CHO-S transcriptome in batch culture
Ontology highlight
ABSTRACT:
ORGANISM(S): Cricetulus griseus
PROVIDER: GSE77800 | GEO | 2016/11/23
SECONDARY ACCESSION(S): PRJNA311594
REPOSITORIES: GEO
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Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 °C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering.
2016-04-03 | GSE72945 | GEO
Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 °C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering. 36 miRNA libraries from three different CHO cell lines and two process condition. In the control run temperature was maintained at 30°C, while temperature was reduced to 30°C after reaching mid exponential phase
2016-04-03 | E-GEOD-72945 | biostudies-arrayexpress
Project description:Small-scale bioreactors used in Chinese hamster ovary (CHO) cell line development allow transcriptomic studies on multiple cell lines. Here we define the CHO cell long non-coding RNA (lncRNA) transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs associated with productivity and growth characteristics, finding that Adapt15, linked to ER stress, GAS5, linked to mTOR signalling/growth arrest, and PVT1, linked to Myc expression, are differentially regulated during fed-batch culture and whose expression relates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported, showing large differences in gene expression between these, and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.
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Project description:Small-scale bioreactors used in Chinese hamster ovary (CHO) cell line development allow transcriptomic studies on multiple cell lines. Here we define the CHO cell long non-coding RNA (lncRNA) transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs associated with productivity and growth characteristics, finding that Adapt15, linked to ER stress, GAS5, linked to mTOR signalling/growth arrest, and PVT1, linked to Myc expression, are differentially regulated during fed-batch culture and whose expression relates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported, showing large differences in gene expression between these, and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.
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Project description:MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from CHO cells provide new tools for the discovery of novel miRNAs in this important production system. The current release of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences. We searched for evolutionary conserved miRNAs in recently published genomic data, derived from hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several dozens novel miRNAs and verify their expression with multiple next-generation sequencing experiments. In addition, a great amount of unexpressed miRNAs could be found, awaiting further confirmation by testing for their transcription in different Chinese hamster tissues. Two batch processes with 6 samples each
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Project description: Not available
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Project description:Chinese hamster ovary (CHO) cells are widely used host cells for recombinant protein production and currently the most commonly utilized mammalian organism in large scale biopharmaceutical production. Since the discovery of exosomes as a new type of small extracellular vesicles (EVs) by Johnstone et al., interest in EV research greatly increased in recent years. However, they yet pose a blank space in CHO research. Exosomes are 30 – 150 nm small vesicles, that derive from the endosomal network and can therefore be distinguished from plasma membrane-shed microvesicles (100 – 1000 nm in diameter) and apoptotic vesicles (50 – 5000 nm), which are secluded over the course of programmed cell death. It turned out exosomes are not only vehicles of cellular waste disposal, as was initially assumed, but also a conserved mechanism of cellular communication. This work aims to outline possible separation techniques followed by a differential proteomic and transcriptomic characterization of CHO EVs over the course of a bioreactor batch cultivation. Therefore, a protocol yielding sEVs with a strong exosomal marker enrichment is compared with HCP, lEVs and whole cell lysate (WCL) from the same batch process. This may allow for further studies in this field to have reference data for evaluation of exosome isolation techniques, separation purity and CHO EV composition in general.
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Project description:Motivation: Sample source, procurement process, and other technical variations introduce batch effects into genomics data. Algorithms to remove these artifacts enhance differences between known biological covariates, but also carry potential concern of removing intra-group biological heterogeneity and thus any personalized genomic signatures. As a result, accurate identification of novel subtypes from batch corrected genomics data is challenging using standard algorithms designed to remove batch effects for class comparison analyses. Nor can batch effects be corrected reliably in future applications of genomics-based clinical tests, in which the biological groups are by definition unknown a priori. Results: Therefore, we introduce new algorithm, personalized-SVA (pSVA), blind to biological covariates corrected technical artifacts while retaining biological heterogeneity in genomic data. This algorithm facilitated accurate subtype identification in head and neck cancer from gene expression data in both formalin fixed and frozen samples. When applied to predict HPV status, pSVA improved cross- study validation even if the sample batches were highly confounded with HPV status in the training set. Availability: All analyses were performed using R version 2.15.0. The code and data used to generate the results of this manuscript is available from https://sourceforge.net/projects/psva.
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Project description:The majority of recombinant protein therapeutics are produced with Chinese Hamster Ovary (CHO) cells. Productivity depends on the initial cell line engineering in terms of integration site or choice of an appropriate promotor for the recombinant gene expression, as well as media and process parameter optimization. Here, proteomic profiling is used to identify optimization targets for a pharmaceutical relevant cell line system. Triplicates of CHO-K1 cell 2 L bioreactor fedbatch cultivations were performed and daily sampled for nLC-MS/MS proteomic analysis. Collected data from day 3 up to day 11 showed high Pearson correlation of 93.7 ± 4 % with ca. 2500 proteins quantified. The different growth phases were separated by principal component analysis and hierarchical clustering approaches. Subsequent statistical analysis revealed distinct protein profiles, where steady increase or decrease over time were the most prominent clusters. Fisher exact enrichment tests yielded in significantly enriched protein annotations which were successfully mapped to growth and metabolic changes during fedbatch cell cultivation. Major improvements in cellular and process understanding were achieved and yielded in the identification of promising new targets, like strong endogenous promotors, for cellular engineering and process optimization of this biopharmaceutical relevant cell line.
2021-01-25 | PXD018439 | Pride