This document provides an overview of the process for producing Bluetongue virus antigen through cell culture. Vero cells are grown and infected with Bluetongue virus serotype 1. When cytopathic effects are observed, the cell culture fluid is harvested and centrifuged to remove viral particles and debris. The inactivated antigen is then concentrated using dialysis and optimized for use in an Agar Gel Immunodiffusion test to detect Bluetongue virus antibodies. The presentation describes preparing and maintaining the Vero cell cultures, infecting them with BTV-1, and harvesting and processing the antigen to be used in diagnostic tests.

1. Prepared by:
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by Debbra Marcel
for July 2013 R&D Meeting

2. List of activities
1. Preparation of the virus (presented by Naama in Aug 2012)
2. Preparation of Vero cells
3. Inoculation of BTV (BTV-Serotype 1) into Vero cells
4. Identification of virus isolates based on the type of
cytopathic effect (CPE).
5. Harvesting & Inactivation of virus
6. Concentration of inactivated BTV
7. Optimization of Bluetongue Antigen by AGID
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3. Objective
The objective of this presentation:
 To explain the cell culture process as one of the important
methods in the production of Bluetongue Antigen (BT Antigen)
The objective of performing the production of BT Antigen:
 To continuously supply the needs of the antigen in
performing the serological identification of BTV by Agar Gel
Imunodiffusion (AGID) test to the samples received in VRI
(disease surveillance)
 To meet our purpose in developing the AGID test kit
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4. Overview
 In this process, Bluetongue Virus Serotype-1 (BTV-1) is used as the
antigen for the kit. Preparation of the virus antigen will be done by
propagating them in African green monkey kidney (VERO) cells and
isolates will be identified based on the type of cytopathic effect (CPE).
The cell culture fluid will be harvested after the CPE is reached and then
centrifuged to remove complete viral particles and cellular debris. The
formalinized BTV antigen will be purified by using dialysis method
 This method involves the withdrawal of fluids through dialysis
membranes using polyethylene glycol to produce an increasingly
concentrated sample within the dialysis membrane.
 The preparation of BTV soluble antigen, regardless of serotype, by the
method described gave excellent results in both the micro and macro-
AGID tests
 Original procedure adapted from: J. Clin. Microbiol. 1983 (modified by UMA)
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5. Activity 2
(Preparation of BTV Antigen AGID)
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6. Introduction
Cell culture
 the removal of cells from an animal or plant and their subsequent growth in a favorable
artificial environment.
 may be removed from the tissue directly and disaggregated by enzymatic or mechanical
means before cultivation, or they may be derived from a cell line or cell strain that has
already been established.
Vero cell:
 established cell line derived from the kidney of an African green monkey (Cercopithecus
aethiops) in the 1960s (Y. Yasumura and Y. Kawakita at the Chiba University in Japan).
 one of the most common mammalian continuous cell lines used in research & virology
studies, vaccine & antigen production, propagation and study of intracellular bacteria (e.g.,
Rickettsia spp.; UNIT 3A.4) and parasites (e.g., Neospora), and assessment of the effects of
chemicals, toxins and other substances on mammalian cells at the molecular level.
Bluetongue (BT) Antigen :
 Soluble antigen used in Agar Gel Immunodiffusion (AGID) test to detect the presence of
BTV antibodies in animal sera.
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7. Requirements of a Tissue Culture Laboratory
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√
√
Culture of Animals Cell, 5th Edition, R. Ian Freshney, page 56

8. Preparing an aseptic environment by GLP
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Culture of Animals Cell, 5th Edition, R. Ian Freshney, page 80

9. Preparation of Cell Culture Medium
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*antibiotics:
Penicillin, Streptomycin, and kanamycin = 100 unit; 100 unit; 50 ug
Maintenance Medium (MEM MM) – 2% FBS
1) Tryptose Phosphate Broth - 10%
2) Fetal Bovine Serum (FBS) - 2 %
3) 7.5% NaHCO3 - 1.5
4) PSK (*antibiotics) - 1%
5) MEM - up to 100%
MEM Minimal Essential Media (basal medium)
 Preparation of powdered media:
1. Dissolve powder into roughly 95% of total
volume to be made.
2. Add the required amount
3. of sodium bicarbonate.
4. Adjust pH to 0.2 to 0.3 units below the desired
working pH (7.0-7.4) by adding 1N NaOH or 1N
HCl with stirring.
5. QS to final volume. Keep container closed until
ready to filter.
6. Filter through a 0.2 micron filter (see Picture 1)
Picture 1 Picture 3Picture 2
Picture 1: The sterilization of MEM (by filtration)
Picture 2: MEM before supplemented
Picture 3: MEM after supplemented
The MEM basal medium then supplemented with other nutrients according to the proportion desired as follow:
Growth Medium (MEM MM) – 10% FBS
1) Tryptose Phosphate Broth - 10%
2) Fetal Bovine Serum (FBS) - 10 %
3) 7.5% NaHCO3 - 1.5
4) PSK (*antibiotics) - 1%
5) MEM - up to 100%

