CryoStor®是Biolife公司特别优化的细胞冻存液，在极低温度 (-70ºC to -196ºC)准备和保存细胞。预混DMSO，为细胞和组织的冷冻、储存和解冻过程提供安全的保护环境。CryoStor®通过调控冻存过程的分子生物学反应，无需血清、蛋白或具有高细胞毒性的试剂，就能增强细胞活力和功能。
1. 待冷冻细胞配制悬液（机械法或酶法解离）。
2. 细胞悬液离心获得沉淀。
3. 去除上清液 - 注意：尽可能多地移除培养基，以降低CryoStor®冻存液被稀释程度。
4. 分离：加入预冷的（2-8°C）CryoStor®冻存液。
a. 细胞密度：0.5-10×106 细胞/ mL，常规细胞培养方案（细胞密度可以更高）。
b. CryoStor®冻存液已经混合了DMSO，不需要再添加其他任何冻存剂。
5. 预冷：细胞/CryoStor®混合悬液2-8°C孵育约10分钟。
6. 成核：-70°C冷冻样品（许多方案交替使用-70°C和-80°C）
a. 大多数哺乳动物细胞，使用程序降温法 (-1°C/min)或类似方法。
b. 程序降温盒或异丙醇容器需要预冷到 2-8°C。
c. 约-5℃时 （-70°C冷冻约15-20min后）启动样品内的冰核形成（seeding），启动方法可以是程序降温仪控制的液氮喷放或是施加机械力搅动样本（轻弹或轻拍）。
d. 使用异丙醇容器的冷冻时间（-70°C）建议为3-4小时。
7.样本保存
a. 长时保存温度液氮-130°C以下。
b. -80°C 保存建议只能用于短时期保存，几周到几月。
8. 解冻复苏：37°C水浴快速解冻样本。
a. 解冻时轻轻摇晃，直到看不到任何冰块。1mL冻存管样本解冻时间大约3分钟。
b. 样本不能在结冰点以上加热（0-10°C）。水浴中拿出来时，冻存管触感是冷的。不推荐被动解冻。
9. 立即用培养基稀释细胞/CryoStor®混合物。
a. 稀释步骤可以一步完成。
b. 稀释用培养基温度为20°C到 37°C。
c. 推荐稀释比例≥1:10 （样本：培养基）
10.合适的条件下培养细胞。
11. 使细胞进入培养环境或立即使用细胞。
12. 解冻后24h进行细胞活力检测。*
注意：细胞活力检测应该在解冻后24h进行，以保证结果准确，并且以非冷冻样本作为对照。
*解冻后立即使用细胞膜完整性指示剂进行样本检测，如台盼蓝，分析样本细胞产量和活力，结果一般会高于正常值。
推荐使用更准确的活力检测方法，活死细胞荧光分析或代谢分析（MTT or alamarBlue®）。
同时推荐视觉观察方法，细胞贴壁和“漂浮”情况。
CryoStor冻存液使用说明书翻译：红荣微再编辑，推荐配合英文原版使用。
Cryostor操作视频可咨询获得。
华雅再生医学旗舰公司：红荣微再（上海）生物工程技术有限公司 ：1500 1904 520。红荣微再-客服: 经销商专员
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 106cells/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.
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*
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.
