Biology Reference
In-Depth Information
Fig.
1
Different modes of culture operation used in adenovirus production. (
a
) Batch mode: easiest approach as
no extra feeding is required and the risk of contamination is low; (
b
) Fed-batch: primarily used to extend culture
lifetime by supplementing limiting nutrients and/or reducing the accumulation of toxic metabolites; (
c
) Medium
exchange at infection: ensures removal of toxic metabolites supplying fresh nutrients at the time of infection; (
d
)
Perfusion mode: cells are retained inside the bioreactor at a relatively high cell concentration, while fresh medium
is continuously supplied, allowing replenishing of nutrients and removal of toxic metabolites
in suspension and CAV-2 using MDCK-E1 cells in microcarriers are
described to exemplify productions of AdV with cell lines adapted to
suspension and anchorage-dependent, respectively.
2
Materials
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. 500 mL Erlenmeyer flasks (Corning Life Sciences or equivalent).
4. Orbital shaker (IKA model HS 260 or equivalent).
2.1
Cell Culture
2.1.1
HEK 293 Cells
Culture
1. MDCK-E1 cell line [
20
] derived from MDCK (ECACC, Nr
84121903) (
see
Note 1
).
2. Optipro™ SFM supplemented with 4 mM of glutamine (both
from Gibco) (
see
Note 2
).
3. Trypsin-EDTA 0.25 %.
4. Dulbecco's phosphate-buffered saline without calcium and
magnesium (D-PBS).
2.1.2
MDCK-E1 Cells
Culture
1. D-PBS.
2. 0.05 % Trypsin-EDTA.
3. 0.1 % (v/v) Trypan blue solution in PBS.
4. Fuchs-Rosenthal
2.2 Monitoring
and Characterization
of Cells and AdV
2.2.1 Determination
of Cell Concentration
and Viability
hemocytometer
(Brand,
Wertheim,
Germany) (
see
Note 3
).