10. Thawing Vero cells from liquid nitrogen/-80°c
Materials
 Vero cell stock, frozen in
liquid nitrogen/at -80°C
 MEM GM (10% FBS)
 15mL conical tubes,
sterile
 25cm2/50cm2 tissue
culture flasks with vented
caps, sterile
 Serological pipettes,
sterile
 70% ethanol solution
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Method (Protocol 1, Current protoc. Microb., NIH, 2009 = Maintenance of Vero cell culture)
1. Quickly thaw vial (cryovial) of Vero cells by gently
swirling in a 37°C water bath. (Keep the O-ring and
cap of the cryovial out of the water)
2. In a laminar flow hood, decontaminate the vial by
spraying with 70% ethanol
3. Transfer the Vero cell suspension from the cryovial
into a 15mL conical tube containing 10mL of MEM
Growth Medium (10%FBS) (after thawing the cells,
dilute and remove the DMSO before transferring the
cells to tissue culture flasks)
4. Pellet cells by centrifugation at 200 × g for 5 minutes at
room temperature.
5. Remove and discard supernatant; resuspend cells in 5-
10mL MEM Growth Medium.
6. Transfer Vero cell suspension to tissue culture flask.
7. Incubate flasks in 37°C incubator with 5% CO2.
8. Monitor cells daily or every other day. Change media
every 3-4 days. (When cells reach a >90% confluent
monolayer, passage cells into new tissue culture flasks)
IMPORTANT NOTES:
 All equipment and
solutions coming into
contact with cells must be
sterile, and proper sterile
technique should be used.
 All cell culture incubations
are performed in a
humidified 37°C incubator,
with 5% CO2.
 All solutions should be
warmed to room
temperature or 37°C before
contacting cells.
Table A.1
Cell medium volume requirements

11. Checking cells
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1. Cells should be checked microscopically daily to ensure they are healthy
and growing as expected. As an attachment cells, Vero cells should be
mainly attached to the bottom of the flask, round and plump or
elongated in shape and refracting light around their membrane. Media
should be pinky orange in colour. If media
2. Discard cells if:
• They are detaching in large numbers (attached lines) and/or look
shrivelled and grainy/dark in colour.
• They are in quiesence (do not appear to be growing at all).
subconfluent confluent
Observing cells
Inverted microscope
with phase contrast
detaching quiesence (no growth)
Unhealthy cellsHealthy cells

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Sub-culturing/Passaging Cells
1. When the cells are 80-90% confluent , they
should still be in the log phase of growth and
need to be sub-cultured/passaged into new sterile
culture vessel/flask until we acquire the desired
amount of Vero cells for the viral infection
process.
2. Warm the fresh culture medium at 37°C water
bath or incubator for at least 30 minutes before
subculture/passage cells. Then carry out the
appropriate procedures (Refer protocol 2, Current
protoc. Microb., NIH, 2009 = Maintenance of Vero
cell culture)
3. After the sub-culture/passage of cells process
done, make sure the flasks are labeled
appropriately (cell line, passage level, date, etc)
4. Place flask(s) straight into 37°C CO2 incubator.
5. Write down the details of the sub-culturing in the
culture record log sheet. There should be a
separate log sheet for each vial of cells
resuscitated and in use.
Figure 1. The growth & maintenance of cell culture.
A. Lag phase = cells grow slowly while recovering from the
stress of sub-culturing.
B. Log or exponential phase = cells enter a period of
exponential growth, lasts until the entire growth surface is
occupied/cell concentration exceeds the capacity of the
medium
C. Stationary phase = Cell proliferation slows and stops.

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CELL COUNTING FORMULA:-
C = n x d x 10-4/ml
c = cell concentration (cells/ml)
n = number of cells counted
v = volume counted (ml)
d = dilution factor
Cell Counting Indication:
a) Clean hemocytometer slide andcoverslip before use.
b) Pressing coverslip down onto slide.
c) Adding a cell suspension to an assembled slide.
d) Longitudinal section of the slide, showing the
position of the cell sample in a 0.1-mm-deep chamber.
e) Viewing slide on microscope.
f) Magnified view of the total area of the grid. The
central area enclosed by the dotted circle is that area
which would be covered by the average 10× objective.
This area covers approximately the central 1 × 1 mm
square of the grid.
g) Low-power (10× objective) microphotograph showing
the 25 smaller 200 × 200-μm squares of a slide, which
make up the central 1 × 1-mm square, loaded with
cells pretreated with Naphthalene Black (Amido
Black).
h) High-power (40× objective) microphotograph of one
of the smaller 200-μm squares, bounded by three
parallel lines and containing 16 of the smallest (50 ×
50 μm) squares. Viable cells are unstained and clear,
with a refractile ring around them; nonviable cells are
dark and have no refractile ring.
(Complete cell counting procedures by CSIRO, AAHL, 2008)
Fig. 21.1. Using a Hemocytometer Slide
Courtesyof:CultureofAnimalsCell,5thEdition,R.IanFreshney
Viable cells=>90%

14. Cell contamination
Main cause of contamination:-
 Contact with non-sterile supplies, media, or solutions
 Particulate or aerosol fallout during culture manipulation, transportation/incubation
 Swimming, crawling, growing into cell culture (exposed vessel)
 Accidents and mistake
Steps for reducing contamination problems:-
 Use good aseptic techniques
 Reduce accidents
 Keep the laboratory clean/in sterile condition
 Routinely monitor for contamination
 Use frozen cell repository strategically
 Use antibiotic sparingly
Most common contamination
fungus mycoplasmayeastbacteria

15.  If severe cells contamination occur , Vero cells are to be
discarded appropriately (separated & autoclaved) and
new cell stock from LN2/-80ºc need to be culture again
(start all over again) to reduce spreading of infection &
cell mutation (minimise the use of antibiotic/fungizone)
 After the Vero cells successfully propagated into the
desired amount and reach confluency (monolayer),
the cells are ready to proceed to the next step:
“Inoculation of BTV1 (BTV-Serotype 1) into Vero
cells” ~ next presentation
Conclusion

16. Acknowledgement …
Monoclonal Antibody Unit
Ali A.R., Nurul Fatiha A. S., Naama T., Fauad T.R.