Scalable Production of Adenovirus Vectors - Adenovirus: Methods and Protocols - page 173

2.2.2 Virus Titration

1. Sterile flat bottom cell culture 24 wells microplates with lid
(BD Falcon™ or equivalent).
2. Sterile tubes of 3.5 mL for viral dilutions and Flow cytometry
analysis.
3. D-PBS.
4. Cell lysis solution: 10 mM Tris pH 8, 2 mM MgCl 2 , 0.01 %
(w/v) Triton X-100 ( see Note 4 ).
5. DMEM supplemented with 10 % (v/v) fetal bovine serum,
FBS (Gibco).
6. Flow cytometer instrument (PARTEC or equivalent).
7. HEK 293 cells (ATCC: CRL-1573) in DMEM with 10 %
(v/v) FBS.
8. MDCK-E1 cells maintained in DMEM with 10 % (v/v) FBS
9. 0.25 % Trypsin-EDTA and 0.05 % Trypsin-EDTA.
10. HAdV and CAV-2 sample(s) to titrate ( see Note 4 ).

1. STBs with 2 and 5 L vessels (Biostat DCU-3 Bioreactors,
Sartorius Stedium, Biotech GmbH or equivalent) ( see Note 5 ).
2. Addition flasks for culture medium, cells, virus and harvesting.
3. Base solution: 1 M NaHCO 3 .

2.3 Production
in STB

2.3.1 Bioreactor
Preparation

1. HEK 293 cells (ATCC: CRL-1573) adapted to suspension
growth ( see Note 1 ).
2. Ex-Cell ® 293 serum-free medium (Sigma-Aldrich Co. LLC)
supplemented with 4 mM of glutamine (Gibco) ( see Note 2 ).
3. HEK 293 cells grown in bioreactor; cell concentration at
infection (CCI) should be at 1 × 10 6 cells/mL.
4. Ex-Cell ® 293 serum-free medium supplemented with 4 mM
of glutamine.
5. Purified first generation HAdV vectors with GFP transgene or
other gene of interest ( see Note 6 ).

2.3.2 HAdV Production

1. Washing solution: KOH in ethanol.
2. Dimethildichorosilane.
3. Toluene.
4. Cytodex™-1 microcarriers (GE Healthcare).
5. Inoculation flask.
6. D-PBS.
7. MDCK-E1 cells with 90 % confluence in T-Flasks.
8. Inoculation culture medium: Optipro™ SFM supplemented
with 4 mM glutamine ( see Note 2 ) and 5 % (v/v) FBS ( see
Note 8 ).

2.3.3 CAV-2 Production
(See Note 7 )