柠檬酸铁铵对悬浮HEK293细胞转染的影响机制探究

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

摘要/Abstract

摘要：

基于聚乙烯亚胺（polyethyleneimine, PEI）的悬浮人胚胎肾细胞（human embryonic kidney 293 cells, HEK293）转染技术前景广阔，但培养基组分会影响转染效率。简单的离心换液存在污染风险，且影响细胞状态。探究抑制转染效率的关键组分对转染过程的影响机制，有利于从根本上解决该问题。首先通过探究培养基中对转染效率具有潜在影响的组分确定关键组分柠檬酸铁铵（ferric ammonium citrate, FAC）的抑制作用，然后通过考察柠檬酸铁铵添加对细胞状态、PEI与DNA形成的复合物（PEI-DNA complex, PEI-DNA复合物）结合情况、目的基因表达情况的影响，分析其抑制转染效率的机制。结果表明，高浓度的柠檬酸铁铵对细胞转染存在明显抑制作用，且该抑制作用随着柠檬酸铁铵浓度的升高而逐渐增强。当柠檬酸根和铁离子同时存在时才会抑制细胞转染过程。过高浓度的柠檬酸铁铵使PEI-DNA复合物的粒径显著增加，复合物进入细胞更加困难，导致进入细胞的复合物数量减少，最终引起细胞转染效率的大幅下降。柠檬酸铁铵通过影响PEI-DNA复合物的大小限制DNA进入细胞，从而抑制PEI介导的悬浮HEK293细胞转染过程。控制转染时柠檬酸铁铵浓度在20 μmol/L内可解决其对转染过程的抑制作用。本研究深入认识了柠檬酸铁铵对PEI介导的悬浮HEK293细胞转染过程的影响及其机制，为悬浮HEK293细胞转染培养基的开发和理性设计提供有力参考。

关键词: 悬浮HEK293细胞, 转染效率, 柠檬酸铁铵, PEI介导的转染, 哺乳动物细胞瞬时表达系统

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

陈中元, 王玉红, 代为俊, 张艳敏, 叶倩, 刘旭平, 谭文松, 赵亮. 柠檬酸铁铵对悬浮HEK293细胞转染的影响机制探究[J]. 生物技术通报, 2023, 39(9): 311-318.

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.

图/表 8

图1 添加柠檬酸铁铵对HEK293细胞转染的影响 A：细胞在含柠檬酸铁铵的Celers101培养基中转染结果；B：细胞在不含柠檬酸铁铵的Celers101培养基中的转染结果。1：荧光；2：明场

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

图2 浓度柠檬酸铁铵对转染效率的影响与10 μmol/L实验组的转染效率相比，*P<0.05，**P<0.01，***P<0.001，下同

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

图3 不同柠檬酸根供体和铁供体对转染效率的影响 A：对照（不加铁元素）；B：转铁蛋白；C：柠檬酸钠；D：柠檬酸；E：氯化铁；F：柠檬酸钾；G：EDTA钠铁；H：柠檬酸铁；I：柠檬酸铁铵

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

图4 柠檬酸铁铵对细胞周期（A）和细胞凋亡（B）的影响

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

图5 柠檬酸铁铵对PEI与DNA结合的影响 DNA：仅添加质粒DNA，PEI和DNA添加比例为0∶1；0.2：PEI和DNA添加比例为0.2∶1；1.2：PEI和DNA添加比例为1.2∶1；3：PEI和DNA添加比例为3∶1

