Biotechnology has been regarded as a high technology by all countries in the world, occupying a special and prominent position in the whole science and technology, especially the development of life science is inseparable from biotechnology, the development of life science is highly valued by all countries. China has established life sciences colleges in many universities. Modern biotechnology is generally considered to include genetic engineering technology, cell engineering technology, enzyme engineering technology and fermentation engineering technology, and the development of these technologies is almost closely related to cell culture, especially in the development of the pharmaceutical field, cell culture has a special Role and value. For example, many genetic engineering drugs or vaccines are realized through cell culture in the research and production process. Many genetic engineering hepatitis B vaccines use CHO cells as a carrier; cell engineering is inseparable from cell culture. Hybridoma monoclonal antibodies are completely achieved through cell culture, even now the rapid development of genetically engineered antibodies Open cell culture. Gene therapy and somatic cell therapy, which are receiving much attention, can only be achieved through the process of cell culture. Some fermentation engineering and enzyme engineering are also closely related to cell culture. In short, cell culture has played a key core role in the development of the entire biotechnology industry. For the development of cell culture, the quality of the medium is the key, and the animal serum in the main components of the medium plays an important or even irreplaceable role in the growth and reproduction of the cells. Bovine serum is the most widely used in the application of animal serum, so bovine serum is one of the important raw materials in medical biotechnology products. Ensuring the quality of bovine serum is also an important link to improve the quality of biological products.
1. Main functions of bovine serum in cell culture medium Bovine serum is the natural medium with the largest dosage in cell culture. It contains rich nutrients necessary for cell growth and has extremely important functions.
1. Provide hormones for maintaining the exponential growth of cells, no or few nutrients in the basic medium, and the main low molecular nutrients.
2. Provide binding proteins that can recognize vitamins, lipids, metals and other hormones, and can bind or modulate the vitality of the substances they bind.
3. In some cases, the binding protein can combine with toxic metals and pyrogens to play a role in detoxification.
4. It is the source of factors needed for cells to adhere to the wall and spread on the plastic medium.
5. It acts as a pH buffer.
6. Provide protease inhibitors to inactivate remaining trypsin during cell passage and protect cells from harm.
2. The main components of bovine serum Serum is a very complex mixture. Although most of its components are known, some of them are still unclear, and the composition and content of serum often depend on the sex, age, and physiology of the blood donor. Conditions and nutritional conditions vary.
1. Protein is the main ingredient in bovine serum. In addition to carrying metal ions, fatty acids and hormone proteins, there are mainly albumin and globulin.
Fibronectin cells promote cell attachment;
α2 macroglobulin inhibits the role of trypsin; fetal bovine serum contains fetuin to promote cell attachment; transferrin can bind iron ions, reducing its toxicity and being used by cells.
2. Peptide: Platelet growth-promoting factor can promote cell division, is one of the main members of the peptide family, is the main cell-promoting factor; fibroblast growth factor, epidermal cell growth factor, nerve cell growth factor, etc. Although it is rare, it also has a certain effect on cell growth.
3. Hormones: Hormones have many effects on cells.
Insulin: Promotes cells to take up glucose and amino acids, which is related to promoting cell division.
Insulin-like growth factor: It can bind to the insulin receptor expressed by cells, and thus has the same effect as insulin.
Somatotropin: Promotes cell proliferation.
Hydrocortisone: The serum contains a quantitative amount of this component, which may have both cell adhesion and proliferation effects, but it has been shown that hydrocortisone in serum, such as high cell density, may have the effect of inhibiting cells and inducing it Differentiation occurs.
4 Other ingredients such as amino acid, glucose, keto acid, etc. are of little significance to the synthetic medium of various nutrients. The trace elements combined with protein are meaningful for cell culture.
3. The quality requirements of bovine serum With the development of science and technology and the continuous improvement of living standards, the quality requirements of biopharmaceutical products are getting higher and higher, so the requirements for the quality standards of various raw materials involved in the preparation process are also increasing. The higher the level, the higher the quality requirement of bovine serum, the main natural ingredient used in cell culture media. Bovine serum includes fetal bovine serum, newborn bovine serum and adult bovine serum.
(1) The requirements of the "Procedures for the Production of Biological Products Using Animal Cells in Vitro Culture" published by WHO:
1. Bovine serum must be from a herd or country that has documented no bovine spongiform encephalopathy. And should have an appropriate monitoring system.
