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Direct Adaptation
The main advantage of this method is time saving. The cells should be
adapted to HyQ SFX-Insect in 4-8 passages. However, there are some cell
lines and clones that are more sensitive to the physiochemical and nutritional
changes, and in some cases it may take longer than 8 passages for the
adaptation. If viabilities decrease to <50%, or if the cultures are growing
slowly (population doubling time is >40 hours) for more than 3-4 consecutive
passages, use the sequential adaptation.
| 1. |
Prewarm HyQ
SFX-Insect to 25-28°C. The medium also can be left overnight at
room temperature providing that it is shaded from the light.
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| 2. |
Transfer the
cells grown in conventional medium supplemented with serum directly
into prewarmed HyQ SFX-Insect. The planting cell density should be
~0.5-1.0 x 106 viable cells/mL. |
| 3. |
When the viable
cell density reaches 1-3 x 106 viable
cells/mL, subculture the cells to 0.5-1.0 x 106
viable cells/mL. |
| 4. |
Subculture stock
cultures of HyQ SFX-Insect adapted cells 1-2 times per week when viable
cell counts reach 2-5 x 106 viable
cells/mL with at least 85% viability in about 4-7 days. |
Sequential Adaptation
| 1. |
Subculture insect
cells growing exponentially in a conventional medium into ratio 1:1
of the HyQ SFX-Insect and serum-supplemented medium with the cell
density between 0.5-1.0 x 106 viable
cells/mL. |
| 2. |
Incubate the
cultures until viable cells double (about one to two population doublings).
Subculture cells by mixing equal volume of the cell suspension in
a condition medium and fresh HyQ SFX-Insect (1:1). |
| 3. |
Continue to subculture
the cells in this manner until the serum concentration is reduced
below 0.05%, cell viability is >85% and the viable cell concentration
is between 2-4 x 106 cells/mL or
higher is achieved. |
| 4. |
Subculture cells
when viable cell concentration is increased from 0.5 x 106
to 2 x 106 cells/mL, or when 1 x
106 to 3 x 106
cells/mL or better is achieved. |
| 5. |
For cryopreservation
of the HyQ SFX-Insect adapted cells, follow instruction described
in step 6 under Direct Adaptation to HyQ SFX-Insect. |
| 6. |
Recover the cells
from cryopreservation, expand and check recombinant protein expression. |
| 7. |
Expend recovered
HyQ SFX-Insect adapted cells from cryopreservation, and prepare a
Master Cell Bank (MCB) using the lowest possible passage available. |
Cryopreservation
| 1. |
When the cells
are adapted to HyQ SFX-Insect, viable cell counts of most insect
cells should routinely exceed about 2-4 x 106
viable cells/mL. After 3-6 days in culture, the viabilities should
be >85%. At this time, the HyQ SFX-Insect adapted culture should
be cryopreserved as a master seed stock. The cells should be cryopreserved
in a medium consisting of 7.5% DMSO in 50% fresh HyQ SFX-Insect
and 50% condition HyQ SFX-Insect.
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| 2. |
Recover the
cells from cryopreservation, expand and check recombinant protein
expression.
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| 3. |
Expand recovered
HyQ SFX-Insect adapted cells from cryopreservation and prepare a
Master Cell Bank (MCB) using the lowest passage available.
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