic Activity of Isoprenylated Coumarin Derivatives under Nutrient-deprived Conditions.

Pancreatic cancer is one of the most devastating forms of human cancer. The lack of effective clinical treatments for pancreatic cancer has led to one of the lowest five-year survival rates among all cancers. Recently, our laboratory has developed a novel series of isoprenylated coumarin derivatives that have exhibited anti-pancreatic cancer activity exclusively under nutrient-deprived conditions. In this study, we report the effect of the various cell culture medium components on the preferential cytotoxicity of our lead isoprenylated coumarin compound against the pancreatic adenocarcinoma cell line PANC-1. In particular, our findings show a clear link between observed cytotoxicity and glucose deprivation, suggesting that our compound targets a salvage pathway when glycolysis is no longer an option for cancer cell survival. The cytotoxicity of our lead compound was also examined in vitro against two other pancreatic cancer cell lines, BxPC-3 and Capan-2 under both nutrient-rich and nutrient-deprived conditions.

Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.

One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.

Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation.

Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

Tailoring recombinant protein quality by rational media design

Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function.

Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media

An unexpected 6–9% drop of viability in suspension CHO cell cultures 24–48h post-inoculation in sparged bioreactors, paired with reduced overall cell growth by an average of 43%, occurred upon switching to new serum and hydrolysate-free chemically defined media (CDM) lots. An investigation was undertaken to identify sources of variability. First, chemical/biological contaminants, including cleaning procedures, gas source/quality, compromised bioreactor parts and mycoplasma contamination, were ruled out. Second, we tested a gradient of sparging rates and observed that early post-inoculation cell death increased with the gas flow rate. While bubbles are known to cause cell damage, this effect was not previously observed at the high sparging rate used. Third, because hydrodynamic damage appeared to be responsible for cell death, we tested the addition of increased levels of surfactant Pluronic F-68 (PF-68), which reduces cell-to-bubble attachment and lowers dynamic surface tension, to the new CDM lots. This restored the viable cell density and viability profiles. Measurement of the dynamic surface tension of the CDM lots employed in this study indicated variability up to 8% between CDM lots supplemented with different PF-68 lots. In summary, our observations suggest that the raw material quality of PF-68 lots can alter the ability to lower the surface tension in chemically-defined cell culture media and significantly impact performance of CHO cell cultures in sparged bioreactors.

Evaluating the impact of high Pluronic® F68 concentrations on antibody producing CHO cell lines.

Pluronic® F68 (P-F68) is an important component of chemically-defined cell culture medium because it protects cells from hydrodynamic and bubble-induced shear in the bioreactor. While P-F68 is typically used in cell culture medium at a concentration of 1 g/L (0.1%), higher concentrations can offer additional shear protection and have also been shown to be beneficial during cryopreservation. Recent industry experience with variability in P-F68-associated shear-protection has opened up the possibility of elevated P-F68 concentrations in cell culture media, a topic that has not been previously explored in the context of industrial cell culture processes. Recognizing this gap, we first evaluated the effect of 1-5 g/L P-F68 concentrations in shake flask cultures over ten 3-day passages for cell lines A and B. Increase in terminal cell density and cell size was seen over time at higher P-F68 concentrations but protein productivity was not impacted. Results from this preliminary screening study suggested no adverse impact of high P-F68 concentrations. Subsequently fed-batch bioreactor experiments were conducted at 1 and 5 g/L P-F68 concentrations with both cell lines where cell growth, viability, metabolism, and product quality were examined under process conditions reflective of a commercial process. Results from these bioreactor experiments confirmed findings from the preliminary screen and also indicated no impact of elevated P-F68 concentration on product quality. If additional shear protection is desired, either due to raw material variability, cell line sensitivity, or a high-shear cell culture process, our results suggest this can be accomplished by elevating the P-F68 concentration in the cell culture medium without impacting cell culture performance and product quality.

Development of a novel micro-ablation system to realise micrometric and well-defined hydrogel structures for tissue engineering applications

