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Direct Adaptation
The following adaptation
procedure is recommended for transferring adherent cells from a serum-containing
medium (SCM)into HyQCDM4Retino. In all adaptation processes, it is recommended
that a reserve culture be maintained in the stock medium.
| 1. |
Pre-warm SCM
to 25°C.
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| 2. |
Subculture existing
adherent cells using standard trypsinization protocols. |
| 3. |
Seed a T-flask
at 4.0 x 104 cells/cm2
(recommended seeding density) using SCM with 1/2 the previous serum
concentration. |
| 4. |
Once the culture
becomes 70-95% confluent (72-96 hours post seeding), subculture the
cells using the recommended seeding density. However, again reduce
by half the concentration of serum. |
| 5. |
Continue the
sequential reduction of serum until the cells have successfully
completed a culture period at 2.5% serum.
NOTE:If at any
time the cells fail to become 79% confluent by 96 hours post-seeding,
subculture the cells at the same serum concentration another culture
period.
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| 6. |
Subculture the
cells using SCM (with 2.5%serum).
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| 7. |
After a 24-hour
incubation period to allow attachment and growth, remover the SCM;
replace the SCM with HyQ CDM 4 Retino.
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| 8. |
Subculture the
cells in HyQ CDM4Retino every 48-72 hours. The cells should no longer
require trypsin to detach. If the cells aggregate, gently pipet
to disrupt prior to counting and subculturing. The cells should
be maintained in static culture (T-flasks) using HyQ CDM4Retino
for three passages prior to transfer into suspension cultures.
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| 9. |
Cells in suspension
cultures using alternative serum-free media may be directly adapted
by gently pelleting the cells (centrifuge 5 minutes at 100 x g)
and resuspending them in HyQ CDM4Retino.
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Cryopreservation
Low passage, mid-log
phase cells cultures in HyQ CDM4Retino can be cryopreserved using the
following procedure (cell viability should be >90%).
| 1. |
Centrifuge a
know concentration of cells at 100 x g for 5 minutes. Aseptically
transfer supernatant (conditional medium) into a sterile container. |
| 2. |
Gently resuspend
the cell pellet at a concentration of 10 x 106
cells/mL using the conditioned medium previously harvested. |
| 3. |
Slowly add an
equal volume of fresh HyQ CDM4Retino supplemented with 20% DMSO to
adjust the cell concentration to 5.0 x 106
cells/mL (final DMSO concentration:10%). |
| 4. |
Transfer this
cell suspension at a volume of 1.0 mL per 1.8 mL cryovial. |
| 5. |
Cells should
be frozen at a rate of -1°C/ minute until reaching -70°C. Within
24 hours, the cells should be transferred into a liquid nitrogen
tank for further storage.
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