Details
| Original language | English |
|---|---|
| Pages (from-to) | 730-740 |
| Number of pages | 11 |
| Journal | Engineering in life sciences |
| Volume | 19 |
| Issue number | 11 |
| Early online date | 22 Aug 2019 |
| Publication status | Published - 4 Nov 2019 |
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-β1) in a shake flask. The purified TGF-β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.
Keywords
- CHO cells, EGFP transfection efficiency, PEI transient transfection, TGF-β1 purification and bioactivity
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Environmental Science(all)
- Environmental Engineering
- Chemical Engineering(all)
- Bioengineering
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In: Engineering in life sciences, Vol. 19, No. 11, 04.11.2019, p. 730-740.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - High cell density transient transfection of CHO cells for TGF-β1 expression
AU - Elshereef, Abdalla A.
AU - Jochums, André
AU - Lavrentieva, Antonina
AU - Stuckenberg, Lena
AU - Scheper, Thomas
AU - Solle, Dörte
N1 - Funding information: The author would like to thank the Deutscher Akademischer Austausch Dienst 57076387 (DAAD) for financial support. Thanks are also extended to the Egyptian Knowledge Bank (EKB), group of nature research for the language editing of the manuscript.
PY - 2019/11/4
Y1 - 2019/11/4
N2 - 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-β1) in a shake flask. The purified TGF-β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.
AB - 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-β1) in a shake flask. The purified TGF-β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.
KW - CHO cells
KW - EGFP transfection efficiency
KW - PEI transient transfection
KW - TGF-β1 purification and bioactivity
UR - http://www.scopus.com/inward/record.url?scp=85071652518&partnerID=8YFLogxK
U2 - 10.1002/elsc.201800174
DO - 10.1002/elsc.201800174
M3 - Article
AN - SCOPUS:85071652518
VL - 19
SP - 730
EP - 740
JO - Engineering in life sciences
JF - Engineering in life sciences
SN - 1618-0240
IS - 11
ER -