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There are many ways
to adapt cell lines to serum-free media. Outlined below are five methods
that have worked well in our laboratories. However, the method you choose
should be based on your knowledge of, and experience with, your cell line
or lines.
The protocols below
assume that you are starting with a suspension cell line. While serum-free
formulas will support the growth and attachment of adherent cells, most
formulas are designed for use in a suspension environment. Attaching cells
can be adapted to suspension growth and protocols are available for doing
so.
The protocols below
are designed for adapting hybridomas to a protein-free medium. These protocols
may require some modifications for your particular cell line and conditions.
Adaptations require
routine monitoring of cell density and pH. You will find that cell viability
may drop to very low levels. This is to be expected and is an essential
part of the adaptation process. As with any new process, commitment, careful
observation, and patience are essential.
Serum Halving Method
| 1. |
Grow cells in
a basal medium supplemented with 10% FBS until the cells reach the
peak of the linear log phase.
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| 2. |
Subculture the
cells at your normal subculture (split) ratio (usually 1 mL cells/
5 mL fresh medium) into serum-free medium containing 5% (v/v) FBS. |
| 3. |
When the cells
have reached saturation density, subculture as above in the serum-free
medium containing 1% FBS. |
| 4. |
At each subsequent
subculture, reduce the FBS by 50% until the FBS concentration is below
0.06%. At this point the cells can be maintained in a serum-free medium. |
| 5. |
If cell growth
declines at any point during adaptation, return the serum concentration
to that promoting cell growth. All the cell growth to stabilize
at that serum concentration before proceeding with the serum reduction
schedule.
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| 6. |
During adaptation,
cell density should not be allowed to fall below 2 x 105
cells/mL nor climb to more than 1.4 x 106
cells/mL. |
Subculture (Split)
Method
| 1. |
Grow the cells
to 90% saturation density, usually about 106 cells/mL, in their
normal medium (basal medium containing 5–10% serum).
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| 2. |
In a T-25 flask,
add 2.5 mL of cell seed stock and 2.5 mL of fresh serum-free medium
at a 1:1 subculture (split) level. |
| 3. |
Twenty four hours
later, subculture (split) the cells 1:1 (2.5 mL cells and 2.5 mL fresh
serum-free medium) into a new flask, feeding the original flask with
3 mL of fresh serum-free medium. |
| 4. |
At some point,
the cell doubling will decrease and the time interval between cell
cultures will increase. |
| 5. |
Continue to
subculture the cells 1:1 as necessary, until such time that the
cells must again be subcultured on a daily basis.
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| 6. |
At this point,
the cells will be fully adapted and can be adjusted to a normal subculturing
program. Assuming doubling times have normalized, subculture within
the linear log portion of the growth curve twice a week. |
Immersion Method
| 1. |
Harvest cells
from serum-containing cultures by centrifugation
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| 2. |
Gently resuspend
cells in serum-free medium to a density of 4–7 x 105
cells/mL. |
| 3. |
Monitor the cells
daily. Remove culture medium when it becomes acidic and add an appropriate
volume of fresh serum-free medium. Maintain the cell density between
2 x 105 cells/mL and 1.4 x 106
cells/mL. If the cell density shifts below the lower limit of above
the upper limit, adjust the medium volume to a level that will reestablish
mid-log phase cell density. |
| 4. |
When the cells
reach the point at which they require daily replacement of at least
50% medium volume, maintain the cells on a routine, semi-weekly subculturing
program. |
Constant Density Method
| 1. |
From a healthy,
actively dividing cell culture, seed a new T-75 flask to a final
density of 5 x 103 cells/mL in
20 mL on the serum-free medium.
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| 2. |
Maintain, on
a daily basis, the viable cell count at 5 x 105
cells/mL by removing an appropriate volume of medium and cells, and
then adding back fresh serum-free medium to maintain the viable count.
Note that many cells may die during this particular adaptation process
which could reduce the volume of the medium as viability is maintained.
Reducing culture vessel size may be required to maintain the proper
viability. |
| 3. |
Once the cell
culture has been adapted to serum-free conditions, it should be cultured
in ever-increasing volumes until ready for standard subculturing techniques. |
Serum-Free Method
| 1. |
This method
is for use with protein-free media and is useful when the above
protocols are unsuccessful.
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| 2. |
Using one of
the methods above, adapt the cell line to a low protein, serum-free
medium. |
| 3. |
After the cells
have adapted to the serum-free conditions, use one of the methods
above to select a population that will grow in the protein-free
medium.
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