Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Aline Do Minh
,
Alexandra T. Star
ORCID logo ,
Jacek Stupak
,
Jean-Francois Gélinas
ORCID logo ,
Jianjun Li
,
Susan M. Twine
,
Amine A. Kamen
*
Version 1 : Received: 28 March 2021 / Approved: 29 March 2021 / Online: 29 March 2021 (13:53:53 CEST)

A peer-reviewed article of this Preprint also exists.

Do Minh, A.; Star, A.T.; Stupak, J.; Fulton, K.M.; Haqqani, A.S.; Gélinas, J.-F.; Li, J.; Twine, S.M.; Kamen, A.A. Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures. Viruses 2021, 13, 797. Do Minh, A.; Star, A.T.; Stupak, J.; Fulton, K.M.; Haqqani, A.S.; Gélinas, J.-F.; Li, J.; Twine, S.M.; Kamen, A.A. Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures. Viruses 2021, 13, 797.

Abstract

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purify with LVs during downstream processing, becoming “impurities” in the context of cell therapy. To characterize EVs from an inducible lentivirus producing cell line, two conditions were studied: non-induced and induced. EVs’ identity was confirmed by transmission electron microscopy and western blot. Seven proteins were identified by mass spectrometry as potential EV markers. Lipid composition of EVs and LVs showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in genes involved in viral processes and binding functions. Flow virometry, GTA and ddPCR results also confirmed the heterogenic nature of EVs and LVs populations. These findings provide insights on the product profile of lentiviral preparation and could help develop separation strategies of co-produced EVs.

Keywords

extracellular vesicles; enveloped viruses; lentiviral vectors; exosome; proteomic; lipidomic; transcriptomic

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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