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

图6 柠檬酸铁铵对复合物的粒径分布（A）、表面电位分布（B）、平均粒径（C）变化和平均电势（D）变化的影响

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

图7 柠檬酸铁铵对转染后细胞中复合物数量的影响

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

图8 柠檬酸铁铵在转染后对细胞的转染效率（A）和平均荧光强度（B）的影响

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

参考文献 26

[1]	Zhu JW. Mammalian cell protein expression for biopharmaceutical production[J]. Biotechnol Adv, 2012, 30(5): 1158-1170. doi: 10.1016/j.biotechadv.2011.08.022 pmid: 21968146
[2]	Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine)and its role in gene delivery[J]. J Control Release, 1999, 60(2/3): 149-160. doi: 10.1016/S0168-3659(99)00090-5 URL
[3]	Coll JL, Chollet P, Brambilla E, et al. In vivo delivery to tumors of DNA complexed with linear polyethylenimine[J]. Hum Gene Ther, 1999, 10(10): 1659-1666. pmid: 10428211
[4]	Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells[J]. Nat Biotechnol, 2004, 22(11): 1393-1398. doi: 10.1038/nbt1026 pmid: 15529164
[5]	Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future[J]. Anal Bioanal Chem, 2010, 397(8): 3173-3178. doi: 10.1007/s00216-010-3821-6 pmid: 20549496
[6]	Lai WF. In vivo nucleic acid delivery with PEI and its derivatives: current status and perspectives[J]. Expert Rev Med Devices, 2011, 8(2): 173-185. doi: 10.1586/erd.10.83 URL
[7]	Tan E, Chin CSH, Lim ZFS, et al. HEK293 cell line as a platform to produce recombinant proteins and viral vectors[J]. Front Bioeng Biotechnol, 2021, 9: 796991. doi: 10.3389/fbioe.2021.796991 URL
[8]	Gutiérrez-Granados S, Cervera L, Kamen AA, et al. Advancements in mammalian cell transient gene expression(TGE)technology for accelerated production of biologics[J]. Crit Rev Biotechnol, 2018, 38(6): 918-940. doi: 10.1080/07388551.2017.1419459 pmid: 29295632
[9]	Torabfam GC, Yetisgin AA, Erdem C, et al. A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells[J]. Cytotechnology, 2022, 74(6): 635-655. doi: 10.1007/s10616-022-00551-1 pmid: 36389283
[10]	Cervera L, Gutiérrez-Granados S, Berrow NS, et al. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement[J]. Biotechnol Bioeng, 2015, 112(5): 934-946. doi: 10.1002/bit.25503 pmid: 25421734
[11]	Dekevic G, Tasto L, Czermak P, et al. Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures[J]. J Biotechnol, 2022, 346: 23-34. doi: 10.1016/j.jbiotec.2022.01.004 URL
[12]	Park JY, Lim BP, Lee K, et al. Scalable production of adeno-associated virus type 2 vectors via suspension transfection[J]. Biotechnol Bioeng, 2006, 94(3): 416-430. pmid: 16622883
[13]	Abbott WM, Middleton B, Kartberg F, et al. Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale[J]. Protein Expr Purif, 2015, 116: 113-119. doi: 10.1016/j.pep.2015.08.016 URL
[14]	Mercedes SM, Alain G, Yves D, et al. Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification[J]. Biotechnol Bioeng, 2007, 98(4): 789-99. pmid: 17461423
[15]	Haldankar R, Li DQ, Saremi Z, et al. Serum-free suspensin large-scale transient transfection of CHO cells in WAVE bioreactors[J]. Mol Biotechnol, 2006, 34(2): 191-199. pmid: 17172664
[16]	van Gaal EVB, van Eijk R, Oosting RS, et al. How to screen non-viral gene delivery systems in vitro?[J]. J Control Release, 2011, 154(3): 218-232. doi: 10.1016/j.jconrel.2011.05.001 URL
[17]	Pham PL, Perret S, Doan HC, et al. Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency[J]. Biotechnol Bioeng, 2003, 84(3): 332-342. pmid: 12968287
[18]	Vega MC. Advanced technologies for protein complex production and characterization[J]. Anticancer Research, 2016, 36(8): 4375. pmid: 27466587
[19]	Eberhardy SR, Radzniak L, Liu Z. Iron(III)citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium[J]. Cytotechnology, 2009, 60(1): 1-9. doi: 10.1007/s10616-009-9198-8 URL
[20]	Yu Y, Kovacevic Z, Richardson DR. Tuning cell cycle regulation with an iron key[J]. Cell Cycle, 2007, 6(16): 1982-1994. pmid: 17721086
[21]	Bai YL, Wu CJ, Zhao J, et al. Role of iron and sodium citrate in animal protein-free CHO cell culture medium on cell growth and monoclonal antibody production[J]. Biotechnol Prog, 2011, 27(1): 209-219. doi: 10.1002/btpr.v27.1 URL
[22]	Capella Roca B, Lao NT, Clynes M, et al. Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells[J]. Biotechnol Prog, 2020, 36(3): e2954. doi: 10.1002/btpr.2954 URL
[23]	Jorge AF, Röder R, Kos P, et al. Combining polyethylenimine and Fe(III)for mediating pDNA transfection[J]. Biochim Biophys Acta Gen Subj, 2015, 1850(6): 1325-1335. doi: 10.1016/j.bbagen.2015.02.007 URL
[24]	Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts: molecular control of mammalian iron metabolism[J]. Cell, 2004, 117(3): 285-297. doi: 10.1016/s0092-8674(04)00343-5 pmid: 15109490
[25]	Prabha S, Zhou WZ, Panyam J, et al. Size-dependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles[J]. Int J Pharm, 2002, 244(1/2): 105-115. doi: 10.1016/S0378-5173(02)00315-0 URL
[26]	van Asbeck AH, Beyerle A, McNeill H, et al. Molecular parameters of siRNA-cell penetrating peptide nano complexes for efficient cellular delivery[J]. ACS Nano, 2013, 7(5): 3797-3807. doi: 10.1021/nn305754c URL