Limits...
Bioreactor cultivation of CHO DP-12 cells under sodium butyrate treatment - comparative transcriptome analysis with CHO cDNA microarrays.

Klausing S, Krämer O, Noll T - BMC Proc (2011)

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld, Germany.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Sodium butyrate (NaBu) is not only known to inhibit proliferation but also to increase the specific productivity in cultivation of Chinese hamster ovary (CHO) cells – the most commonly used mammalian cell line for pharmaceutical protein production... Here we show an application of our CHO cDNA microarray to identify genes associated with increased productivity during cultivation of CHO cells under sodium butyrate treatment... The custom designed 2 x 105 k cDNA microarray (Agilent Technologies) was spotted with 94,580 probes designed from CHO cDNA sequenced in-house. 38,310 of 41,039 sequenced contigs were used for the microarray, each covered by 2-4 probes... The control culture reached a maximum viable cell density of 1·10 cells/mL while NaBu treated cells reached a plateau at about 6·10 cells/mL and retained a viability above 90 % four days longer than untreated cells (Figure 1A)... The three biological replicates of NaBu cultures yielded results with similar general trends... For analysis, the following filtering settings were chosen to identify differentially expressed genes: adjusted p-value ≤ 0.05, log-ratio < -1 or > 1 (equals fold change < -2 or > 2) and log-intensity ≥ 6 (equals ≥ 64 raw intensity)... From a total of 1461 genes found to be differentially expressed under NaBu treatment, 771 genes were upregulated and 690 genes were downregulated (derived from EC numbers in KEGG pathways, Figure 1B)... Many differentially expressed genes from pathways involved in carbohydrate, lipid, amino acid and glycan metabolism are upregulated which is most likely linked to higher productivity... A large portion of genes from pathways associated with cell growth and death are downregulated and most of these genes originate from cell cycle processes... Microarray analysis revealed a high number of regulated genes under sodium butyrate treatment in pathways like carbohydrate metabolism, cell cycle and signal transduction... Some of the regulated genes are promising targets for overexpression or knockdown/knockout experiments and we will further investigate the knockdown effect of selected genes using a siRNA approach in CHO cells... Our in-house microarray is suitable for further transcriptomic analysis of CHO cells under various conditions.

No MeSH data available.


(A): Concentration of viable cells and cell viabilities for the time course of CHO DP-12 batch processes. Error bars represent the standard deviation of triplicate measurements with the Cedex system (Roche Diagnostics). Red and orange lines represent biological replicates of cultures treated with 2 mM NaBu, the control process is shown in green. Dashed lines show viabilities. The grey arrow indicates the addition of NaBu, the grey circles show the sample points compared later in the microarray analysis (72 h of NaBu Treatment). (B): Number of up- and downregulated genes of selected KEGG pathway categories with a detailed view of four pathways from the Cell Growth and Death KEGG category. Results show only those found as differentially expressed after filtering. Red: downregulated in NaBu cultures; green: upregulated in NaBu cultures (compared to control culture).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3285020&req=5

Figure 1: (A): Concentration of viable cells and cell viabilities for the time course of CHO DP-12 batch processes. Error bars represent the standard deviation of triplicate measurements with the Cedex system (Roche Diagnostics). Red and orange lines represent biological replicates of cultures treated with 2 mM NaBu, the control process is shown in green. Dashed lines show viabilities. The grey arrow indicates the addition of NaBu, the grey circles show the sample points compared later in the microarray analysis (72 h of NaBu Treatment). (B): Number of up- and downregulated genes of selected KEGG pathway categories with a detailed view of four pathways from the Cell Growth and Death KEGG category. Results show only those found as differentially expressed after filtering. Red: downregulated in NaBu cultures; green: upregulated in NaBu cultures (compared to control culture).

Mentions: The control culture reached a maximum viable cell density of 1·107 cells/mL while NaBu treated cells reached a plateau at about 6·106 cells/mL and retained a viability above 90 % four days longer than untreated cells (Figure 1A). The three biological replicates of NaBu cultures yielded results with similar general trends. The maximum antibody concentration of the control culture was 110 mg/L whereas cells treated with NaBu reached a maximum of 175 mg/L antibody. 72 hours after addition of NaBu the specific antibody production rate was increased by a factor of 3.6 (NaBu culture: 4.5 pg/(cell·d)) compared to control culture (1.2 pg/(cell·d)).


