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

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Abstract

Insect cell lines such as Sf9 and High Five (TM) 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 (TM) 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 (TM) 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. (C) 2013 Elsevier B.V. All rights reserved.
Original languageUnknown
Pages (from-to)436-439
JournalJournal of Biotechnology
Volume168
Issue number4
DOIs
Publication statusPublished - 1 Jan 2013

Cite this

@article{ecfc24782bfe431bae986f9ac039bdf4,
title = "A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five (TM) cells",
abstract = "Insect cell lines such as Sf9 and High Five (TM) 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 (TM) 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 (TM) 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. (C) 2013 Elsevier B.V. All rights reserved.",
keywords = "RMCE, FACS, Pluronic acid, High Five (TM) cells, Fluorescence tagged clones, Sf9 cells",
author = "Vidigal, {Jo{\~a}o Miguel Nunes} and Alves, {Paula Maria} and Manuel Carrondo",
year = "2013",
month = "1",
day = "1",
doi = "10.1016/j.jbiotec.2013.10.020",
language = "Unknown",
volume = "168",
pages = "436--439",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier Science B.V., Inc",
number = "4",

}

TY - JOUR

T1 - A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five (TM) cells

AU - Vidigal, João Miguel Nunes

AU - Alves, Paula Maria

AU - Carrondo, Manuel

PY - 2013/1/1

Y1 - 2013/1/1

N2 - Insect cell lines such as Sf9 and High Five (TM) 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 (TM) 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 (TM) 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. (C) 2013 Elsevier B.V. All rights reserved.

AB - Insect cell lines such as Sf9 and High Five (TM) 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 (TM) 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 (TM) 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. (C) 2013 Elsevier B.V. All rights reserved.

KW - RMCE

KW - FACS

KW - Pluronic acid

KW - High Five (TM) cells

KW - Fluorescence tagged clones

KW - Sf9 cells

U2 - 10.1016/j.jbiotec.2013.10.020

DO - 10.1016/j.jbiotec.2013.10.020

M3 - Article

VL - 168

SP - 436

EP - 439

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - 4

ER -