Will repeated freezing and thawing of cell cryopreservation solution after packaging affect its effectiveness? Commonly used large packaging in laboratories

Aliquoting large packages into 50 ml tubes, and then having to freeze-thaw again each time before use, carries considerable risk. Our lab used to do the same, but later switched to aliquoting directly in a biosafety cabinet into 15 ml or 5 ml working tubes upon arrival, stored at -20. Take one tube at a time, thaw at 4 degrees, mix well, and use. HepG2 and 293T are relatively tolerant, but if you are doing transfection or drug screening, poor cell condition will affect the results. If precipitation, phase separation, or color change is observed after freeze-thaw, discard directly. After opening a large package, do not store it at 4 degrees for an extended period; the risk of bacterial growth in serum-free liquid increases. It is best to refer to the shelf life after opening stated in the instructions; we usually do not exceed 2 weeks.

The impact of repeated freeze-thaw cycles on the formula depends on the ingredients. Pure DMSO combined with basic culture medium is relatively stable, but many serum-free cryopreservation solutions contain polymers or proteins, and repeated freeze-thaw cycles may damage the protective components. After packaging, it is recommended to freeze thaw no more than once and prioritize using smaller sizes. You can conduct a small experiment: store the same batch of HepG2 cells separately in new packaging, freeze thaw once, and freeze thaw three times. After recovery, observe the viability and 24-hour adhesion rate, so that you have a sense of confidence. We tested a domestic system and found that there was little change after one freeze-thaw cycle, but the recovery rate significantly decreased after three freeze-thaw cycles. Therefore, we strictly followed the single use packaging method later on. Do not repeatedly freeze the entire bottle.

Your concern is reasonable, but it also depends on the storage conditions. -The 20% frozen storage solution is afraid of temperature fluctuations. If the refrigerator is frost free, automatic defrosting will cause temperature fluctuations, which may exacerbate the repeated freeze-thaw effect. Suggest placing it deep in the refrigerator, not against the door. Seal the pipe opening properly during packaging to prevent moisture from entering. Our laboratory does not use a large amount, so we directly purchase 100ml small packages. After arrival, they are divided into 10ml tubes and stored at -20 ° C. Each tube is disposable. This way, there is no need to worry about 4-degree pollution or repeated freezing and thawing. Although large packaging is cheap, it causes greater pollution or loss due to failure.

Packaging is correct as it can avoid repeated freezing and thawing of the entire bottle, as well as long-term contamination from opening at 4 degrees Celsius. However, it is not recommended to repeatedly freeze and thaw after packaging, especially in serum-free systems that contain proteins, polymers, and cytokines. Repeated freezing and thawing may lead to degradation or phase separation. It is generally recommended to pack according to a single dose and use each tube once; If placed at -20 degrees, slowly melt at 4 degrees in advance and do not heat with a 37 degree water bath. Freezing and thawing once is usually not a big problem, and I do not recommend using repeated freezing and thawing for more than 3 times for critical experiments on HepG2 and HEK293T. You don't need much, why don't you just buy 50ml small packages to save the trouble of packaging.