Insect cell culture

This presentation covers the introduction to Insect Cell Culture. Also covers its general information about cell culture practices followed in the lab. It covers culture media, the source of cells for culture and examples of the cell line with their culture conditions.

1. Insect cell culture Presented by- Sushant Balasaheb Jadhav Roll No. – 18PBT206 Pharmaceutical Biotechnology Institute of Chemical Technology, Mumbai

2. Introduction The use of  insect cell lines  as production hosts is an emerging technology for the production of bio pharmaceuticals. There are currently more than 100 insect cell lines available for recombinant protein production with lines derived from  Bombyx mori ,  Mamestra brassicae ,  Spodoptera frugiperda ,  Trichoplusia ni , and  Drosophila melanogaster  being of particular interest. Insects cell lines are commonly used in place of prokaryotic ones because post-translational modifications of proteins are possible in insect cells whereas this mechanism is not present in prokaryotic systems .

3. Introduction cont. Commonly used cell lines are sf9 & sf21 derived from the pupal ovarian tissue of the fall army worm spodoptera frugiperda and high five derived from the ovarian cells of the cabbage looper . Baculovirus is a lytic, dsDNA virus , routinely amplified in cells of the insects belonging to Lepidoptera family. It is noninfectious in vertebrates and its promoters are inactive in mammalian cells

4. Insect cell expression systems

5. Expression of protein using baculovirus /insect cells involves the following steps: U se of competent   E. coli  cells to take up DNA sequence of interest I ntegration of the DNA into bacterial genome or circularization of the DNA sequence to exist as a plasmid S election of transformed  E. coli  using a selection marker (antibiotic ) E xpansion of selected  E. coli  in appropriate culture media I solation of DNA or plasmid P reparation of a second plasmid containing viral genes required for multiplication and formation of virus particles C o-transfection of the expression plasmid and the second plasmid into  Sf 9 or  Sf 21 insect cells P urification of the recombinant viral stock A mplification of the virus and additional plaque assays to increase the titer of the recombinant viral stock I nfection of the insect cells with high-titer recombinant virus stock I solation and purification of intracellular/secreted proteins

6. Culture media Old Standards Grace’s Schneider’s Mitsuhashi and Maramorosch Commercial Serum-Free Ex- Cell TM 400 Series Sf-900 II Insect-XPRESS TM SFX- Insect TM Drosophila-SFM

7. Source of cells Eggs (Embryos) Many cell types are actively dividing and undifferentiated Whole larvae (Neonate) All cell types Some (or Most?) are already terminally differentiated Larval tissues (from older larvae) Specific cell types Many terminally differentiated Adult tissues Reproductive tissues

8. Source of cells Cont. Successful for Cell Lines Reproductive Ovaries Testes Hemocytes Fat Body Imaginal discs Midguts Nerves Not previously used for cell lines Malphigian tubules Tracheoles Salivary glands Muscles/Aorta Endocrine glands

9. Types of Cell culture Monolayer culture Suspension culture

10. Methods of sub culturing adherent cells Three methods to dislodge monolayers in adherent cell culture - Sloughing - Trypsinization -Tapping the layer until monolayer loosens

11. Procedure of monolayer sub culture Monolayer should reach to confluency in 2-4 days . Serum supplemented cultures do not adhere to surface tightly where as serum free attach very tightly to substrates Aspirate medium & floating cells from a confluent monolayer & discard them. Add 4ml of complete growth medium to each 25cm 2 flask(12 ml to a 75 cm 2 flask) Resuspend cells by pipetting the medium across the monolayer with a Pasteur pipette . (Enzymatic dissociation is not recommended) Observe cell monolayer using an inverted microscope to ensure adequate cell detachment

12. Procedure of monolayer sub culture cont. Perform viable cells count on harvested cells. Inoculate cells at 2 x 10 5 viable cells/ml into respective culture vessels. Inoculate cultures kept at 25-28 °C with loose caps to allow gaseous exchange On day 4 post-planting, aspirate the spent medium from one side of the monolayer & subculture the flask With slower growing cell lines , it may be necessary to feed the flasks on day 3-4 post planting Subculture the flasks when the monolayer reaches 80-100% confluency , approx 2-3 days post planting