Bioreactor cultivation of CHO DP-12 cells under sodium butyrate treatment - comparative transcriptome analysis with CHO cDNA microarrays.

Klausing S, Krämer O, Noll T - BMC Proc (2011)

(A): Concentration of viable cells and cell viabilities for the time course of CHO DP-12 batch processes. Error bars represent the standard deviation of triplicate measurements with the Cedex system (Roche Diagnostics). Red and orange lines represent biological replicates of cultures treated with 2 mM NaBu, the control process is shown in green. Dashed lines show viabilities. The grey arrow indicates the addition of NaBu, the grey circles show the sample points compared later in the microarray analysis (72 h of NaBu Treatment). (B): Number of up- and downregulated genes of selected KEGG pathway categories with a detailed view of four pathways from the Cell Growth and Death KEGG category. Results show only those found as differentially expressed after filtering. Red: downregulated in NaBu cultures; green: upregulated in NaBu cultures (compared to control culture).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285020&req=5

Figure 1: (A): Concentration of viable cells and cell viabilities for the time course of CHO DP-12 batch processes. Error bars represent the standard deviation of triplicate measurements with the Cedex system (Roche Diagnostics). Red and orange lines represent biological replicates of cultures treated with 2 mM NaBu, the control process is shown in green. Dashed lines show viabilities. The grey arrow indicates the addition of NaBu, the grey circles show the sample points compared later in the microarray analysis (72 h of NaBu Treatment). (B): Number of up- and downregulated genes of selected KEGG pathway categories with a detailed view of four pathways from the Cell Growth and Death KEGG category. Results show only those found as differentially expressed after filtering. Red: downregulated in NaBu cultures; green: upregulated in NaBu cultures (compared to control culture).
Mentions: The control culture reached a maximum viable cell density of 1·107 cells/mL while NaBu treated cells reached a plateau at about 6·106 cells/mL and retained a viability above 90 % four days longer than untreated cells (Figure 1A). The three biological replicates of NaBu cultures yielded results with similar general trends. The maximum antibody concentration of the control culture was 110 mg/L whereas cells treated with NaBu reached a maximum of 175 mg/L antibody. 72 hours after addition of NaBu the specific antibody production rate was increased by a factor of 3.6 (NaBu culture: 4.5 pg/(cell·d)) compared to control culture (1.2 pg/(cell·d)).

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld, Germany.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Sodium butyrate (NaBu) is not only known to inhibit proliferation but also to increase the specific productivity in cultivation of Chinese hamster ovary (CHO) cells – the most commonly used mammalian cell line for pharmaceutical protein production... Here we show an application of our CHO cDNA microarray to identify genes associated with increased productivity during cultivation of CHO cells under sodium butyrate treatment... The custom designed 2 x 105 k cDNA microarray (Agilent Technologies) was spotted with 94,580 probes designed from CHO cDNA sequenced in-house. 38,310 of 41,039 sequenced contigs were used for the microarray, each covered by 2-4 probes... The control culture reached a maximum viable cell density of 1·10 cells/mL while NaBu treated cells reached a plateau at about 6·10 cells/mL and retained a viability above 90 % four days longer than untreated cells (Figure 1A)... The three biological replicates of NaBu cultures yielded results with similar general trends... For analysis, the following filtering settings were chosen to identify differentially expressed genes: adjusted p-value ≤ 0.05, log-ratio < -1 or > 1 (equals fold change < -2 or > 2) and log-intensity ≥ 6 (equals ≥ 64 raw intensity)... From a total of 1461 genes found to be differentially expressed under NaBu treatment, 771 genes were upregulated and 690 genes were downregulated (derived from EC numbers in KEGG pathways, Figure 1B)... Many differentially expressed genes from pathways involved in carbohydrate, lipid, amino acid and glycan metabolism are upregulated which is most likely linked to higher productivity... A large portion of genes from pathways associated with cell growth and death are downregulated and most of these genes originate from cell cycle processes... Microarray analysis revealed a high number of regulated genes under sodium butyrate treatment in pathways like carbohydrate metabolism, cell cycle and signal transduction... Some of the regulated genes are promising targets for overexpression or knockdown/knockout experiments and we will further investigate the knockdown effect of selected genes using a siRNA approach in CHO cells... Our in-house microarray is suitable for further transcriptomic analysis of CHO cells under various conditions.

No MeSH data available.