2. Some countries also require bovine serum from cows that have not used ruminant protein feed.
3. To prove that the used bovine serum does not contain inhibitors to the vaccine virus produced.
4. Serum should be filtered and sterilized through the filter membrane to ensure sterility.
5. No pollution by bacteria, mold, mycoplasma and virus. Some countries require no pollution by bacteria and bacteriophage.
6. It has a good function of supporting reproduction of cells.
(2) China has put forward stricter standard requirements in the 2000 edition of "Quality Control Standards for Main Raw and Auxiliary Materials of Chinese Biological Products" for the quality of bovine serum. Including protein content, bacteria, fungi, mycoplasma, bovine virus, E. coli bacteriophage, bacterial endotoxin, support cell proliferation examination.
(3) Quality requirements for bovine serum in the United States: The bovine serum from two major U.S. bovine serum suppliers, Gibco and Sigma, has entered the Chinese market.
They are extremely strict with their quality standards, with clear regulations from the source of serum to product quality.
1) Serum source: Fetal bovine serum can be collected from countries all over the world, but it must meet his quality standards and US Department of Agriculture import requirements. The local authorities are not clear whether there is any serum in the local area where the bovine spongiform virus epidemic is not collected, and there is proof of the quality of the serum: including all the data of the collection process, test results and delivery status.
2) Collection of serum: fetal bovine serum is collected by cardiac puncture. Newborn cows (10-14 days) and calves (within 10 months) are taken intravenously. Serum collection conditions must meet the standards of industrial production: collection at low temperature, one-time separation, and mixing and freezing immediately after separation. Serum that meets the requirements is inactivated in a 56 ° C water bath for 30 minutes.
3) Preparation of serum:
a. Ultra-low IgG fetal bovine serum: remove the gamma globulin in fetal bovine serum by affinity chromatography process to minimize the content of FBS gamma globulin, generally IgG≤5ug / ml, and the biological activity remains unchanged.
b. Serum dialysis: dialyze with a dialysis membrane with a molecular weight of 12000-14000 in 0.15M NaCl until the glucose content is <0.5mg / ml (measured by glucose oxidase / peroxidase method).
c. γ-ray irradiation: γ-ray irradiation has been proven to effectively inactivate viruses and mycoplasma that may be contaminated in serum. The irradiation dose is 30 ～ 45KG.
And this dose of γ-ray irradiation will not change the physical and chemical properties of serum and the impact on cell culture.
4) Sterilization and filtration: The crude serum after the above treatment is then filtered through a series of sterilization filters including 0.2 micron and 0.1 micron filters. FBS should pass through three layers of 0.1 micron filter membrane, and other serum can pass through 0.2 micron filter membrane.
4. Serum quality of the final product
1) Chemical verification: osmotic pressure
pH
Protein content-electrophoresis method albumin globulin hemoglobin increases with the age of cattle. The total protein content in the serum increases accordingly. The total serum protein content can determine the product specifications and age.
2) Microbiological examination: bacteria and fungi meet USP standards.
Mycoplasma: cultivated for 3 to 4 weeks on mycoplasma professional culture medium, the culture temperature is 36 ℃ ± 2 ℃
It is carried out under aerobic and anaerobic conditions, and some of them also need to be uploaded 4 times on mycoplasma agar culture dishes. Hoechst fluorescein DNA staining is used to check those mycoplasmas that cannot be detected by the culture method, such as Mycoplasma hominis.
Virus check:
Bovin blue tongue
Bovine Adenovirus
Bovine Parvovirus
Bovine Viral Diarhea Virus
Rabies virus Rabies
Reovirus
Bovine respiratory syncytial virus BRSV
Parainfluenza â…¢
Inspection Method:
Bovine cell cultures that have been proven to be free of contamination by exogenous factors are supplemented with 15% serum to be tested in the cell growth fluid and passed on for 3 consecutive days for 21 days. Negative control cell culture fluid was added with 15% bovine serum known to be free of virus contamination, under the same conditions as the test group. Pay attention to changes in cell morphology or cytopathy during the observation period. On the 14th day of the observation period, the samples to be tested and the control samples were checked by standard virus detection methods.
For example, the cell culture to be tested is digested with trypsin and then divided into 6 containers with coverslips. Negative control samples follow the same method. Then, bovine diarrhea virus, bovine parvovirus, bovine adenovirus, rabies virus and reovirus were added to the coverslip container of the culture to be tested as a positive control, and cultured for 7 more days, and then checked with fluorescent antibody technology.
