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High cell density transient transfection of CHO cells for TGF-?1 expression.

ABSTRACT: High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO-K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10 × 106 cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor-beta 1 (TGF-?1) in a shake flask. The purified TGF-?1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.

SUBMITTER: Elshereef AA

PROVIDER: S-EPMC6999575 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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High cell density transient transfection of CHO cells for TGF-β1 expression.

Elshereef Abdalla A AA Jochums André A Lavrentieva Antonina A Stuckenberg Lena L Scheper Thomas T Solle Dörte D

Engineering in life sciences 20190902 11

High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A line ...[more]

PMID: 32624966

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Project description:BackgroundTransforming growth factor-beta (TGF-β) signaling is essential in initialization and progression of hepatocellular carcinoma (HCC). Therefore, a treatment targeting TGF-β pathway may be a promising option for HCC control.MethodsFirst, publicly available RNA-seq datasets and clinical characteristics of 374 HCC patients in The Cancer Genome Atlas (TCGA) database were downloaded. Then, Cox regression analysis and LASSO analysis were used to construct a prognostic model for TGF-β family genes. The area under the curve (AUC) of the risk signature was calculated to evaluate the predictive power of the model. Cox regression analysis was applied to predict whether TGF-β1 can be an independent prognosis factor for HCC. Next, hazard ratio and survival analyses were performed to investigate the correlation between TGF-β1 expression and survival time. Furthermore, differential expression level of TGF-β1 in HCC tissues and cells was determined. In addition, Gene Set Enrichment Analysis (GSEA) identified the top significantly activated and inhibited signal pathways related to high expression of TGF-β1. Finally, the CIBERSORT tool was adopted to correlate the tumor-infiltrating immune cells (TICs) with TGF-β1 expression in HCC cohorts.ResultsCox regression analysis and LASSO analysis revealed that seven TGF-β family members (including TGF-β1) could be used as prognostic factors for HCC. Interestingly, TGF-β1 was demonstrated to be an independent prognostic factor of HCC. RT-qPCR and immunofluorescence staining confirmed the high expression of TGF-β1 in HCC cell lines and tissues, which is significantly related to pathological classifications, poor prognosis, and short survival time. Finally, GSEA and CIBERSORT analyses suggested that TGF-β1 may interact with various immune cells and influence the prognosis of HCC patients through Tregs and γδ T cells.ConclusionWe established a novel prognostic prediction method to predict the risk scores of TGF-β genes in HCC prognosis. TGF-β1 is highly expressed in HCC cell lines and tissues, correlates to poor prognosis, and thus can be used as a potential biomarker to predict HCC prognosis. We showed that TGF-β1 may play its roles in HCC prognosis by modulating the immune microenvironment of tumor cells. Our data may shed more light on better understanding the role of TGF-β1 in HCC prognosis.

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High cell density transient transfection of CHO cells for TGF-?1 expression.

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High cell density transient transfection of CHO cells for TGF-β1 expression.

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