Vendors sell one serum-free medium for, say, Chinese hamster ovary cells, an epithelial cell line that is often used to produce therapeutic proteins, and others to expand particular types of blood ...cells. Concrete data, like good microscope images or expression data, can help researchers recognize when the cells used in their experiment have changed, says Anne Plant, a division chief at the US National Institute of Standards and Technology in Gaithersburg, Maryland, who hopes to find quantitative ways of making cellculture experiments comparable across laboratories.
Nowadays, chemically defined cell culture media (CCM) have replaced serum‐ and hydrolysate‐based media that rely on complex ingredients, such as yeast extracts or peptones. Benefits include a ...significantly lower lot‐to‐lot variability, more efficient manufacturing by reduction to essential components, and the ability to exclude components that may negatively influence growth, viability, or productivity. Even though current chemically defined CCMs provide an excellent basis for various mammalian biotechnological processes, vitamin instabilities are known to be a key factor contributing to the variabilities still present in liquid CCM as well as to short storage times. In this review, the chemical degradation pathways and products for the most relevant vitamins for CCM will be discussed, with a focus on the effects of light, oxygen, heat, and other CCM compounds. Different approaches to stabilize vitamins in solution, such as replacement with analogs, encapsulation, or the addition of stabilizing compounds will also be reviewed. While these vitamins and vitamin stabilization approaches are presented here as particular for CCM, the application of these concepts can also be considered relevant for pharmaceutical, medical, and food supplement purposes. More precise knowledge regarding vitamin instabilities will contribute to stabilize future formulations and thus decrease residual lot‐to‐lot variability.
The use of chemically‐defined cell culture media in place of serum‐ or hydrolysate‐based media has significantly enhanced the robustness of cell culture in the biomanufacturing of therapeutic proteins. However, some of these essential chemical compounds, such as vitamins, still contribute to cell culture inconsistency via chemical degradation upon storage. This review highlights the likely chemical degradation pathways for vitamins in cell culture media, factors contributing to this degradation, and potential strategies to stabilize these vitamins in complex cell culture media.
Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of ...compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.
A variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells in spin tubes and the ambr® system. Ehret et al. summarized successful modulators for their impact on the main glycosylation species of mannosylation, fucosylation, galactosylation and sialylation as well as highlighting their impact on the main cell culture parameters.
Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the ...alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
The Rise of Physiologic Media Cantor, Jason R.
Trends in cell biology,
11/2019, Letnik:
29, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Developed decades ago, traditional culture media were not intended to resemble the metabolic composition of human blood, and indeed poorly do so. Yet, despite what is now a clear recognition that ...environmental factors influence metabolism, such media remain standard to in vitro studies across virtually all areas of biological research. The recent development of physiologic media, like other efforts designed to address the modeling capacity of cell culture, holds immense potential to improve understanding and interpretation of diverse biological and pharmacological studies.
Environmental factors impact metabolic phenotypes and intertwined physiologic processes in mammalian cells.Complete media that remain the workhorses of cell culture studies typically consist of a basal medium that poorly resembles conditions encountered in the human body and a largely undefined serum supplement.The systematic and bottom-up design of new physiologic media represents an important advance toward improving the modeling capacity of cell culture methods.The development and selection of culture media should now be guided by distinct applications or modeling-based objectives.The immense promise for incorporation of physiologic media into basic and translational research must be balanced by careful considerations critical to the design, use, and continued evolution of experimental model systems.
Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been evaluated for the treatment of immunological diseases. However, the animal-derived growth supplements utilized for ...MSC manufacturing may lead to clinical complications. Characterization of alternative media formulations is imperative for MSC therapeutic application. Human BMMSC and AdMSC were expanded in media supplemented with either human platelet lysates (HPL), serum-free media/xeno-free FDA-approved culture medium (SFM/XF), or fetal bovine serum (FBS) and the effects on their properties were investigated. The immunophenotype of resting and IFN-γ primed BMMSC and AdMSC remained unaltered in all media. Both HPL and SFM/XF increased the proliferation of BMMSC and AdMSC. Expansion of BMMSC and AdMSC in HPL increased their differentiation, compared to SFM/XF and FBS. Resting BMMSC and AdMSC, expanded in FBS or SFM/XF, demonstrated potent immunosuppressive properties in both non-primed and IFN-γ primed conditions, whereas HPL-expanded MSC exhibited diminished immunosuppressive properties. Finally, IFN-γ primed BMMSC and AdMSC expanded in SFM/XF and HPL expressed attenuated levels of IDO-1 compared to FBS. Herein, we provide strong evidence supporting the use of the FDA-approved SFM/XF medium, in contrast to the HPL medium, for the expansion of MSC towards therapeutic applications.
As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour ...microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.
Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which ...led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the "great plate count anomaly," that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27(T) and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.
Abstract
STUDY QUESTION
Is it important that end-users know the composition of human embryo culture media?
SUMMARY ANSWER
We argue that there is as strong case for full transparency concerning the ...composition of embryo culture media intended for human use.
WHAT IS KNOWN ALREADY
Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring.
STUDY DESIGN, SIZE, DURATION
A review of the literature was carried out.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made.
MAIN RESULTS AND THE ROLE OF CHANCE
The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations.
LIMITATIONS, REASONS FOR CAUTION
There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring.
WIDER IMPLICATIONS OF THE FINDINGS
Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the composition of embryo culture media.
STUDY FUNDING/COMPETING INTEREST(S)
This work was funded by The European Society for Human Reproduction and Embryology. No competing interests to declare.
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