CryoStor®是Biolife公司特別優(yōu)化的細(xì)胞凍存液，在極低溫度 (-70ºC to -196ºC)準(zhǔn)備和保存細(xì)胞。預(yù)混DMSO，為細(xì)胞和組織的冷凍、儲(chǔ)存和解凍過(guò)程提供安全的保護(hù)環(huán)境。CryoStor®通過(guò)調(diào)控凍存過(guò)程的分子生物學(xué)反應(yīng)，無(wú)需血清、蛋白或具有高細(xì)胞毒性的試劑，就能增強(qiáng)細(xì)胞活力和功能。

CryoStor使用說(shuō)明書(shū)

準(zhǔn)備細(xì)胞

1. 待冷凍細(xì)胞配制懸液（機(jī)械法或酶法解離）。

2. 細(xì)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(yǎng)基，以降低CryoStor®凍存液被稀釋程度。

細(xì)胞凍存

4. 分離：加入預(yù)冷的（2-8°C）CryoStor®凍存液。

a. 細(xì)胞密度：0.5-10×10⁶  細(xì)胞/ mL，常規(guī)細(xì)胞培養(yǎng)方案（細(xì)胞密度可以更高）。

b. CryoStor®凍存液已經(jīng)混合了DMSO，不需要再添加其他任何凍存劑。

5. 預(yù)冷：細(xì)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(shù)哺乳動(dòng)物細(xì)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(yù)冷到 2-8°C。

c. 約-5℃時(shí) （-70°C冷凍約15-20min后）啟動(dòng)樣品內(nèi)的冰核形成（seeding），啟動(dòng)方法可以是程序降溫儀控制的液氮噴放或是施加機(jī)械力攪動(dòng)樣本（輕彈或輕拍）。

d. 使用異丙醇容器的冷凍時(shí)間（-70°C）建議為3-4小時(shí)。

保存樣本

7.樣本保存

a. 長(zhǎng)時(shí)保存溫度液氮-130°C以下。

b.  -80°C 保存建議只能用于短時(shí)期保存，幾周到幾月。

樣本解凍

8. 解凍復(fù)蘇：37°C水浴快速解凍樣本。

a. 解凍時(shí)輕輕搖晃，直到看不到任何冰塊。1mL凍存管樣本解凍時(shí)間大約3分鐘。

b. 樣本不能在結(jié)冰點(diǎn)以上加熱（0-10°C）。水浴中拿出來(lái)時(shí)，凍存管觸感是冷的。不推薦被動(dòng)解凍。

9. 立即用培養(yǎng)基稀釋細(xì)胞/CryoStor®混合物。

a. 稀釋步驟可以一步完成。

b. 稀釋用培養(yǎng)基溫度為20°C到 37°C。

c. 推薦稀釋比例≥1:10 （樣本：培養(yǎng)基）

10.合適的條件下培養(yǎng)細(xì)胞。

11. 使細(xì)胞進(jìn)入培養(yǎng)環(huán)境或立即使用細(xì)胞。

12. 解凍后24h進(jìn)行細(xì)胞活力檢測(cè)。*

注意：細(xì)胞活力檢測(cè)應(yīng)該在解凍后24h進(jìn)行，以保證結(jié)果準(zhǔn)確，并且以非冷凍樣本作為對(duì)照。

*解凍后立即使用細(xì)胞膜完整性指示劑進(jìn)行樣本檢測(cè)，如臺(tái)盼藍(lán)，分析樣本細(xì)胞產(chǎn)量和活力，結(jié)果一般會(huì)高于正常值。

推薦使用更準(zhǔn)確的活力檢測(cè)方法，活死細(xì)胞熒光分析或代謝分析（MTT or alamarBlue®）。

同時(shí)推薦視覺(jué)觀察方法，細(xì)胞貼壁和“漂浮”情況。

CryoStor凍存液使用說(shuō)明書(shū)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(yī)學(xué)旗艦公司：紅榮微再（上海）生物工程技術(shù)有限公司  ：1500 1904 520。紅榮微再-客服:   經(jīng)銷商專員

CryoStor凍存液英文原版說(shuō)明書(shū)

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(chǔ)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(xì)胞復(fù)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.