HOW TO PREVENT FREEZING BAG BREAKS

如何防止冷凍袋破裂

All freezing bags can be broken while frozen, - EVA, EVO, FEP, every flexible bag material can be broken when frozen solid. Of course, some materials are more rugged than others, but with a bit of care, you can prevent breaks altogether. Of the few breakage complaints we receive, almost all come from relatively new customers. Once they implement the changes below, their breakage problem disappears.

所有冷凍袋在冷凍時都可能破裂，EVA、EVO、FEP，每種柔性袋材料在冷凍時都可能破裂。當(dāng)然，有些材料比其他材料更堅固，但只要小心一點，就可以防止破裂。我們收到的少數(shù)破損投訴幾乎都來自相對較新的客戶。一旦他們實施以下更改，破損問題就會消失。

Here is what we recommend以下是我們的建議:

l                      Seal Donor Tubes Short 密封供體管

l                      Remove Air 排出空氣

l                      Use the Right Cassette  使用正確的盒子

l                      Handle with Care 小心處理

l                      Use the Right Cryoprotectants 使用正確的冷凍保護劑

l                      Be Aware of Cell Metabolism 注意細(xì)胞代謝

SEAL DONOR TUBES SHORT密封供體管短

This is the most common cause we see contributing to bag breaks. The donor tube should be sealed below the spike ports and as close to the stub tube as possible (see photo at right). Sealing it short protects the frozen donor tube from potential impact while frozen if the bag slides within the cassette. Conversely, leaving it long makes it act like a lever, and any impact on the frozen tube can snap it off at the bag joint. Finally, cut, don’t pull, the seal apart. This may work for PVC, but not for EVA tubing used on the Cryostore bag.

這是導(dǎo)致袋子破裂的最常見原因。供體管應(yīng)密封在尖頭端口下方，并盡可能靠近短管（見右圖）。如果袋子在盒內(nèi)滑動，密封時間短可保護冷凍供體管免受冷凍過程中的潛在沖擊。相反，如果密封時間長，則其會像杠桿一樣，任何對冷凍管的沖擊都可能使其在袋子接頭處折斷。最后，將密封剪開，不要拉開。這可能適用于 PVC，但不適用于 Cryostore 袋上使用的 EVA 管。

REMOVE AIR去除空氣

Air is made of 79% nitrogen, which will be liquefied when stored in LN. This liquid then runs down to collect at the lowest open area in the bag. The problem is that during re-warming, nitrogen expansion back to the gas phase will occur while the bag is still brittle, and most likely in different regions than where it was before liquefaction. The resulting rapid expansion can create a bubble of pressure and cause a crack or bursting of the bag.

空氣中 79% 的成分是氮氣，儲存在液氮中時會液化。然后，液體會流下并聚集在袋子中低的開口區(qū)域。問題是，在重新加熱過程中，氮氣會膨脹回到氣相，而袋子仍然很脆，并且很可能發(fā)生在與液化之前不同的區(qū)域。由此產(chǎn)生的快速膨脹會產(chǎn)生壓力氣泡，導(dǎo)致袋子破裂或爆裂。

USE THE RIGHT CASSETTE使用正確的盒子

If the storage cassette is too tight or has projections that distort the bag, that is a problem. The bag expands as it warms, and the chance of the bag shifting to a different area during storage is high.

如果儲存盒太緊或有凸起物使袋子變形，那就有問題了。袋子在變暖時會膨脹，儲存期間袋子移位到不同區(qū)域的可能性很高。

As the bag expands into a restricted space, a pinch can result in a bag break. At the same time, if the cassette is too large, the frozen bag can slide around in the cassette, and the impact of sliding into the metal cassette can cause a crack. Use of the OriGen Overwrap bag may help keep the bag protected and in place.

