Sf9 spodoptera frugiperda insect cells instructions for. We therefore here describe methods for the production of milligram quantities of recombinant mouse kindlin3 in insect cell culture, suitable for extensive structural studies and biochemical. In either case, care should be taken while growing sf9 cells, as they are extremely sensitive to fluctuations in temperature, cell density, and agitation. Gibco sf9 cells in sf900 ii sfm were prepared as serumfree, suspension cultures from sf9 cells that originated at the usda insect pathology laboratory. Kit cells medium insectselect system with sf9 cells sf9 graces insect cell culture medium, unsupplemented contains lglutamine 2 x 500 ml insectselect system with high five cells. Schematic diagram for the production and purification of baculovirus. The original sf9 cells were cloned from the parental iplbsf21 sf21 cell line that was derived from the pupal ovarian tissue of the fall army worm, spodoptera frugiperda. Cell culture basics handbook thermo fisher scientific. Insect cell culture using the new brunswick bioflo. This protocol is a slight modification of expression systems protocols for generating viruses. Culture mode sf9 insect cells can be grown using any of the three. Efficient production and purification of recombinant murine. Cell culture is the process by which cells are grown under controlled conditions.
Sf9 cells are seeded in cell culturetreated plate transfected with bacmid dna. Nov 16, 2017 in the last three decades, the baculovirus expression vector system bev has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Graces insect cell culture medium, supplemented instructions for use product description graces insect cell culture medium is commonly used to support cell growth, protein expression, and baculovirus vector production for spodoptera frugiperda insect cells such as sf9 and sf21. Introduction to sf9 insect culture in a stirredtank bioreactor. A new singlestep protocol for rapid baculovirusdriven protein production in insect cells. A new singlestep protocol for rapid baculovirusdriven.
The following morphological changes are typical of monolayer sf9 cells infected with recombinant acnpv. Tissue culture treated dishes for seeding insect cells appropriate complete insect cell culture medium e. Herein, we used online digital holographic microscopy dhm to monitor bioreactor cultures of sf9 insect cells. We therefore here describe methods for the production of milligram quantities of recombinant mouse kindlin3 in insect cell culture, suitable for extensive structural studies and biochemical analysis. Growth and maintenance of insect cell lines the wolfson centre.
Pdf affordable uniform isotope labeling with 2h, c and. All handling of insect cell lines should be carried out under sterile conditions, as you would for mammalian cell culture. Insect virus latest ally in gene therapy production. Sf9 cells in sf900 ii sfm thermo fisher scientific. Media of the transfected culture were exchanged at 10, 30, 60, 90, 120, 180 minutes posttransfection. Iper insect cell protein extraction reagent extracts cytoplasmic protein from sf9 and sf21 insect cells grown in suspension and adherent cultures. In the highcelldensity culture process, the insect cells were infected. Infected cells are subsequently propagated in serumfree suspension culture. Transient expression of foreign genes in insect cells sf9 for protein functional assay. They were originally established from ovarian tissue. At oet we recommend initially setting up the cells in an adherent. Sf9 cell transfection using lipofectamine and is cmv.
Use vented shake, spinner, or tissue culture flask caps to ensure adequate dissolved oxygen content. The insect cell culture based protein expression system is inexpensive and easy to scale up. Sf9 insect cells can be grown as either a monolayer in a tissue culture flask or in suspension culture in spinner flasks. Insect cell culture using the new brunswick bioflo celligen. Transfection of insect cell in suspension for efficient baculovirus. This protocol describes a heat shockinduced protein expression system pdhspv5his sf9 cell system, which can be used for either expressing foreign proteins or evaluating the antiapoptotic activity of potential foreign proteins and their truncated amino acids in insect cells. May 20, 2002 insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. This protocol is used to generate baculoviruses containing gpcr genes of interest for expression in sf9 cells. Figure 1 vertiga shaker manufactured by thomson instruments co.
Lynn, in molecular and diagnostic procedures in mycoplasmology, 1995. The cells are highly susceptible to baculovirus infection and are used in the production of protein products genetically manipulated into baculovirus vector systems. Insect cell culture prepare 50 ml of cell culture medium. Oet insect cell culture manual oxford expression technologies. Gibco sf9 cells are adapted to serumfree suspension culture in gibco sf900 ii sfm, which saves significant time and expense associated with the adaptation of cultures. For more information about maintaining suspension and adherent cultures, refer to the guide to baculovirus expression vector systems bevs and insect cell culture techniques, which is available for downloading from our web. Introduction to insect cell line strains spodoptera frugiperda sf9 and sf21 insect cells are cell lines commonly used for expression of recombinant proteins using baculovirus systems. The sf9, sf21, and high fivez cell lines are suitable for use in expressing recombinant proteins with. Maintain sf9 cells an established cell culture line derived from spodoptera frugiperda ovarian tissue similarly but using tnmfh insect culture medium supplemented with 10% fetal bovine serum fbs. Types of cell lines there are three commercially available cell lines generally used for baculovirus expression. The standard protocol for generating recombinant baculovirus comprises.
