BioLife Solutions CryoStor CS10 Freeze Media 使用與凍存方法
CryoStor? Usage and 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 for routine cell culture protocols (higher [cell] possible).
b. DMSO is pre-mixed in CryoStor? - no additives are necessary.
5) PRE-FREEZE: Incubate cell suspension at 2-8°C for approximay 10 minutes
6) NUCLEATION: Freeze samples at -70°C (many protocols utlilize -70°C and -80°C interchangeably)
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 approximay -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 approximay15-20 min. at -70°C.
d. Freeze time (-70°C) using isopropanol containers is recommended to be 3-4 hours.
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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 approximay 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 immediay 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 configuration
11) Place cells into culture conditions or utilize immediay
12) Viability assessment 24-hours post-thaw*
BioLife Solutions 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 immediay post-thaw with membrane integrity indicators, such as Trypan Blue, for comparative analysis of sample cell yield and viability often results in significant 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.