BioLife Solutions CryoStor CS10 Freeze Media 使用与冻存方法

CryoStor® Usage and Cryopreservation Protocol

细胞准备

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.

加CryoStor冻存液

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.

BioLife Solutions 储存

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).

细胞复苏解冻

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.