The composition of iper reagent is compatible with downstream processing steps such as 6xhistagged protein purification and ion exchange chromatography. In the highcelldensity culture process, the insect cells were infected when the cell density was around 9. Insect cell sf9 perfusion culture in a 2 l bioflo celligen 115 bioreactor with spin filter impeller. Transfection of sf9 cells in suspension nih common fund. The developed transfection protocol has now been successfully used to transfect.
Jan, 2011 cell culture is the process by which cells are grown under controlled conditions. The metabolic condition of the culture and growth medium used also can. We have described a superior method to transfect insect cells using polyethyleneimine pei in suspension culture for the purpose of recombinant. Sf9 cell line from spodoptera frugiperda pupal ovarian. Production and purification of baculovirus for gene. Sf9 spodoptera frugiperda insect cells instructions for use product description the sf9 insect cell line is a clonal isolate derived from the parental spodoptera frugiperda cell line iplb. We recommend thawing a fresh, lowpassage culture of frozen sf9 cells every 3 months or 30 passages. Invitrogens bactobac and bacnblue expression systems. The optimal temperature for sf9 and sf21 growth and infection is 2729c. Using the sf9 easy titer cell line 21 we have measured p0 titers for.
Sf9 cells lose exponential growth, lack stationary phase. Inoculum inoculum should be grown to a density of 4. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. Adherent culture of sf9 cells was maintained in plate cultures containing graces insect medium gibco supplemented with 10% fbs, at 28c. Purifying histagged proteins from insect and mammalian cells. A new singlestep protocol for rapid baculovirusdriven protein. The sf9 cells are adapted to serum free suspension culture in esf 921. The cells were inoculated from ml shaker flask culture. In this protocol we make use of an engineered knockout bacmid bac10 ko.
Sf9 cell line from spodoptera frugiperda pupal ovarian tissue. Cells are maintained at 2728 c in both monolayer and suspension culture co2 atmosphere is not necessary. For largescale applications, the baculovirus expression vector system bevs is particularly advantageous. Baculovirus supernatant is harvested and used to infect new sf9 cells in adherent culture. Cell line description derived from pupal ovarian tissue of spodoptera frugiperda. Insect cell growth medium t25t75 monolayer flasks 125 ml cell culture shake flask 1ml and 10 ml sterile pipettes incubator at 2728c and a shaking platform 100110 rpm water bath at 37c best to use a temporary bath such as a clean beaker with warm clean water rather than a dirty water bath protocol. Graces insect cell culture medium, supplemented instructions. Excell 420 serumfree medium for insect cells with l. Suspension culture of sf9 cells was maintained in shake flask cultures containing sf900ii sfm gibco, at 28c. Cell culture is a vital technology used in life science research and in biotechnology laboratories. The cells are highly susceptible to baculovirus infection and are used in the production of protein products. Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. The sf9 insect cell line is a clonal isolate derived from the parental. Cell culture was harvested at 96 hpi and processed for aav recovery and analysis.
This application note presents a simple protocol for achieving highdensity culture of sf9 insect cells using an eppendorf benchtop, autoclavable stirredtank bioreactor. Gibco sf9 cells are commonly used to isolate and propagate recombinant baculoviral stocks and to produce recombinant proteins. Insect cell culture media historically have been based on hemolymph characteristics of the insect of interest. Confluency confluency is a marker for when to subculture your cells. Full protocols can be downloaded from our insect cell culture user guide at. We recommend starting the cells in adherent culture and. Application excell 420 is a complete medium developed and optimized for the serumfree growth of sf9 and sf21 insect cell lines. Excell 420 serumfree medium for insect cells with lglutamine, sterilefiltered, suitable for insect cell culture. At 96 hpi, the culture was harvested, cells were lysed, and the clarified lysate was collected for subsequent analysis. To make the complete growth medium, add the following. Sf9 spodoptera frugiperda insect cells instructions for use product description the sf9 insect cell line is a clonal isolate derived from the parental spodoptera frugiperda cell line iplbsf21ae. Insect cells are biohazard level 1, but are susceptible to bacterial infection if sterile technique is poor. Maintain sf9 cells an established cell culture line derived from spodoptera.
