What is the reason for the formation of flocculent sediment during rapid thawing of fetal bovine serum in a 37 ℃ water bath

This bottle does not necessarily need to be discarded. If HUVEC or 293T cells show no abnormalities in a small-scale test, it can be labeled as a rapid-thaw batch and preferentially used for tolerant cells, preliminary experiments, or routine maintenance, while normal-thaw serum is used for critical primary experiments. For future thawing, it is recommended to transition from -80 to -20 for a few hours, then place at 4°C overnight; if urgent, it can be placed in a 15-25°C water bath with gentle shaking, avoiding temperatures above 30°C. Retain a sample and record this thawing process, contact the supplier to explain the situation, and ask whether other users of the same lot have reported similar feedback.

When transferring directly from -80°C to a 37°C water bath, the temperature inside the bottle rises unevenly. The regions near the bottle wall heat up rapidly, and fibrinogen and lipoproteins tend to aggregate and precipitate. The cloudy appearance is mostly related to the formation of these large particles. Overnight thawing at 4°C is slow and uniform, which is why less precipitate formed previously. The precipitate produced by rapid thawing is mostly not bacterial contamination, but if the bottle opening is not well sealed during the water bath or water enters the bottle, contamination may also be introduced. This needs to be judged in combination with the supernatant color, pH, and sterility testing.

Directly filtering the entire bottle through a 0.22 μm filter is not recommended, as flocculent material will quickly clog the membrane, and filtration can only remove insoluble matter, not restore already denatured protein. Allow it to stand overnight at 4°C, then aseptically collect the supernatant and aliquot; or centrifuge at low speed, collect the supernatant, and then sterilize by 0.22 μm filtration. HUVEC are relatively sensitive to endothelial cell culture conditions. It is recommended to first use this serum to prepare DMEM for small-scale test culture for 2-3 passages, observing attachment, proliferation, and morphology. If HUVEC show vacuolation, elongation, or significantly slowed proliferation, use it only for general cells or preliminary experiments.