當(dāng)袋子膨脹到有限的空間時，擠壓可能會導(dǎo)致袋子破裂。同時，如果盒太大，冷凍袋可能會在盒中滑動，滑入金屬盒的沖擊力可能會導(dǎo)致袋子破裂。使用 OriGen 外包裝袋可能有助于保護袋子并將其固定在位。

HANDLE WITH CARE小心處理

If you handle the bag like it is fragile when frozen, your extra care will result in fewer bag breaks. On the other hand, if you transport the frozen bag rattling around loose in a hard container as you push it across the parking lot, you should not be surprised to find a break. Bag breaks that develop during freezing and/or thawing occur due to impact, crushing, or bending of the frozen CryoStore bag. When frozen below -60°C, the EVA acts like a pane of glass and can crack if enough force is applied. Use care when removing, transporting, re-racking, and thawing all frozen bags.

如果您像處理冷凍易碎袋一樣處理袋子，那么您的額外小心將減少袋子破裂的幾率。另一方面，如果您將冷凍袋放在硬容器中松散地推過停車場，那么發(fā)現(xiàn)袋子破裂也就不足為奇了。冷凍和/或解凍過程中出現(xiàn)的袋子破裂是由于冷凍 CryoStore 袋受到撞擊、擠壓或彎曲造成的。當(dāng)冷凍溫度低于 -60°C 時，EVA 就像一塊玻璃，如果施加足夠的力，就會破裂。取出、運輸、重新裝架和解凍所有冷凍袋時要小心。

USE THE RIGHT CRYOPROTECTANTS使用正確的冷凍保護劑

Different cell cultures and different cryoprotectants can behave differently and actually expand during freezing. Water goes through several different phase changes on its way down to LN temperatures, some of those phase changes expand dramatically. We all know icebergs float, so we shouldn’t be surprised to see expansion happen during freezing. Know what to expect from your solutions and maintain consistency in your processing.

不同的細(xì)胞培養(yǎng)物和不同的冷凍保護劑在冷凍過程中會有不同的表現(xiàn)，并且實際上會膨脹。水在降至液氮溫度的過程中會經(jīng)歷幾種不同的相變，其中一些相變會急劇膨脹。我們都知道冰山會漂浮，因此看到冷凍過程中發(fā)生膨脹我們不應(yīng)該感到驚訝。了解您的解決方案會產(chǎn)生什么效果，并在處理過程中保持一致性。

CELL METABOLISM細(xì)胞代謝

This is the one that is trickiest of all, and the most maddening to figure out. If your cells are actively growing just before freezing, they are expelling a lot of CO2. Carbon Dioxide liquefies, seeks the lowest level, and then freezes at -78°C (“ Dry Ice”). The volume decreases dramatically as it goes from gas to solid. As the bag is subsequently warmed above -78°C, the solid CO2 formed during cryopreservation goes suddenly to gas, and increases in volume about 1000 times. Since it has likely trickled to a new location, is it cannot expand back into its original area. The gas pressure inside the bag causes it to crack if still frozen, or explode with a ‘ POP’ if the bag material is warmed. We have seen both happen.

這是最棘手的，也是最令人抓狂的。如果你的細(xì)胞在冷凍前正在活躍生長，它們會排出大量的二氧化碳。二氧化碳液化，尋找低水平，然后在 -78°C 凍結(jié)（“干冰”）。當(dāng)它從氣體變成固體時，體積急劇減小。當(dāng)袋子隨后被加熱到 -78°C 以上時，冷凍保存期間形成的固體二氧化碳突然變成氣體，體積增加約 1000 倍。由于它可能已經(jīng)滴流到新的位置，因此它無法膨脹回其原始區(qū)域。如果袋子仍然凍結(jié)，袋子內(nèi)的氣壓會導(dǎo)致袋子破裂，如果袋子材料變暖，則會發(fā)出“砰”的一聲爆炸。我們都見過這兩種情況。

We believe that if you are aware of these few safety measures and use care in handling frozen cells and tissues, you will join the hundreds of users who have never had a bag break.

我們相信，如果您了解這幾項安全措施，并在處理冷凍細(xì)胞和組織時小心謹(jǐn)慎，您將加入從未遇到過袋子破裂的數(shù)百名用戶。