Other changes caused by cytopathy or virus were observed by hematoxylin or eosin staining. Take another cell culture flask to be tested with human “O†red blood cells,
Guinea pig erythrocytes and fresh chicken erythrocytes were tested for hemocytosis virus. The test was conducted at 2-8 ° C and 20-24 ° C. Negative control cells were pre-infected with parainfluenza III virus as a positive control. Uninfected cells served as negative controls.
3) Limulus reagent for endotoxin detection.
4) Bacteriophage inspection mainly checks E.Coli (C300 or K-12) bacteriophage.
5) Determination of hormone content Each batch of FBS must be measured for certain hormone content, including: estradiol, insulin, pregnancy, testosterone, thyroxine and so on.
6) Detection of hemoglobin The ratio of hemoglobin to oxyhemoglobin ≥70%, hemoglobin content ≤mg15 / dl.
7) Determination of cell growth effect
a. Cell cloning effect determination Cell selection: Sp20 / Ag-14 or P3X63-Ag8.653
Serum concentration and cell number:
10% serum / 1 cell / well
4% serum / 5 cells / well cell culture medium RPMI-1640, 96-well culture plate 36 ℃ ± 2 ℃, 5% carbon dioxide culture 10 ～
15 days. In the test, a known reference bovine serum was selected as a control.
Cloning efficiency calculation:
Cloning efficiency = (average number of positive wells / total number of culture wells) X100%
Relative cloning efficiency = test serum cloning efficiency / reference serum cloning efficiency
b. Adhesive efficiency test:
Using human passage cells for the adherence efficiency test of each batch of serum, A549 (human lung cancer cells) cells can reflect the adherence changes of low concentration serum. A 6-well culture plate was used to inoculate each batch of serum with two concentrations and two different types of cells, namely 10% serum-100 cells / well and 4% serum 200 cells / well. Incubate at 36 ° C ± 2 ° C and humidify 5% carbon dioxide incubator for 10 to 14 days, calculate the coloring cell clones, and determine the adhesion efficiency. From these two different serum concentrations, the level of experimental serum supporting cell adhesion growth was established, and the average adhesion efficiency under the two test conditions was analyzed.
Adherence efficiency (%) = (average number of clones per well / average number of cultured cells per well) X100%
Relative attachment efficiency = measured serum attachment efficiency / reference serum attachment efficiency
c. Diploid fibroblast growth promotion test human diploid cell line WI28 MRc5
25cm2 cell culture flask, 5% serum, each flask is inoculated with 1.5X105 cells for 3 consecutive generations, the serum concentration of each generation is the same as the number of inoculated cells, each generation is cultured for 7 days, each generation is inoculated with two cell flasks, and cultured at 36 ℃ ± 2 ℃ . Parallel culture was performed with known reference serum under the same conditions. Count the harvested cells of each generation and calculate the relative growth rate (RGR) of each batch of test serum and reference serum.
RGR = (average number of cells per flask of test serum / average number of cells per flask of reference serum) X100%
Main quality requirements of bovine serum (Sigma)
Fetal bovine serum, neonatal bovine serum, bovine serum, fetal bovine fetus, 10 to 14 days, <10 months, sterile---
Virus---
Mycoplasma---
Phage---
Endotoxin (ng / ml) ≤1.0 ≤10.0 ≤10 (15)
Hemoglobin (g%) 3.0 ～ 4.5 3.5 ～ 6.0 5.0 ～ 8.5
pH 6.7 ～ 8.0 7.0 ～ 8.0 7.0 ～ 8.0
Osmotic pressure (mom / Kg H2O) 240 ～ 340 240 ～ 340 240 ～ 340
The new drug management law is about to be announced, and the new drug management law imposes stricter quality requirements on biological products. In particular, China is about to join the WTO, and more foreign biological products will enter the Chinese market. China ’s biological products also hope to enter the international market more, so the quality of products will be higher, and the market competition of products should mainly rely on quality. competition. Therefore, the quality requirements of the main raw materials in the products will become more and more strict. For example, SDA has put forward strict quality standards for laboratory animals, and put forward the deadline for the various national departments to meet the standards. In order to implement the 2000 edition of the "Chinese Biological Products Regulations", the Standard Committee revised the "Quality Control Standards for Main Raw and Auxiliary Materials of Chinese Biological Products", which includes the quality control requirements for bovine serum for production. This is a minimum requirement, and the requirements for foreign products There is still a gap. I hope that each bovine serum production unit and user unit can work out higher and stricter standards to further improve the quality of biological products in China.
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