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Direct Adaptation
The main advantage
of this method is time saving. The cells should be adapted to SFM4MAb
or SFM4MAb-Utility 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.
It is also recommended
to start adaptation with higher cell densities, in particular if the cultures
are adapting slow (e.g., start with up to 1.0 x106
cells/mL). 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.
The cultures can be
grown and maintained in conventional T-flasks, roller bottles, shaker
flasks (~110-120 rpm), and spinner flasks (~65-75 rpm) in a CO2 gased
incubator (caps 1/3 loose).
| 1. |
Prewarm SFM4MAb
or SFM4MAb-Utility to ~37°C using a conventional waterbath.
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| 2. |
Transfer the
cells grown in current medium, serum-free or serum-containing, directly
into prewarmed SFM4MAb or SFM4MAb-Utility. The seeding cell density
should be ~0.50 x 106 viable cells/mL. |
| 3. |
When the viable
cell density reaches 1-3 x 106 viable
cells/mL, subculture the cells to 0.50 x 106
viable cells/mL. |
| 4. |
Subculture stock
cultures of SFM4MAb or SFM4MAb-Utility 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 3-4 days. |
| 5. |
When the cells
are adapted to SFM4MAb or SFM4MAb-Utility, viable cell counts of
most hybridoma cells should routinely exceed about 2-4 x 106
viable cells/mL after 3-6 days in culture (viabilities should be
>85%). At this time, the SFM4MAb or SFM4MAb-Utility adapted culture
should be cryopreserved as a master seed stock.
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Sequential Adaptation
| 1. |
Subculture hybridoma
cells growing exponentially in current medium into ratio 1:1 of the
SFM4MAb or SFM4MAb-Utility and current medium with the cell density
~0.50 x 106 viable cells/mL. It is
also recommended to start adaptation with higher cell densities, in
particular if the cultures are adapting slowly (e.g., start with up
to 1.0 x 106 cells/mL). The cultures
can be grown and maintained in conventional T-flasks, shaker flasks
(~110 –120 rpm), and spinner flasks (~65 –75 rpm) in a 5% CO2 gased
incubator (caps 1/3 loose). |
| 2. |
Incubate the
cultures until viable cells double (one to two population doublings).
Subculture cells by mixing equal volumes of the cell suspension in
a conditioned medium and fresh SFM4MAb or SFM4MAb-Utility medium 1:1.
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| 3. |
Continue to subculture
the cells in this manner until the current medium and/or 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. |
| 4. |
Subculture cells
when viable cell concentration is increased from 0.5 x 106
to 2 x 106 cells/mL. |
Cryopreservation
| 1. |
The adapted cells
should be cryopreserved in a medium consisting of 7.5% DMSO in 50%
fresh SFM4MAb or SFM4MAb-Utility and 50% condition medium (final concentration
of DMSO should be 7.5%). |
| 2. |
Prior to relying
on the frozen cells as a Master Seed Stock, recover the cells from
cryopreservation expand and check for cell growth and monoclonal antibody
production. |
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