The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

Arigony, Ana Lúcia Vargas; Oliveira, Iuri Marques de; Machado, Miriana S.; Bordin, Diana Lilian; Bergter, Lothar; Prá, Daniel; Henriques, João Antônio Pêgas

doi:10.1155/2013/597282

Cited by 83 publications

(56 citation statements)

References 181 publications

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“…Several media (N2, DMEM/F12) supplemented with serum, most commonly from fetal bovine (FBS) or horse origin, are commonly used for neuronal cultures. However, the chemical composition of the animal serum is not fully defined and includes some factors not present in the brain; moreover, the production of commercial serum is prone to batch to batch variability 3,4 . For these reasons, the use of a chemically defined, serum-free medium is recommended for studies in which a complete control of the environment in the nutrient medium is desired 5 .…”

Section: Introduction *Manuscript (With Page Numbers) Click Here To Dmentioning

confidence: 99%

An improved method for growing neurons: Comparison with standard protocols

Pozzi

Ban

Iseppon

et al. 2017

Journal of Neuroscience Methods

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Highlights An improved protocol for primary hippocampal cell cultures is proposed.  The method relies on serum-free astrocytes conditioned medium (ACM).  The ACM method is extensively compared with other two commonly used protocols.  ACM improved morphology and function of both short-and long-term cultures. AbstractBackground: Since different culturing parameters -such as media composition or cell density -lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons -both in vivo and in vitro -is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes.New Method: Our culturing protocol is based on a novel serum-free preparation of astrocyteconditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods: Neurobasal/B27-and FBS-based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1,7,14 and 60 days in vitro (DIV).Results: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone's size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. Comparison with Existing Method(s):ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. Conclusions:We propose our ACM-based culture protocol as an improved and more suitable method for both short-and long-term neuronal cultures.

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Section: Introduction *Manuscript (With Page Numbers) Click Here To Dmentioning

confidence: 99%

An improved method for growing neurons: Comparison with standard protocols

Pozzi

Ban

Iseppon

et al. 2017

Journal of Neuroscience Methods

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“…Galactose is another factor which can improve galactosylation, either alone or in combination with Mn and uridine, and has also been reported to increase cell growth and productivity of CHO cells . Metal ions, including iron, copper, zinc and selenium are cofactors of many enzymes and are essential for cell growth and metabolism . They are involved in several post‐translational modifications of proteins, including oxidation, deamidation, aggregation, and fragmentation …”

Section: Introductionmentioning

confidence: 99%

Modulation of mAb quality attributes using microliter scale fed‐batch cultures

Rouiller

Périlleux

Vesin

et al. 2014

Biotechnology Progress

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A high-throughput DoE approach performed in a 96-deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO-S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed-batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N-glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines - some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small-scale bioreactors. Being fast and resource effective, this integrated high-throughput approach can provide information which is particularly useful during early stage cell culture development.

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“…Previous studies have shown that the addition of Cbl back to growth medium that is initially devoid of both Cbl and folate, results in an enhanced rate of cellular proliferation [46–48]. In these studies the enhanced rate of proliferation was generally in the order of an approximately 10% increase in cell number [46].…”

Section: Discussionmentioning

confidence: 99%

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum

Zhao

Ruberu

et al. 2016

PLoS ONE

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Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines.

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The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

Cited by 83 publications

References 181 publications

An improved method for growing neurons: Comparison with standard protocols

An improved method for growing neurons: Comparison with standard protocols

Modulation of mAb quality attributes using microliter scale fed‐batch cultures

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum

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