If the insect culture medium used employs a phosphate buffering system, a co2 incubator is not required e. Sop oppf insect cell expression last updated 6th october 2016 4 2. Graces insect cell culture medium, supplemented instructions for use product description graces insect cell culture medium is commonly used to support cell growth. Despite the significant improvements introduced during the past years, the bev system still has major drawbacks, primarily the time required to generate recombinant virus and virus. These cell lines were generated from the parental cell line ipblsf21, which was derived from the pupal ovarian tissue of the fall army worm vaughn, et al. Since the development of the baculovirus expression vector system smith et al. Growth and maintenance of insect cell lines user guide pub. Transfection of sf9 cells in suspension december, 2008 version 1. Application sf9 cell line has been used for baculovirus viral studies. In either case, care should be taken while growing sf9 cells, as they. Study of the improved sf9 transient gene expression process. The sf9 insect cell line is a clonal isolate derived from the parental spodoptera frugiperda cell line iplbsf21ae1,2, and is a suitable host for expression of recombinant proteins from baculovirus expression systems3,4 e. Specialized media, transfection reagents, and vectors have been developed in response to recent advances in insect cell culture and molecular biology methods.
Sf9 cells are a clonal isolate from spodoptera frugiperda fall armyworm iplbsf21ae cells. Sf9 cells, a clonal isolate of spodoptera frugiperda sf21 cells iplbsf21ae, are commonly used in insect cell culture for recombinant protein production using baculovirus. The base medium for this cell line is graces insect medium supplemented gibcoinvitrogen cat. Instructions iper insect cell protein extraction reagent. Sf9 or sf21 trans it insect transfection reagent poet transfer plasmid or other compatible vector containing gene of interest. Production and purification of baculovirus for gene therapy. Insect cell cultures are now commonly used in insect physiology. Introduction to sf9 insect culture in a stirredtank. To culture sf9 and high five cells, refer to the insect cell lines manual included with each kit. Sf9 cells lose exponential growth, lack stationary phase jan272012 hey there, ive been maintaining shaker flasks of sf9 insect cells for the past 2. Sf9 spodoptera frugiperda insect cells instructions for use.
The sf9 insect cell line is a clonal isolate derived from the parental spodoptera frugiperda cell line iplbsf21ae1,2, and is a suitable host for expression of recombinant proteins from. Transfection of insect cell lines using polyethylenimine. Efficient production and purification of recombinant. Whilst many labs use sf9 for all protocols, sf21 cells are superior for plaque assays and monitoring virus cytopathic effects and are more tolerant to sub optimal. Types of cell lines there are three commercially available cell lines generally used for. Recombinant protein production in baculovirusinsect. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. A sf9 cells were transfected with egfpcoding plasmid dna and pei at a starting cell density of 4. Arid land agricultural research center, agricultural. A confluent monolayer is an adherent cell culture dish, plate or flask in which the cells have formed a single layer over the entire surface area available for growth. Insect cell culture an overview sciencedirect topics. This paper describes methods that are effective for maintaining various insect cell lines. Whilst many labs use sf9 for all protocols, sf21 cells are superior for plaqueassays and monitoring virus cytopathic effects and are more tolerant to suboptimal. The metabolic condition of the culture and growth medium used also can affect the timing of baculovirus infection.
Sf9 insect cells have become the workhorse of the baculovirus expression system. This manual is included with sf9, sf21, and high five insect cells and. Determine cell density electronically using a coulter. Transient expression of foreign genes in insect cells sf9. Affordable uniform isotope labeling with 2h, c and 15n in insect cells. Being eukaryotic, insect cells allow correct folding and posttranslation modification, and. Based system for enhanced protein and virus production in. The sf9, sf21, and high fivez cell lines are suitable for use in expressing recombinant proteins with baculovirus and other insect expression systems e. Sf9 cells are seeded in cell culture treated plate transfected with bacmid dna. These advantages make insect cell culture an important tool in the expression of eukaryotic proteins. Guide to baculovirus expression vector systems bevs and. Batches of baculovirusinfected sf9 cells producing recombinant adenoassociated virus aav and noninfected cells were used to evaluate dhm prediction capabilities for viable cell concentration, culture viability and aav titer.
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