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:
• Use of competent E. coli cells to take up DNA sequence of interest
• Integration of the DNA into bacterial genome or circularization of the DNA sequence to
exist as a plasmid
• Selection of transformed E. coli using a selection marker (antibiotic)
• Expansion of selected E. coli in appropriate culture media
• Isolation of DNA or plasmid
• Preparation of a second plasmid containing viral genes required for multiplication and
formation of virus particles
• Co-transfection of the expression plasmid and the second plasmid into Sf9 or Sf21
insect cells
• Purification of the recombinant viral stock
• Amplification of the virus and additional plaque assays to increase the titer of the
recombinant viral stock
• Infection of the insect cells with high-titer recombinant virus stock
• Isolation and purification of intracellular/secreted proteins

6. CULTURE MEDIA
Old Standards
• Grace’s
• Schneider’s
• Mitsuhashi and
Maramorosch
Commercial Serum-Free
• Ex-CellTM 400 Series
• Sf-900 II
• Insect-XPRESSTM
• SFX-InsectTM
• 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
o Ovaries
o 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
25cm2 flask(12 ml to a 75 cm2 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 105 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 106 cells/ml
they should be diluted to no less than 7 x
105 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 106 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
Antibiotic
s
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
Mimi
c 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.Me
l2
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
• Recombinant protein is highly expressed during the
last phases of lytic cycle before cell lysis
• Suitable to generate both cytoplasmic and secreted
proteins
• Disulfide bonds in proteins are efficiently generated
• Provide 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