There are many areas where researchers need to be aware of the process of cell culture. As one of the most basic technologies in biological research, cell culture permeates all aspects of life science research. This post is shared with the role of fetal bovine serum, selection, and preservation, as well as freeze-thaw method.
Brief Introduction to Serum and its effect
Serum, that is the yellowish clear liquid isolated from fibrin in plasma or plasma from which fibrin has been removed. And its main effects involve:
. As a provider: nutrition and energy sources; growth and adhesion factors; peptide and steroid hormones; carrier protein.
. Protect cells from proteases, toxic substances, PH value fluctuations, mechanical shear stress, oxidative damage
Characteristics of Fetal bovine serum
. Collection Method: Ensure the quality of serum, low endotoxin, and low hemoglobin collection system through a closed system
. Filtering Method: Usually filtered with 3 consecutive 100 nm filters
. Detection system: Each batch of products are subject to strict testing of bacteria, mycoplasma, virus, endotoxin, and hemoglobin
The difference between fetal bovine serum（FBS） and neonatal bovine serum（NBS） or calf serum（NCS）
1. Different serum types
.Gestational age of FBS is 3-8 months of pregnancy
.Gestational age of NCS is within 10 days newborn
.Gestational age of BCS is between 16-22 weeks
2. Different blood collection methods
. Fetal Bovine Serum: Caesarean section closed heart puncture method to collect blood while the cow is 3-8 months of pregnancy,
. Newborn bovine serum, bovine serum, donor bovine serum: respectively taken from newborn cow (calf), adult cow blood.
3. Difference in Serum composition
Fetal bovine serum has not yet been exposed to the outside, the serum contains antibodies, complement and other harmful components at the least; cell growth factor, promoting adhesion molecule, hormones and other active substances and other components are different from other types of serum.
4. Different application areas
. Some specific cells must be cultured with fetal bovine serum, such as stem cells, lymphocytes, nerve cells, astrocytes, etc.
. While some cell cultures require only bovine serum to complete, such as tumor cells. Serum concentrations are also different, generally between 10% -15%, with special requirements at a concentration of 20%.
5. Different color – the serum performance color will differ according to the different hemoglobin content,
. True fetal bovine serum usually is with poor coagulation, erythrocytes will rupture so that the general appearance is reddish or orange, and[but?] newborn cattle (calf) serum will perform as wax yellow or partial yellow
6. Different clarity and consistency
Fetal bovine serum will show as thin and light as albumin and lipid content is low, but newborn (calf) serum is more viscous than serum
7. Tips on choosing proper serum
.In these types of serum, the quality of fetal bovine serum is undoubtedly the highest, if your cells more delicate and difficult to support, in order to get a better culture, please select fetal bovine serum.
. If your cells are more susceptible to get a better effect, the use of newborn calf serum is enough.
. The general choice of serum species can be based on ATCC and experimental needs.
How to Preserve the Serum
1. Preservation Method
Serum should be stored at -20 °C. If you cannot run out of a bottle, it should be aseptically packaged and then cryopreserved. Be careful not to freeze and thaw repeatedly.
2. What will make the serum stored in the refrigerator get precipitated?
Some fetal bovine serum products are not pre-aged and various proteins and lipoproteins (such as cold agglutinin, fibrinogen, vitronectin, etc.) in the serum may aggregate to form a precipitate or visible opacity while stored at 2-8 °C. This should not affect the quality of the serum. It is recommended to store fetal bovine serum at -20 ° C to[And] avoid repeated freezing and thawing.
Correct thawing method of Serum
Place the serum in a refrigerator at 2-8 °C for 12-24 hours, slowly dissolving it partially, and then allow it to fully dissolve at room temperature.
Note: Swing the serum slowly and gently at intervals to dissolve it.
Note: Avoid 37 °C water bath.
Common problems and solutions
1. What is the sediment in serum?
. Fibrin – The large precipitate that often appears. As serum are collected and rapidly processed at low temperatures, some fibrinogen remains in solution during processing and coagulates to form fibrin precipitates after the final filtration.
. Calcium phosphate - a common precipitate which will make the serum become turbid and will increase when cultured at 37 °C. The sediment will be like a black spot while observing under an inverted microscope. As it appears to be movable due to Brownian motion and is therefore frequently mistaken as microbial contamination.
