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Real-time transfer of lentiviral particles by producer cells using an engineered coculture system

  • Lauren M. Timmins
  • Riya S. Patel
  • Matthew S. Teryek
  • Biju ParekkadanEmail author
Original Article
  • 15 Downloads

Abstract

Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6 days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.

Keywords

Lentivirus transduction Engineered cell therapy Scalable engineering systems 

Notes

Acknowledgements

This work was supported in part by the National Institutes of Health Grants (R01GM127353 and R01EB012521).

Author contributions

LMT and BP formulated the research study design, LMT conducted experiments, acquired data, analyzed data, wrote manuscript. RP assisted in conducting experiments and acquiring data. MT assisted in conducting experiments.

Supplementary material

10616_2019_343_MOESM1_ESM.docx (4.9 mb)
Supplementary material 1 (DOCX 5075 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringRutgers UniversityPiscatawayUSA

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