13. Working with suspension culture Insect cells are not generally anchorage dependent & can be well adapted to suspension culture Prior to establish a spinner culture, cells are maintained firstly as healthy adherent cells . Cell density reaches to 2-2.5 x 10 6 cells/ml they should be diluted to no less than 7 x 10 5 cells/ml Use a spinner flask with a vertical impeller Culture volume should not exceed half of the volume of the flask Use of surfactant to decrease shearing e.g. Pluronic F-68

14. Working with suspension culture cont. Not necessary to change medium regularly . Sub culturing requires the removal of cell suspension & the addition of medium Impeller should be rotating regularly Impeller should be submerged 1 cm or more to ensure adequate aeration Cell viability of 95% is required Minimum density of 1 x 10 6 cells/ml is required Keep record of the passage number . After 30 passage or more (2-3 months), cells doubling time increased and also loose their viability and infectivity. Keep a cell log , to do so one should have a knowledge of following; date of initiation of culture, lot number date of passage & passage number density & viability at passage comment on cell appearance medium & its lot number

15. Do and Don'ts Check cells daily until a confluent monolayer is formed. Passage cells at confluency only , as cells will be easy to dislodge & shows better viability Do not overgrow cells , it results in decreased viability Do not splits cells too far . Densities lower than 20% confluency inhibit growth Passage the cells only in log phase , log phase growth can be maintained by splitting cells in 1:5 dilution

16. Types of Insect cell lines Cells Doubling time Cell appearance Medium Origin Type of culture Sf 9 72 hrs Spherical, granular, regular in size, firm attachment to surface TNM-FH IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda Grow well as monolayer and suspension Sf 21 24 hrs Spherical, granular, different in size, firm attachment to surface TNM-FH IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda Grow well as monolayer and suspension High-five 18 hrs Spherical, granular, regular in size, loose attachment to surface Express five SFM Ovarian cells of cabbage looper Grow well as monolayer, also as suspension

17. Cell culture conditions Temp Media with serum SFM Antibiotics Sf9 27°C ± 1°C Grace’s supplemented (TNM-FH) with 10% heat-inactivated (HI) FBS; Add 0.1% Pluronic F-68 for suspension cultures Sf-900 II SFM Sf-900 III SFM Pen/Strep Mimic Sf9 27°C ± 1°C Grace’s supplemented (TNM-FH) with 10% HI FBS; Add 0.1% Pluronic F-68 for suspension cultures No Pen/Strep Sf21 27°C ± 1°C Grace’s supplemented (TNM-FH) with 10% HI FBS; Add 0.1% PluronicF-68 for suspension cultures Sf-900 II SFM Sf-900 III SFM Pen/Strep High Five 27°C ± 1°C No Express Five SFM with the addition of glutamine Pen/Strep S2 22°–24°C Schneider’s media supplemented with HI FBS Drosophila SFM Pen/Strep D.Mel2 22°–24°C No Drosophila SFM Pen/Strep

18. Basic aseptic conditions If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger Work either left to right or vice versa, so that all material goes to one side, once finished Clean up spills immediately & always leave the work place neat & tidy

19. Basic aseptic conditions cont. Possibly keep cultures free of antibiotics in order to be able to recognize the contamination Never use the same media bottle for different Insect cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Switch on the laminar flow cabinet 20 mts prior to start working Cell cultures which are frequently used should be subcultered & stored as duplicate strains

20. Features of insect cell line R ecombinant protein is highly expressed during the last phases of lytic cycle before cell lysis S uitable to generate both cytoplasmic and secreted proteins D isulfide bonds in proteins are efficiently generated P rovide majority of post-translational modifications found in mammalian cells

21. References Techniques for the Development of New Insect Cell Lines – SIVB Baculovirus expression system - Paras Yadav et. al. https:// www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-expression-handbook/pex-handbook-insect-cell-based-protein-expression.html https://www.sigmaaldrich.com/technical-documents/articles/biology/protein-expression-systems.html

22. Thank you