Purpose – This paper aims to develop a novel micro-ablation system to realise micrometric and well-defined hydrogel structures. To engineer a tissue it is necessary to evaluate several aspects, such as cell-cell and cell-substrate interactions, its micro-architecture and mechanical stimuli that act on it. For this reason, it is important to fabricate a substrate which presents a microtopology similar to natural tissue and has chemical and mechanical properties able to promote cell functions. In this paper, well-defined hydrogel structures embedding cells were microfabricated using a purposely developed technique, micro-laser ablation, based on a thulium laser. Its working parameters (laser power emission, stepper motor velocity) were optimised to produce shaded “serpentine” pattern on a hydrogel film. Design/methodology/approach – In this study, initially, swelling/contraction tests on agarose and alginate hydrogel in different solutions of main components of cell culture medium were performed and were compared with the MECpH model. This comparison matched with good approximation experimental measurements. Once known how hydrogel changed its topology, microstructures with a well-defined topology were realised using a purposely developed micro-laser ablation system design. S5Y5 neuroblastoma cell lines were embedded in hydrogel matrix and the whole structure was ablated with a laser microfabrication system. The cells did not show damages due to mechanical stress present in the hydrogel matrix and to thermal increase induced by the laser beam. Findings – The hydrogel structure is able to reproduce extracellular matrix. Initially, the hydrogel swelling/contraction in different solutions, containing the main components of the most common cell culture media, was analysed. This analysis is important to evaluate if cell culture environment could alter microtopology of realised structures. Then, the same topology was realised on hydrogel film embedding neuronal cells and the cells did not show damages due to mechanical stress present in the hydrogel matrix and to thermal increase induced by the laser beam. The interesting obtained results could be useful to realise well-defined microfabricated hydrogel structures embedding cells to guide tissue formation Originality/value – The originality of this paper is the design and realisation of a 3D microfabrication system able to microfabricate hydrogel matrix embedding cells without inducing cell damage. The ease of use of this system and its potential modularity render this system a novel potential device for application in tissue engineering and regenerative medicine area.

Elucidating the effects of postinduction glutamine feeding on the growth and productivity of CHO cells

Inducible mammalian expression systems are increasingly being used for the production of valuable therapeutics. In such system, maximizing the product yield is achieved by carefully balancing the biomass concentration during the production phase and the specific productivity of the cells. These two factors are largely determined by the availability of nutrients and/or the presence of toxic waste metabolites in the culture environment. Glutamine is one of the most important components of cell culture medium, since this substrate is an important building block and source of energy for biomass and recombinant protein production. Its metabolism, however, ultimately leads to the formation of ammonia, a well known inhibitor of cellular growth and productivity. In this work, we show that nutrient feeding post-induction can greatly enhance the product yield by alleviating early limitations encountered in batch. Moreover, varying the amount of glutamine in the feed yielded two distinct culture behaviors post-induction; whereas excess glutamine allowed to reach greater cell concentrations, glutamine-limited fed-batch led to increased cell specific productivity. These two conditions also showed distinctive lactate metabolism. To further assess the physiological impact of glutamine levels on the cells, a comparative (13) C-metabolic flux analysis was conducted and a number of key intracellular fluxes were found to be affected by the amount of glutamine present in the feed during the production phase. Such information may provide useful clues for the identification of physiological markers of cell growth and productivity that could further guide the optimization of inducible expression systems.

Effect of cell culture medium components on color of formulated monoclonal antibody drug substance

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B‐vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1270–1277, 2013

Thermosensitive behavior in cell culture media and cytocompatibility of a novel copolymer: poly(N-isopropylacrylamide-co-butylacrylate)

Cell sheet technology is a promising step forward in tissue engineering. Cell sheets are usually generated using Poly(N-isopropylacrylamide) hydrogels due to their swelling change around the lower critical solution temperature (LCST). Nevertheless, LCST can be affected by cell culture medium components and therefore it is necessary to ensure that the polymer preserves its thermosensitivity under these conditions. We propose a novel thermosensitive crosslinked-copolymer: Poly(N-isopropylacrylamide-co-butylacrylate). This copolymer is shown to be cytocompatible and thermosensitive under cell culture medium conditions, and besides, it can be synthesized inexpensively. Thermosensitivity was investigated by determining the LCST with differential scanning calorimetry and swelling/ratio measurements. Cytocompatibility and capacity to deliver cell sheets were studied employing 3T3 and human oral epithelial cells. In conclusion, we obtained a thermosensitive copolymer that allows cell sheet formation/detachment by using a simple and low-cost polymerization method. Furthermore, crosslinking allows easy manipulation of cell sheets growing on the copolymer for potential in situ applications.

Cell culture medium improvement by rigorous shuffling of components using media blending

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.

Formation of Nano-Bio-Complex as Nanomaterials Dispersed in a Biological Solution for Understanding Nanobiological Interactions

Information on how cells interface with nanomaterials in biological environments has important implications for the practice of nanomedicine and safety consideration of nanomaterials. However, our current understanding of nanobiological interactions is still very limited. Here, we report the direct observation of nanomaterial bio-complex formation (other than protein corona) from nanomaterials dispersed in biologically relevant solutions. We observed highly selective binding of the components of cell culture medium and phosphate buffered saline to ZnO and CuO nanoparticles, independent of protein molecules. Our discoveries may provide new insights into the understanding of how cells interact with nanomaterials.

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.