. Other: Some lipids or proteins.
2. Serious precipitation is generally caused by improper operation
. Unreasonable[Incorrect] thawing procedure
. Improper storage: repeated freeze-thaw cycles or long-term storage at 2-8 °C, or at room temperature for too long[Long time?]
Which treatment will increase serum sediment?
. Gamma γ-ray irradiation
. Serum was incubated at 37 °C
. Heat-inactivated serum
. Improper thawing operation, no mixing when melted
. Storage improper: repeated freezing and thawing; long-term 2-8 °C storage or room temperature for a long time.
How to avoid precipitation in serum
(1) While thawing serum, please shake it at any time, so that the temperature and composition can be uniformity, reducing the occurrence of precipitation.
(2) Shake evenly regularly (be careful not to cause bubbles) when serum is stored frozen so that the temperature and composition will be uniformity.
(3) Do not directly thaw the serum from -20 °C to 37 °C, the protein tends to coagulate and precipitate if the temperature changes too much.
(4) Avoid putting the serum at 37 °C too long, otherwise, the serum will become turbid, while many of the unstable components of the serum will be destroyed, thus affecting the quality of serum.
(5) Heat inactivation of serum is very easy to cause the increase of sediment, if not necessary, just skip this step.
(6) If the heat of serum inactivation is necessary, need to follow the principle of 56 °C for 30 minutes, otherwise, will cause increased sediment.
How to deal with the flocculent precipitate after thawing?
. Precipitation in serum can occur for a number of reasons, but the most common cause is due to the denaturation of lipoproteins in serum, which is also present in serum after thawing in serum, and one of the main causes of sediment. However, these flocculent sediments do not affect the quality of the serum itself.
. For removing these flocculent sediments, serum can be aliquoted into sterile centrifuge tubes and centrifuged at 400g for a while. The supernatant can be added to the medium for cell culture without affecting the culture process.
. It is not recommended to filter out these flocculent sediments because it may clog the filter membrane.
Is it possible to use different serum types from the original culture conditions?
The answer is NO. The serum is an extremely important source of nutrition in cell culture, so the type and quality of serum have a huge impact on cell growth. Serum from different species differs in the amount or content of some substance or molecule. The wrong use of serum often results in cells that can not survive. Therefore, you can not directly replace the cells used in the culture of serum, even if different batches of the same serum.
For mixed serum, in principle, should be based on the original serum, and gradually increase the proportion of new serum, and finally replace the original serum. The proportion of the general use will be 3: 1-1: 1-1: 3-100% new serum.
What is heat inactivation?
The thawed serum is typically treated at 56 °C for 30 minutes because this heating step can deactivate complement, and the complement is involved in the following activities: cytolytic activity, smooth muscle contraction, release of mast cells and platelet histamine, phagocytosis enhancement, chemotaxis and activation of lymphocytes, macrophages.
Why serum needs to be heat inactivated?
Heating can inactivate the complement system. Activated complement is involved in lysing cellular events, stimulating smooth muscle contraction, releasing histamine from cells and platelets, and activating lymphocytes and macrophages. In immunological studies, ES cell culture, insect cells, smooth muscle cells, the heat-inactivated serum is recommended.
Is it necessary to conduct heat inactivation?
Experiments have shown that heat-inactivated serum, properly processed, is not required for most cells. The treated serum had little or no effect on the growth of the cells, and often resulted in a decrease in the rate of cell growth even though the quality of the serum was usually affected by the high-temperature treatment. And after heat treatment of serum, the formation of sediment will be significantly increased, these sediments observed under an inverted microscope, such as "black spots," researchers often mistaken for serum contamination to place the serum in a 37 °C environment, which will increase the sediment, and eventually led researchers to mistake it as the division of microbial expansion.
The correct method for heat inactivation
Place the melted serum and a beaker of the same volume in the water at 56 °C in a water bath. When the temperature in the beaker reaches 56 ° C and start counting, the inactivation is completed after 30 minutes. The whole process needs to be shaken and mixed at any time.
All the mentioned above is related to the information, note, and tips for Fetal Bovine Serum.
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