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Applied Microbiology and Biotechnology

, Volume 102, Issue 24, pp 10495–10510 | Cite as

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

  • Eduard Puente-Massaguer
  • Martí Lecina
  • Francesc Gòdia
Biotechnological products and process engineering
  • 114 Downloads

Abstract

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (< 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 106 cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/106 transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

Keywords

High Five cells Polyethylenimine Transient gene expression Cryo-electron microscopy Dynamic light scattering Design of experiments 

Notes

Acknowledgments

The authors would like to thank Dr. Paula Alves (Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal) for providing the BTI-TN-5B1-4 cell line and pITV5-eGFP plasmid vector. We also would like to thank Martí de Cabo and Mónica Roldán from Servei de Microscòpia of UAB for his support with the Cryo-EM and confocal microscopy, respectively. The help of Llorenç Badiella (Servei d’Estadística Aplicada, UAB) in developing the R code and on statistical analysis is also acknowledged. The help of José Amable Bernabé (Institut de Ciència de Materials de Barcelona, CSIC), Manuela Costa (Institut de Biotecnologia i Biomedicina, UAB), and Dr. Salvador Bartolomé (Departament de Bioquímica i de Biologia Molecular, UAB) for the assistance with DLS, cytometry, and fluorometry, respectively, are also appreciated. Eduard Puente-Massaguer is a recipient of a FPU grant from Ministerio de Educación, Cultura y Deporte of Spain (FPU15/03577). The research group is recognized as 2017 SGR 898 by Generalitat de Catalunya.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

253_2018_9423_MOESM1_ESM.pdf (2.6 mb)
ESM 1 (PDF 2.55 mb)

