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Background
An
indicator of HIV vaccine immunogenicity is a cytotoxic T cell (CTL) response.
As part of the requirement for the CTL assay, autologous target cells
are required. To fulfill these requirements, B cells from the study subject
are immortalized using Epstein-Barr virus (EBV). The source of EBV is
a cotton-top marmoset cell line B95-8 (ATCC #CRL-1612) chronically infected
with EBV. These cells must be grown to log phase to provide EBV containing
cell supernate, which is then used to transform B lymphocytes derived
from study subjects.
Materials
and Methods:
Use
sterile technique throughout the procedure and universal precautions for
use
and disposal of biohazardous reagents.
1.
Sterile T-25, T-75 and T-225 flasks - Corning or Costar
2. RPMI media
3. Fetal bovine serum (FBS)
4. Penicillin/Streptomycin (P/S)
5. L-Glutamine
6. Sterile pipettes - 5ml, 10ml, 25ml, 50 ml
7. 0.4% trypan blue
8. Cyclosporin A, (Sigma, St Louis, MO) : Cyclosporin A is first dissolved
in 100% ethanol at 5mg/ml, the stock is then diluted 1:10 in CM. The 0.5mg/ml
stock should then be sterile filtered and can be stored in aliquots at
4 í C. Final concentration for use in B cell transformations is 2 ug/ml.
9. Leighton slant tubes (PGC Scientific, Gaithersburg, MD): 10 ml capacity
tubes for tissue culture, has large surface area for cell expansion and
for better examination of cells under the microscope.
Preparation of EBV containing supernate
1. Prepare complete media (CM) using the following formula:
a. Bring media components to room temperature
b. Mix RPMI-1640 + 10% FBS + 1% L-glutamine + 1%P/S
c. Filter through a 0.22 mm filter, and label appropriately.
2. Rapidly thaw a vial of cryopreserved B95-8 in a 37oC water bath.
3. Centrifuge the cells at 200 g for 10 minutes.
4. Decant the supernate and resuspend the cells in 10 ml media and do
a total and viable cell count using trypan blue exclusion
5. Resuspend the cells to a concentration of 0.2 X 106 cells/ml in CM
6. Cells are routinely subcultured at 1:3 or 1:5 at weekly intervals,
monitoring
the media for acidity.
7. Supernate should be harvested when the cells are in log phase - 1 X
106/ml
8. For preparation of EBV containing supernate
Centrifuge the cell suspension at 200X g for 15 minutes at 20 í C
Decant the supernate to a new centrifuge tube
Centrifuge the supernate at 800 X g for 15 minutes at 4 í C
To ensure removal of B95-8 cells, filter through a 0.45 um filter
Aliquot the supernate in 1ml volumes in labeled cryovials - store at -70
í C.
9. The pelleted cells may be subcultured or cryopreserved at 6 X 106 cells
in 1ml aliquots. Cryopreservation Media: RPMI-1640 + 20% FBS + 10% DMSO
+1%P/S
Notes:
1. The cell line must be tested with the Gen-probe mycoplasma detection
kit
2. Adherent cells may be observed
3. Giant cells will be observed - these giant cells are rich sources of
EBV
Immunofluorescent staining of single cells for EBV usually yields 7% positive
Immunofluorescent staining of giant cells for EBV usually yields 100%
positive
4. Keep a log of the passage history of the cells
5. Titration of the EBV supernate is not necessary, however it is advisable
to test the supernate for its ability to transform B cells as described
below.
Transformation of B cells
1.
Separate PBMC by standard Ficoll/Hypaque. Five to ten million isolated
PBMC are needed, frozen PBMC can also be used. Prepare the PBMC at a concentration
of between 5 and 10 x 106 cells/ml in CM. Place 1 ml of the cells in a
10 ml slant tube.
2. Add one vial of EBV containing supernatant prepared above (in 1 ml
volume), 2ml CM (with 20% FBS for the initial transformation) and 2 ug/ml
Cyclosporin A (4 ml total). The culture is incubated for seven days at
37 í C in a humidified CO2 incubator, after which 1 ml of medium is removed
and a further 1ml of EBV containing supernatant along with fresh Cyclosporin
A is added. The tube is incubated for an additional 7-14 days, after which
the culture medium should become acidic and the cells should form macroscopic
clumps. Clumps generally appear after 2-3 weeks, occasionally the culture
will appear static and will not start forming clumps until 6-8 weeks after
initiation of the culture. Transformations can also been done in 24 well
plates or T-25 flasks.
3. Once clumps have started to form, the cells should be expanded by splitting
1:2 or 1:3 in CM and transferring the cells to larger volume flasks as
necessary and then cryopreserving the cells at 10-20 X 106 cells in 1ml
aliquots. Cryopreservation Media: RPMI-1640 + 20% FBS + 10% DMSO + 1%P/S
4. The cell line can be continuously maintained in 75cm 2 flasks by splitting
the cells 1:3 once or twice a week. Confirmation of the immortalization
of the cells is best assessed by the continued growth of the cells in
culture. Additional quality control includes phenotyping by flow cytometry
with a CD 20 monoclonal antibody and demonstration of MHC class I expression
on the cell surface.
NOTES:BLCL
show considerable variation in their growth characteristics, some grow
very slowly and need to be split only once a week, others grow rapidly
and need splitting a couple of times a week. Healthy dividing cultures
will turn the culture media yellow within 2-3 days and large clumps of
cells should form. When splitting the cells, these clumps should be disrupted
by either vigorously shaking the flask or by resuspending the cells with
a pipette. It is possible to maintain some cultures for several months
without compromising their viability or ability to present antigen. However
for the CTL assay, since good target viability is critical it is best
to keep a close eye on the cells and thaw new BLCL if the viability is
poor. BLCL should be thawed out at least two weeks prior to being used
as targets in a CTL assay, to allow a few cell divisions to occur. To
conserve reagents, BLCL can be passaged in 25cm2 flasks in a volume of
10 ml.
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