CryoStorTM Quick Reference Cryopreservation Protocol
1) Place cells to be cryopreserved into suspension (mechanical
or enzymatic dissociation)
2)
Centrifuge cells to obtain cell pellet
3) Remove supernatant
Note: Remove as much culture media as possible, to reduce dilution of CryoStor™
solution.
4) ISOLATION: Add cold (2-8°C)
CryoStor™a. Cell concentrations: 0.5-10 x 106 cells/ml (higher [cell] possible with testing). b. DMSO is pre-mixed in CryoStor™
- no additives are necessary.
5) PRE-FREEZE: Incubate
cell suspension at 2-8°C for approximately 10 minutes6)
NUCLEATION: Freeze samples at -80°C a. Use a controlled rate freeze (-1°C/min) or similar protocol for most
mammalian cell systems.
b. The freezing device or isopropanol
container should be pre-cooled to 2-8°C.
c. Ice nucleation
within the sample (seeding) should be initiated at approximately -5°C
using either a liquid nitrogen burst program setting on a controlled rate freezer or
mechanical agitation (flick or tap) of the cryovial/sample container after approximately15-
20 min. at -80°C.
d.
Freeze time (-80°C) using isopropanol containers is recommended to be 3-4 hours.
7) STORAGE: Place samples into storage a. Store samples
at liquid nitrogen temperatures (below -130°C).
b. Sample
storage at -80°C is only recommended for short-term storage (weeks to months).
8) THAWING: Thaw samples quickly in a 37°C water
bath a.
Sample thawing should be conducted with gentle swirling of sample until all visible ice has
melted. Approximate thaw time for a 1 ml sample in a cryovial is approximately 3 minutes.
b. DO NOT allow sample to warm above chilled temperatures (0-10°C). Cryovials
should
be cool to the touch when removed from bath. Passive
thaw is not recommended.
9) Dilute cell/CryoStor™
mixture immediately with culture media
a. Dilution procedure
can be preformed in a single step.
b. The dilution media
should be between 20°C and 37°C.
c. A dilution ratio
of 1:10 (sample to media) or greater is recommended.
10)
Plate cells in appropriate confi guration 11) Place cells into culture conditions
or utilize immediately
12) Viability assessment 24-hours
post-thaw*
Note: To obtain an accurate measure of cell viability
following cryopreservation, assessment
should be performed
24 hours post-thaw and compared to non-frozen controls.
*Sample
assessment immediately post-thaw with membrane integrity indicators, such as Trypan
Blue, for comparative analysis of sample cell yield and viability often results in signifi cant overestimates of cell survival.
Live/Dead
fluorescent assays or metabolic assays (MTT or AlamarBlue) are recommended
for more accurate viability assessment. Visual inspection
of adherent cells and cells
“floating”
in the media is also recommended.f cytotoxic agents.
