A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells.

Abstract

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.

• Authors: João Vidigal, Mafalda M Dias, Fabiana Fernandes, Marco Patrone, Cláudia Bispo, Cláudia Andrade, Rui Gardner, Manuel J T Carrondo, Paula M Alves, Ana P Teixeira
• Journal: Journal of biotechnology (J Biotechnol) Vol. 168 Issue 4 Pg. 436-9 (Dec 2013) ISSN: 1873-4863 [Electronic] Netherlands
• PMID: 24161917 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013 Elsevier B.V. All rights reserved.

Chemical References

• Recombinant Proteins
• Recombinases
• Green Fluorescent Proteins

Topics

• Animals
• Cell Line
• Flow Cytometry (methods)
• Genetic Vectors
• Green Fluorescent Proteins (chemistry)
• Insecta (cytology)
• Recombinant Proteins (biosynthesis, genetics)
• Recombinases (genetics)