Mechanism Investigation of Ferric Ammonium Citrate on Transfection for Suspended HEK293 Cells

doi:10.13560/j.cnki.biotech.bull.1985.2023-0177

Abstract

Abstract:

Suspended HEK293（human embryonic kidney）cell transfection based on PEI（polyethyleneimine）is a prospective technology. However, the components of the medium often affect the transfection efficiency, which needs to be changed with transfection medium by centrifugation. This process is cumbersome and not only has the risk of contamination, but also affects the cell state. In order to fundamentally solve this problem, the influence mechanism of key components that inhibit the efficiency of transfection on the transition process need to be explored. First we explored that the key composition that have potential effects on transfection efficiency in the medium is ferric ammonium citrate（FAC）, and determined the its inhibitory effect. Then we analyzed its mechanism for inhibiting transit efficiency, including the effects of ferric ammonium citrate on the cell state, PEI-DNA complex formation, and destination gene expression. Results showed that certain amount of FAC had significant inhibitory effect on the transfection that was also gradually enhanced with concentration increasing. Further studies found that the transfection was inhibited only when citrate and iron ions were present at the same time. Over high concentration of FAC increased the particle size of the PEI-DNA complex, making it more difficult to enter the cell. This led to a reduction in the number of complexes entering the cells, and ultimately resulted in a significant drop in cell transfection efficiency. FAC restricted DNA into cells by affecting the size of the PEI-DNA complex, thereby inhibiting PEI-mediated suspended HEK293 cell transfusion process. The inhibitory effect can be eliminated when the FAC concentration was < 20 μmol/L. Through this study, the effect of FAC on the transfection process of PEI-mediated suspended HEK293 cells and its mechanism were deeply understood, providing a solid reference for the development and rational design of the transfection medium for suspended HEK293 cells.

Key words: suspended HEK293 cells, transfection efficiency, ferric ammonium citrate, PEI mediated transfection, mammalian cell transient gene expression technology

CHEN Zhong-yuan, WANG Yu-hong, DAI Wei-jun, ZHANG Yan-min, YE Qian, LIU Xu-ping, TAN Wen-Song, ZHAO Liang. Mechanism Investigation of Ferric Ammonium Citrate on Transfection for Suspended HEK293 Cells[J]. Biotechnology Bulletin, 2023, 39(9): 311-318.

Figures/Tables 8

Fig. 1 Effects of adding ferric ammonium citrate on HEK293 cell transfection A: Transfection efficiency of cells in Celers101 medium containing ferric ammonium citrate. B: Transfection efficiency of cells in Celers101 medium without ferric ammonium citrate. 1: Fluorescence; 2: bright filed

Fig. 2 Effect of ferric ammonium citrate concentration on transfection efficiency Compared with 10 μmol/L group, *P<0.05; **P<0.01; ***P<0.001, the same below

Fig. 3 Effects of different citrate donors and iron donors on transfection efficiency A: Control（Without iron）. B: Transferrin. C: Sodium citrate. D: Citric acid. E: FeCl3. F: Potassium citrate. G: FeNaEDTA. H: Iron（III）citrate. I: Ferric ammonium citrate

Fig. 4 Effect of ferric ammonium citrate on cell cycle（A）and apoptosis（B）

Fig. 5 Effects of ferric ammonium citrate on the binding of PEI to DNA DNA: PEI and DNA added ratio is 0∶1. 0.2: PEI and DNA added ratio is 0.2∶1. 1.2: PEI and DNA added ratio is 1.2∶1. 3: PEI and DNA added ratio is 3∶1

Fig. 6 Changes of particle size distribution（A）, zeta potential distribution（B）, average particle size（C）and average potential（D）after ferric ammonium citrate（FAC）added

Fig. 7 Effects of FAC on the number of complexes in transfected cells

Fig. 8 Effects of FAC on the cell transfection efficiency（A）and mean fluorescence intensity（B）after transfection

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