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-MassaguerEmail author
  • Martí Lecina
  • Francesc Gòdia
Biotechnological products and process engineering


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.


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



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)


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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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