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Direct Adaptation
Vero, MDCK, COS-7
and MDBK cells adapt well to HyQ SFM4MegaVir using the following direct
adaptation method:
| 1. |
Seed flasks
as standard procedure using serum-containing medium. Allow 24 hours
incubation for attachment and spreading.
|
| 2. |
Replace the serum-containing
medium with warmed (25°C) HyQ SFM4MegaVir. |
| 3. |
Observe cell
monolayer and pass culture into fresh HyQ SFM4MegaVir upon reaching
80-90% confluency using the steps outlined in the cell maintenance
procedure below.
|
| 4. |
Repeat this
procedure through at least three complete serum-free cycles. Follow
the cell maintenance procedure outlined below once adaptation is
complete.
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Sequential Adaptation
Sequential adaptation
is neither required nor recommended using HyQ SFM4MegaVir.
Cell Maintenance
The following cell maintenance procedure may be used for maintaining cells
in HyQ SFM4MegaVir:
| 1. |
Remove liquid
medium from each culture vessel.
|
| 2. |
Carefully rinse
cell monolayer once using Magnesium and Calcium-free Phosphate Buffered
Saline. |
| 3. |
Add minimum
amount of HyQ®Tase™ (HyClone
SV30030.01) (enough to just “wet” monolayer) to each vessel and
incubate for 3-5 minutes.
|
| 4. |
Following the
incubation observe monolayer for cell detachment. When cells are
completely detached cell counting may be accomplished using various
methods (i.e., hemacytometer or Coulter).
|
| 5. |
Seed new vessels
with approximately 10,000 cells/cm2.
Incubate at 37°C, 5% CO2 until culture reaches 80-90%
confluency.
|
| 6. |
When initiating
roller bottle cultures the roller bottle apparatus should be set
to 1 rpm until the cells have attached firmly to the roller bottle
surface.
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Trypsin can
be used in place of HyQTase. However, trypsin must be inhibited after
monolayer has been completely dissociated. Add equivalent amount of soybean
trypsin inhibitor (Sigma T6522 @ 1 mg/mL PBS) to each vessel. Removal
of soybean trypsin inhibitorprotease complex is required, and accomplished
by centrifugation at 100x g. Trypsin inhibitor can be left
out of the cell dissociation procedure. However removal of trypsin is
required, either by centrifugation and washing or careful monitoring of
cell monolayer while under trypsin. Once cells begin to round up, but
prior to detachment, the trypsin is removed. Cells are then allowed to
detach due to residual trypsin and counted for reseeding culture vessel.
Using this procedure centrifugation is not required.
Cryopreservation
Cells maintained in
exponential phase growth with viabilities greater than 90% may be cryopreserved
using the following procedure:
| 1. |
Remove the conditioned
medium from culture and store for later use. |
| 2. |
Rinse the cell
monolayer with PBS, followed by dissociation of cell monolayer using
HyQTase (See cell maintenance section above). |
| 3. |
Centrifuge detached
cells at 100x g to produce a cell pellet. Discard supernatant and
gently resuspend pellet at a concentration of 10 x 106
cells/mL in conditioned medium. |
| 4. |
Add an equal
volume of fresh HyQ SFM4MegaVir supplemented with 20% DMSO to adjust
cell density to 5 x 106 cells/mL. |
| 5. |
Transfer this
suspension to plastic Nalge 1.8 mL cryovials at a volume of 1.0 mL
each. Cells should be frozen at a rate of 1.0°C/min to reach a temperature
of -70°C. |
| 6. |
Once the vials
have reached -70°C transfer them to liquid nitrogen storage tanks.
Cells should not remain at -70°C for longer than 24 hrs. |
Thawing Method
| 1. |
Remove vials
from liquid nitrogen storage and place immediately into a 37°C waterbath
with gentle agitation, allowing vial to warm to 25°C. |
| 2. |
Disinfect the
vial using 70% isopropyl alcohol. |
| 3. |
Transfer the
cell contents to a 15 mL centrifuge tube, adding an equal volume of
fresh, pre-warmed (25°C) HyQ SFM4MegaVir drop-wise to the suspension. |
| 4. |
Allow the tube
to stand for 5 min, then add 3 mL of fresh, pre-warmed HyQ SFM4MegaVir
drop-wise to bring the total volume to 5 mL. Again, allow the tube
to stand for 5 min. |
| 5. |
Add an additional
5 mL of fresh, prewarmed HyQ SFM4MegaVir drop-wise to bring the volume
to 10 mL at an expected cell density of 5x105
cells. |
| 6. |
Centrifuge the
cells at 100x g for 10 minutes, pelleting the cells. Remove the supernatant
and gently resuspend the cells in 20 mL of fresh, pre-warmed HyQ SFM4MegaVir
medium. A small sample of the suspension may be counted to determine
actual cell density with the realization that a viability assessment
will not be accurate for at least 24 hrs. |
| 7. |
Place the cell
suspension into a T-75 flask and incubate at 37°C, 5% CO2. |
Initiating Microcarrier
Cultures
Cells can be expanded
in roller bottles to gain sufficient densities for microcarrier cultures.
The following procedure has been used successfully in initiating microcarrier
cultures:
| 1. |
A cell density
of 1-5 x 107 cells/L (final working
volume) should be used with 3 g/L of Cytodex 1. |
| 2. |
Cells and microcarriers
are added to 25% of the final working volume (in this case 250 mL). |
| 3. |
The culture should
be stirred intermittently for 8 hours, or until cell have attached,
at 20 rpm for 2 min every 60 min. |
| 4. |
After 8 hours,
or after cells have attached, an additional 25% of the final working
volume of HyQ SFM4MegaVir should be added. |
| 5. |
The agitation
should be adjusted to a constant speed sufficient to keep the microcarriers
from settling to the bottom of the vessel, typically 20-30 rpm. |
| 6. |
After 24 hours
fresh HyQ SFM4MegaVir is added to bring the volume to 100% final working
volume. |
| 7. |
A 50% medium
exchange should be performed every 3-4 days to maintain cells until
cells are confluent. After reaching confuency the culture can be maintained
by exchanging 50% of the culture medium every 2 days. |
| 8. |
Additional microcarriers
may be added to expand surface area and cell densities once cells
have reached 80-90% confluency. |
| 9. |
Intermittent
agitation at 20 rpm for 2 min every 60 min for a total of approximately
8 hrs should be used to allow migration of cells to new microcarriers.
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