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Medium optimization based on comparative metabolomic analysis of chicken embryo fibroblast DF-1 cells.

ABSTRACT: Chicken embryo fibroblast DF-1 cells are increasingly being used in the production of avian virus vaccines. However, the relatively low proliferative capacity does not meet the requirements of industrial production. In this study, we attempted to improve the proliferative capacity of DF-1 cells. The results of intracellular metabolomics showed that 28 types of metabolites could play roles in DF-1 cell growth based on the variance and timing analysis of intracellular metabolites from DF-1 cells grown in two media with distinct growth difference, DMEM/F12 (1 : 1) and DMEM. By examining the differences in the components in the two media, DOE was used to screen and optimize the growth medium for DF-1 cells. The maximum cell density was 40.72% higher, and the infectious bursal disease virus (IBDV) titer was 2.68 times higher, in the optimized medium than in the control. This study proposes a complete solution from metabolomics to media optimization.

SUBMITTER: Lin J

PROVIDER: S-EPMC9070647 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Medium optimization based on comparative metabolomic analysis of chicken embryo fibroblast DF-1 cells.

Lin Jia J Yi Xiaoping X Zhuang Yingping Y

RSC advances 20190830 47

Chicken embryo fibroblast DF-1 cells are increasingly being used in the production of avian virus vaccines. However, the relatively low proliferative capacity does not meet the requirements of industrial production. In this study, we attempted to improve the proliferative capacity of DF-1 cells. The results of intracellular metabolomics showed that 28 types of metabolites could play roles in DF-1 cell growth based on the variance and timing analysis of intracellular metabolites from DF-1 cells g ...[more]

PMID: 35529190

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Project description:RNA-Seq was used to unveil the transcriptional profile of DF-1 cells at the early stage of cell-adapted Infectious Bursal Disease Virus (caIBDV) infection. Total RNAs were extracted from virus-infected cells at 0, 6 and 12 hpi. RNA-Seq datasets of respective samples mapped to 56.5 - 57.6% of isoforms in the reference genome Galgal4.73. At 6 hpi, 23 isoforms underwent an elevated expression, while 128 isoforms were up-regulated and 5 were down-regulated at 12 hpi in the virus-infected group. Besides, 10 isoforms were exclusively expressed in the virus-infected cells. Though no significant change was detected in cytokine and interferon expression levels at the first 12 hours of infection, modulations of the upstream regulators were observed. In addition to the reported regulatory factors including EIF2AK2, MX, OAS*A, GBP7 and IFIT, IBDV infection also triggered a IFIT5-IRF1/3-RSAD5 pathway in the DF-1 cells which potentially restricted the viral replication cycle in the early infection stage. Over-expression of LIPA and CH25H, together with the suppression of STARD4, LSS and AACS genes implied a modulation of membrane fluidity and lipid raft arrangement in the infected cells. Alternative splicing of the EFR3 homolog A gene was also through to be involved in the lipid membrane regulation, and these cumulative responses projected an inhibition of viral endocytosis. Recognition of viral RNA genomes and intermediates was presumably enhanced by the elevated levels of IFIH1, DHX58 and TRIM25 genes which possess properties on detecting viral dsRNA. On the other hand, the caIBDV arrested the host's apoptotic process by inducing the expression of apoptosis inhibitors including NFKBIA/Z, TNFAIP2/3 and ITA at the first 12 hours of infection. In conclusion, the differential expression landscape demonstrated with RNA-Seq provides a comprehensive picture on the molecular interactions between host cells and virus at the early stage of infection. Two sample groups (infected and mock-infected) at three time points (0, 6, 12 hpi) were analysed.

Project description:Lead (Pb) is a heavy metal that can have harmful effects on the environment, which has severe cytotoxicity in many animal tissues. N-acetylcysteine (NAC) has antioxidant activity, reducing lead-induced oxidative stress and apoptosis, but its role in chicken cells is unknown. The current study explored the antagonistic effect of NAC on lead-induced apoptosis and oxidative stress in chicken embryo fibroblast (CEF). In this study, CEF was used as a model to measure the cytotoxic effects of lead nitrate at different concentrations, demonstrating a dose-dependent effect on CEF activity. Employing inverted microscopy, the investigation of morphological alterations in CEF cells was conducted. Fluorescence staining methodology enabled the assessment of reactive oxygen species (ROS) levels within CEF cells. Moreover, an enzyme-linked immunosorbent assay was utilized to detect the presence of oxidative damage indicators encompassing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activity, malondialdehyde (MDA) content, and total antioxidant capacity (T-AOC) within CEF cells. Furthermore, the determination of the apoptosis rate of CEF cells was accomplished through the utilization of the Hoechst 33258 staining method in combination with the Annexin V-FITC dual staining method. By using RT-qPCR for detection, lead treatment increased expression of pro-apoptotic genes, caspase-3, and caspase-9, and reduced expression of anti-apoptotic genes, Bcl-2, and BI-1. Reduced antioxidant capacity was shown by increased ROS and MDA levels in CEF cells after lead treatment. The results showed that NAC inhibited the expression of caspase-3 and caspase-9 in lead-treated CEF cells, while NAC had a certain inhibitory effect on the relative expression of Bcl-2 and BI-1 mRNA in lead-induced CEF cells. NAC significantly reduced lead-induced oxidative damage and apoptosis. Overall, our results demonstrate a novel protective effect of NAC against lead-induced injury in chicken cells, providing a theoretical basis for future investigations of drugs that are effective in preventing lead poisoning in animals.

Dataset Information

Medium optimization based on comparative metabolomic analysis of chicken embryo fibroblast DF-1 cells.

Publications

Medium optimization based on comparative metabolomic analysis of chicken embryo fibroblast DF-1 cells.

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