Christiane Beer

Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.

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Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. / de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F; Wirth, Manfred; Wagner, Ronald.

In: Cytotechnology, Vol. 66, No. 3, 2014.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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de Los Milagros Bassani Molinas M, Beer C, Hesse F, Wirth M, Wagner R. 2014. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. Cytotechnology. 66(3).

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de Los Milagros Bassani Molinas M, Beer C, Hesse F, Wirth M, Wagner R. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. Cytotechnology. 2014;66(3).

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de Los Milagros Bassani Molinas, Maria ; Beer, Christiane ; Hesse, F ; Wirth, Manfred ; Wagner, Ronald. / Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring. In: Cytotechnology. 2014 ; Vol. 66, No. 3.

Bibtex

@article{91880267b0af4f8d90cc4bc1fdfd6a19,
title = "Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.",
abstract = "Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle{\texttrademark} 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL-1 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.",
author = "{de Los Milagros Bassani Molinas}, Maria and Christiane Beer and F Hesse and Manfred Wirth and Ronald Wagner",
year = "2014",
language = "English",
volume = "66",
journal = "Cytotechnology",
issn = "0920-9069",
publisher = "Springer",
number = "3",

}

RIS

TY - JOUR

T1 - Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring.

AU - de Los Milagros Bassani Molinas, Maria

AU - Beer, Christiane

AU - Hesse, F

AU - Wirth, Manfred

AU - Wagner, Ronald

PY - 2014

Y1 - 2014

N2 - Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL-1 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.

AB - Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL-1 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.

M3 - Journal article

VL - 66

JO - Cytotechnology

JF - Cytotechnology

SN - 0920-9069

IS - 3

ER -