References

  1. Backliwal G, Hildinger M, Hasija V, Wurm FM (2008) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99:721–727CrossRefGoogle Scholar
  2. Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76:965–977CrossRefGoogle Scholar
  3. Bleckmann M, Schürig M, Chen FF, Yen ZZ, Lindemann N, Meyer S, Spehr J, Van Den Heuvel J (2016) Identification of essential genetic baculoviral elements for recombinant protein expression by transactivation in Sf21 insect cells. PLoS One 11:1–19CrossRefGoogle Scholar
  4. Bollin F, Dechavanne V, Chevalet L (2011) Design of experiment in CHO and HEK transient transfection condition optimization. Protein Expr Purif 78:61–68CrossRefGoogle Scholar
  5. Cervera L, Gutiérrez-granados S, Martínez M, Blanco J, Gòdia F, Segura MM, Mercedes M (2013) Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. J Biotechnol 166:152–165CrossRefGoogle Scholar
  6. Chan LCL, Reid S (2016) Development of serum-free media for lepidopteran insect cell lines. Humana Press, New York, pp 161–196Google Scholar
  7. Chen L, Xiao S, Zhu H, Wang L, Liang H (2016) Shape-dependent internalization kinetics of nanoparticles by membranes. Soft Matter 12:2632–2641CrossRefGoogle Scholar
  8. Chu Z, Zhang S, Zhang B, Zhang C, Fang C-Y, Rehor I, Cigler P, Chang H-C, Lin G, Liu R, Li Q (2015) Unambiguous observation of shape effects on cellular fate of nanoparticles. Sci Rep 4:4495CrossRefGoogle Scholar
  9. Clément N, Grieger JC (2016) Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev 3:16002.  https://doi.org/10.1038/mtm.2016.2 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Derringer G, Suich R (1980) Simultaneous optimization of several response variables. J Qual Technol 12:214–219CrossRefGoogle Scholar
  11. Fernandes F, Vidigal J, Dias MM, Prather KLJ, Coroadinha AS, Teixeira AP, Alves PM (2012) Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression. Biotechnol Bioeng 109:2836–2844CrossRefGoogle Scholar
  12. Fernandes F, Teixeira AP, Carinhas N, Carrondo MJT, Alves PM (2013) Insect cells as a production platform of complex virus-like particles. Expert Rev Vaccines 12:225–236CrossRefGoogle Scholar
  13. Fuenmayor J, Cervera L, Gutiérrez-Granados S, Gòdia F (2018) Transient gene expression optimization and expression vector comparison to improve HIV-1 VLP production in HEK293 cell lines. Appl Microbiol Biotechnol 102:165–174CrossRefGoogle Scholar
  14. Glaeser RM (2016) How good can cryo-EM become? Nat Methods 13:28–32CrossRefGoogle Scholar
  15. Gutierrez-Granados S, Cervera L, Segura Mde L, Wolfel J, Godia F (2016) Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells. Appl Microbiol Biotechnol 100:3935–3947CrossRefGoogle Scholar
  16. Gutierrez-Granados S, Cervera L, Kamen AA, Godia F (2018) Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics. Crit Rev Biotechnol 38:1–23CrossRefGoogle Scholar
  17. Hacker DL, Kiseljak D, Rajendra Y, Thurnheer S, Baldi L, Wurm FM (2013) Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expr Purif 92:67–76CrossRefGoogle Scholar
  18. Islam R, Sparling R, Cicek N, Levin D (2015) Optimization of influential nutrients during direct cellulose fermentation into hydrogen by Clostridium thermocellum. Int J Mol Sci 16:3116–3132CrossRefGoogle Scholar
  19. Kuhnt S, Rudak N (2013) Simultaneous optimization of multiple responses with the R package JOP. J Stat Softw 54:1–23CrossRefGoogle Scholar
  20. Liu F, Wu X, Li L, Liu Z, Wang Z (2013) Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expr Purif 90:104–116CrossRefGoogle Scholar
  21. Lu M, Johnson RR, Iatrou K (1996) Trans-activation of a cell housekeeping gene promoter by the IE1 gene product of baculoviruses. Virology 218:103–113CrossRefGoogle Scholar
  22. Lu M, Farrell PJ, Johnson R, Iatrou K (1997) A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells. J Biol Chem 272:30724–30728CrossRefGoogle Scholar
  23. Montgomery DC (2012) Design and analysis of experiments, 5th edn. Wiley, ArizonaGoogle Scholar
  24. Mori K, Hamada H, Ogawa T, Ohmuro-matsuyama Y, Katsuda T, Yamaji H (2017) Efficient production of antibody Fab fragment by transient gene expression in insect cells. J Biosci Bioeng xx:3–8Google Scholar
  25. Osz-Papai J, Radu L, Abdulrahman W, Kolb-Cheynel I, Troffer-Charlier N, Birck C, Poterszman A (2015) Insect cells–baculovirus system for the production of difficult to express proteins. Humana Press, New York, pp 181–205Google Scholar
  26. Paillet C, Forno G, Soldano N, Kratje R, Etcheverrigaray M (2011) Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero). Vaccine 29:7212–7217CrossRefGoogle Scholar
  27. Palomares LA, Realpe M, Ramírez OT (2015) An overview of cell culture engineering for the insect cell-baculovirus expression vector system (BEVS). Springer, Cham, pp 501–519Google Scholar
  28. Peixoto JL (1987) Hierarchical variable selection in polynomial regression models. Am Stat 41:311Google Scholar
  29. Radner S, Celie PHN, Fuchs K, Sieghart W, Sixma TK, Stornaiuolo M (2012) Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells. J Struct Biol 179:46–55CrossRefGoogle Scholar
  30. Raup A, Wang H, Synatschke CV, Jérôme V, Agarwal S, Pergushov DV, Müller AHE, Freitag R (2017) Compaction and transmembrane delivery of pDNA: differences between l-PEI and two types of amphiphilic block copolymers. Biomacromolecules 18:808–818CrossRefGoogle Scholar
  31. Roest S, Kapps-Fouthier S, Klopp J, Rieffel S, Gerhartz B, Shrestha B (2016) Transfection of insect cell in suspension for efficient baculovirus generation. MethodsX 3:371–377CrossRefGoogle Scholar
  32. Román R, Miret J, Scalia F, Casablancas A, Lecina M, Cairó JJ (2016) Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide. J Biotechnol 239:57–60CrossRefGoogle Scholar
  33. Royl P (1995) Transient expression in insect cells using a recombinant baculovirus synthesising bacteriophage T7 RNA polymerase. Nucleic Acids Res 23:188–191CrossRefGoogle Scholar
  34. Sander L, Harrysson A (2007) Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein. Cytotechnology 54:35–48CrossRefGoogle Scholar
  35. Sang Y, Xie K, Mu Y, Lei Y, Zhang B, Xiong S, Chen Y, Qi N (2015) Salt ions and related parameters affect PEI-DNA particle size and transfection efficiency in Chinese hamster ovary cells. Cytotechnology 67:67–74CrossRefGoogle Scholar
  36. Shen X, Hacker DL, Baldi L, Wurm FM (2013) Virus-free transient protein production in Sf9 cells. J Biotechnol 171:61–70.  https://doi.org/10.1016/j.jbiotec.2013.11.018 CrossRefPubMedGoogle Scholar
  37. Shen X, Pitol AK, Bachmann V, Hacker DL, Baldi L, Wurm FM (2015) A simple plasmid-based transient gene expression method using High Five cells. J Biotechnol 216:67–75CrossRefGoogle Scholar
  38. Shoja Z, Tagliamonte M, Jalilvand S, Mollaei-Kandelous Y, De stradis A, Tornesello ML, Buonaguro FM, Buonaguro L (2015) Formation of self-assembled triple-layered rotavirus-like particles (tlRLPs) by constitutive co-expression of VP2, VP6, and VP7 in stably transfected high-five insect cell lines. J Med Virol 87:102–111CrossRefGoogle Scholar
  39. Thompson BC, Segarra CRJ, Mozley OL, Daramola O, Field R, Levison PR, James DC (2012) Cell line specific control of polyethylenimine-mediated transient transfection optimized with “design of experiments” methodology. Biotechnol Prog 28:179–187CrossRefGoogle Scholar
  40. van Gaal EVB, van Eijk R, Oosting RS, Kok RJ, Hennink WE, Crommelin DJA, Mastrobattista E (2011) How to screen non-viral gene delivery systems in vitro? J Control Release 154:218–232CrossRefGoogle Scholar
  41. Vera Candioti L, De Zan MM, Cámara MS, Goicoechea HC (2014) Experimental design and multiple response optimization. Using the desirability function in analytical methods development. Talanta 124:123–138CrossRefGoogle Scholar
  42. Vidigal J, Fernandes B, Dias MM, Patrone M, Roldão A, Carrondo MJT, Alves PM, Teixeira AP (2018) RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles. Appl Microbiol Biotechnol 102:655–666CrossRefGoogle Scholar
  43. Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departament d’Enginyeria Química, Biológica i Ambiental, Escola d’EnginyeriaUniversitat Autònoma de BarcelonaBarcelonaSpain
  2. 2.IQS School of EngineeringUniversitat Ramón LlullBarcelonaSpain

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