title: 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.affiliation: IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: João
authors.initials: J
authors.lastname: Vidigal
authors.forename: Mafalda M
authors.initials: MM
authors.lastname: Dias
authors.forename: Fabiana
authors.initials: F
authors.lastname: Fernandes
authors.forename: Marco
authors.initials: M
authors.lastname: Patrone
authors.forename: Cláudia
authors.initials: C
authors.lastname: Bispo
authors.forename: Cláudia
authors.initials: C
authors.lastname: Andrade
authors.forename: Rui
authors.initials: R
authors.lastname: Gardner
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.forename: Ana P
authors.initials: AP
authors.lastname: Teixeira
mesh_terms: D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007313:Insecta / Q000166:cytology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D045522:Recombinases / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D045522:Recombinases; D049452:Green Fluorescent Proteins
keywords: FACS; Fluorescence-tagged clones; High Five™ cells; Pluronic acid; RMCE; Sf9 cells