HOW TO PREVENT FREEZING BAG BREAKS

如何防止冷冻袋破裂

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.

所有冷冻袋在冷冻时都可能破裂，EVA、EVO、FEP，每种柔性袋材料在冷冻时都可能破裂。当然，有些材料比其他材料更坚固，但只要小心一点，就可以防止破裂。我们收到的少数破损投诉几乎都来自相对较新的客户。一旦他们实施以下更改，破损问题就会消失。

Here is what we recommend以下是我们的建议:

l                      Seal Donor Tubes Short 密封供体管

l                      Remove Air 排出空气

l                      Use the Right Cassette  使用正确的盒子

l                      Handle with Care 小心处理

l                      Use the Right Cryoprotectants 使用正确的冷冻保护剂

l                      Be Aware of Cell Metabolism 注意细胞代谢

SEAL DONOR TUBES SHORT密封供体管短

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.

这是导致袋子破裂的最常见原因。供体管应密封在尖头端口下方，并尽可能靠近短管（见右图）。如果袋子在盒内滑动，密封时间短可保护冷冻供体管免受冷冻过程中的潜在冲击。相反，如果密封时间长，则其会像杠杆一样，任何对冷冻管的冲击都可能使其在袋子接头处折断。最后，将密封剪开，不要拉开。这可能适用于 PVC，但不适用于 Cryostore 袋上使用的 EVA 管。

REMOVE AIR去除空气

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.

空气中 79% 的成分是氮气，储存在液氮中时会液化。然后，液体会流下并聚集在袋子中低的开口区域。问题是，在重新加热过程中，氮气会膨胀回到气相，而袋子仍然很脆，并且很可能发生在与液化之前不同的区域。由此产生的快速膨胀会产生压力气泡，导致袋子破裂或爆裂。

USE THE RIGHT CASSETTE使用正确的盒子

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.

如果储存盒太紧或有凸起物使袋子变形，那就有问题了。袋子在变暖时会膨胀，储存期间袋子移位到不同区域的可能性很高。

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.

当袋子膨胀到有限的空间时，挤压可能会导致袋子破裂。同时，如果盒太大，冷冻袋可能会在盒中滑动，滑入金属盒的冲击力可能会导致袋子破裂。使用 OriGen 外包装袋可能有助于保护袋子并将其固定在位。

HANDLE WITH CARE小心处理

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.

如果您像处理冷冻易碎袋一样处理袋子，那么您的额外小心将减少袋子破裂的几率。另一方面，如果您将冷冻袋放在硬容器中松散地推过停车场，那么发现袋子破裂也就不足为奇了。冷冻和/或解冻过程中出现的袋子破裂是由于冷冻 CryoStore 袋受到撞击、挤压或弯曲造成的。当冷冻温度低于 -60°C 时，EVA 就像一块玻璃，如果施加足够的力，就会破裂。取出、运输、重新装架和解冻所有冷冻袋时要小心。

USE THE RIGHT CRYOPROTECTANTS使用正确的冷冻保护剂

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.

不同的细胞培养物和不同的冷冻保护剂在冷冻过程中会有不同的表现，并且实际上会膨胀。水在降至液氮温度的过程中会经历几种不同的相变，其中一些相变会急剧膨胀。我们都知道冰山会漂浮，因此看到冷冻过程中发生膨胀我们不应该感到惊讶。了解您的解决方案会产生什么效果，并在处理过程中保持一致性。

CELL METABOLISM细胞代谢

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.

这是最棘手的，也是最令人抓狂的。如果你的细胞在冷冻前正在活跃生长，它们会排出大量的二氧化碳。二氧化碳液化，寻找低水平，然后在 -78°C 冻结（“干冰”）。当它从气体变成固体时，体积急剧减小。当袋子随后被加热到 -78°C 以上时，冷冻保存期间形成的固体二氧化碳突然变成气体，体积增加约 1000 倍。由于它可能已经滴流到新的位置，因此它无法膨胀回其原始区域。如果袋子仍然冻结，袋子内的气压会导致袋子破裂，如果袋子材料变暖，则会发出“砰”的一声爆炸。我们都见过这两种情况。

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.

我们相信，如果您了解这几项安全措施，并在处理冷冻细胞和组织时小心谨慎，您将加入从未遇到过袋子破裂的数百名用户。