The heat inactivation of serum is a topic of interest to many culturers. The expensive serum contains precious substances such as growth factors, vitamins, amino acids, etc. It is completely unnecessary to place them at temperatures above 50 °C for up to 30 minutes. of. Despite this, the thermal inactivation of serum in most laboratories is performed routinely. Most of the experimenters did not consider the negative effects of heat treatment on serum growth factors, amino acids and other components in our technical hotline. The most frequently mentioned is whether the serum should be heat inactivated. Let us discuss and explain the heat inactivation of fetal calf serum.
The purpose of heat inactivation is to remove heat sensitive substances such as complement in serum, but inactivation of complement in fetal bovine serum is clearly unnecessary. Triglia and Linscott [1] have measured the complement components in commercial fetal bovine serum. They found that the C1 and C6 contained in the fetal bovine serum only reached 1-3% of the annual animal serum, while the other complement components were only adult animals. 5-50%, as the main component of complement C3 is almost impossible to detect in fetal bovine serum. Through the complement fixation experiment, we also obtained similar results in several different batches of fetal bovine serum. Even in undiluted serum, no significant hemolysis was observed. In addition, most laboratories pre-heat the culture solution before incubation, and also have a deactivation effect on the thermosensitive complement.
According to the survey, at least 70% of the researchers' inactivation of serum is only due to routine operations, or is taken for granted. Conventional inactivation recommends temperatures between 45 ° C and 62 ° C, while times range from 15 minutes to 60 minutes. The most common method is heat treatment at 56 ° C for 30 minutes. With the improvement of serum collection, processing and processing techniques, many of the reasons previously thought to be heat inactivation are no longer valid. Only a few researchers who performed heat inactivation on serum confirmed the effectiveness and necessity of this step in the experiment. Sex.
Pinyopummintr et al. demonstrated that the serum removal inactivation step did not affect the developmental differentiation of bovine embryos [2]; the results of the study [3] confirmed that the heat inactivation step attenuated the adhesion of fetal bovine serum and calf serum to cells, using SV -BHK, BALB-3T3, CV-1, FS-4 cells in the cell adhesion experiment, the effect of heat inactivation on fetal bovine serum is less than the effect on calf serum.
We investigated the effects of heat inactivation on fetal bovine serum and on the growth of different cell lines. By comparing 11 different cell lines, it was found that heat inactivation negatively affected the growth of 6 cell lines (HBAE, MDBK, Vero, fibroblast, MRC-5), three cell lines (FOX-NY, MDCK) And CHO-K1) were not affected by heat inactivation, while only two cell lines (Balb/3T3, Sp2/0Ag14 hybrid) showed a slight improvement in cell growth after heat inactivation. Therefore, under normal operation, heat inactivation usually does not significantly promote cell growth.
In addition to complement inactivation, heat treatment also has an inactivation effect on mycoplasma that may be present in the serum. Many years ago, when a 450 nm pore size filter was used to process serum, contamination of mycoplasma in serum occurred. In response to this problem, HyClone was the first to use 100 nm three-layer filter continuous filtration technology. Since the adoption of this technology and the subsequent 40 nm filtration technology, no contamination of mycoplasma has been found in our serum products. Another reason why inactivation becomes unnecessary.
Under normal operation, heat inactivation often has a negative impact on serum products, and heating of the serum often results in the production of precipitates in the serum, which is often considered to be microbial contamination. After noticing this phenomenon, in order to verify the presence of contamination, or to promote the dissolution of the precipitate, many culturers tend to incubate the serum at 37 °C. This often makes the situation worse, the protein in the serum is further precipitated, in order to make sure that the serum is not contaminated, the microscopic examination, the sterile culture test, and the Gram stain test are a waste of time for the experimenter. And energy.
In conclusion, heat inactivation of serum in most cell cultures is not necessary. In many cases, heat inactivation does not improve cell growth, but reduces the ability to support cell growth. Even in the case of a few promotions, the rate of promotion is negligible. In addition, precipitation caused by heat inactivation of serum is often considered to be microbial contamination, causing unnecessary trouble to both users and suppliers. We recommend that users who inactivate serum should be tested to confirm their necessity and determine the appropriate conditions for inactivation; during inactivation, strict monitoring is required and a reproducible protocol is selected.
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