Biology Reference
In-Depth Information
14. Harvest medium and cells. Distribute it among six 50-mL
Falcon tubes. Centrifuge at room temperature for 5 min at
620 ×
g
. Keep 19 mL of the supernatant and discard the rest.
15. Resuspend the pellets with the reserved 19 mL of supernatant.
16. Freeze/thaw three times to release adenovirus from cells.
17. Centrifuge at room temperature for 5 min at 1,200 ×
g
. Discard
the pellet and keep the supernatant (crude lysate).
18. This crude lysate can be tested for
Mycoplasma
contamination
as a quality control (
see
Note 10
).
19. Store at −80 °C.
3.3 Purifi cation
of Recombinant
Adenovirus
Protocols for purifi cation of adenovirus vectors have evolved over
the last decade. The most classical and easy method for a non-
specialized laboratory remains the ultracentrifugation on a CsCl
gradient, although this purifi cation method is limited by the vol-
ume of cell lysate that can be processed. However, this procedure
is still widely used and most of the time is suffi cient for fundamen-
tal studies and/or early in vivo preclinical evaluation of the vectors.
More complex techniques based on column chromatography and
membrane techniques are now well developed for the generation
of high purity grade and up-scaled production suitable for human
clinical applications [
10
,
11
].
1. In a 38.5 mL Ultra-clear SW32 polyallomer centrifuge tube,
add 10 mL of 1.25 g/mL of CsCl.
2. Add 10 mL of 1.4 g/mL of CsCl by placing the tip of a 10 mL
pipette at the bottom of the tube (and under the fi rst CsCl
solution), then carefully and slowly dispensing solution to cre-
ate two phases.
3. Gently add ~19 mL of crude lysate on top of the 1.25 g/mL
CsCl layer. Leave about 0.1 cm at the top of the tube. If a
counterbalance tube is required, use the same total volume of
D-PBS.
4. Balance tubes by weighing and load in rotor.
5. Centrifuge for 1 h 42 min at 125,500 ×
g
18 °C in a Beckman
SW32 rotor, maximum brake.
6. Remove tubes from rotor with forceps.
7. The adenovirus appears as an opaque band at the interface of
the 1.25 and the 1.4 g/mL CsCl layers (
see
Fig.
3a
). Remove
the band by piercing the tube about 1 cm below the vector
with a 10 mL syringe with a 18 G needle.
8. Collect all the vector bands in a 15-mL Falcon tube and add
1.34 g/mL CsCl to obtain a fi nal volume of 13 mL.
3.3.1 Initial
Step Gradient
