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CryoStor®是Biolife公司特別優化的細胞凍存液，在極低溫度 (-70ºC to -196ºC)準備和保存細胞。預混DMSO，為細胞和組織的冷凍、儲存和解凍過程提供安全的保護環境。CryoStor®通過調控凍存過程的分子生物學反應，無需血清、蛋白或具有高細胞毒性的試劑，就能增強細胞活力和功能。
1. 待冷凍細胞配制懸液（機械法或酶法解離）。
2. 細胞懸液離心獲得沉淀。
3. 去除上清液 - 注意：盡可能多地移除培養基，以降低CryoStor®凍存液被稀釋程度。
4. 分離：加入預冷的（2-8°C）CryoStor®凍存液。
a. 細胞密度：0.5-10×106 細胞/ mL，常規細胞培養方案（細胞密度可以更高）。
b. CryoStor®凍存液已經混合了DMSO，不需要再添加其他任何凍存劑。
5. 預冷：細胞/CryoStor®混合懸液2-8°C孵育約10分鐘。
6. 成核：-70°C冷凍樣品（許多方案交替使用-70°C和-80°C）
a. 大多數哺乳動物細胞，使用程序降溫法 (-1°C/min)或類似方法。
b. 程序降溫盒或異丙醇容器需要預冷到 2-8°C。
c. 約-5℃時 （-70°C冷凍約15-20min后）啟動樣品內的冰核形成（seeding），啟動方法可以是程序降溫儀控制的液氮噴放或是施加機械力攪動樣本（輕彈或輕拍）。
d. 使用異丙醇容器的冷凍時間（-70°C）建議為3-4小時。
7.樣本保存
a. 長時保存溫度液氮-130°C以下。
b. -80°C 保存建議只能用于短時期保存，幾周到幾月。
8. 解凍復蘇：37°C水浴快速解凍樣本。
a. 解凍時輕輕搖晃，直到看不到任何冰塊。1mL凍存管樣本解凍時間大約3分鐘。
b. 樣本不能在結冰點以上加熱（0-10°C）。水浴中拿出來時，凍存管觸感是冷的。不推薦被動解凍。
9. 立即用培養基稀釋細胞/CryoStor®混合物。
a. 稀釋步驟可以一步完成。
b. 稀釋用培養基溫度為20°C到 37°C。
c. 推薦稀釋比例≥1:10 （樣本：培養基）
10.合適的條件下培養細胞。
11. 使細胞進入培養環境或立即使用細胞。
12. 解凍后24h進行細胞活力檢測。*
注意：細胞活力檢測應該在解凍后24h進行，以保證結果準確，并且以非冷凍樣本作為對照。
*解凍后立即使用細胞膜完整性指示劑進行樣本檢測，如臺盼藍，分析樣本細胞產量和活力，結果一般會高于正常值。
推薦使用更準確的活力檢測方法，活死細胞熒光分析或代謝分析（MTT or alamarBlue®）。
同時推薦視覺觀察方法，細胞貼壁和“漂浮”情況。
CryoStor凍存液使用說明書翻譯：紅榮微再編輯，推薦配合英文原版使用。
Cryostor操作視頻可咨詢獲得。
華雅再生醫學旗艦公司：紅榮微再（上海）生物工程技術有限公司 ：1500 1904 520。紅榮微再-客服: 經銷商專員
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.
儲存
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).
細胞復蘇解凍
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.
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