CN111304143A - Method for extracting fetal calf serum - Google Patents
Method for extracting fetal calf serum Download PDFInfo
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- CN111304143A CN111304143A CN202010169061.6A CN202010169061A CN111304143A CN 111304143 A CN111304143 A CN 111304143A CN 202010169061 A CN202010169061 A CN 202010169061A CN 111304143 A CN111304143 A CN 111304143A
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 108091003079 Bovine Serum Albumin Proteins 0.000 title claims abstract description 16
- 239000012894 fetal calf serum Substances 0.000 title claims abstract description 14
- 210000002966 serum Anatomy 0.000 claims abstract description 65
- 238000003860 storage Methods 0.000 claims abstract description 15
- 210000004369 blood Anatomy 0.000 claims abstract description 12
- 239000008280 blood Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 239000011521 glass Substances 0.000 claims abstract description 11
- 244000309466 calf Species 0.000 claims abstract description 9
- 238000010257 thawing Methods 0.000 claims abstract description 7
- 239000002158 endotoxin Substances 0.000 claims abstract description 5
- 238000011146 sterile filtration Methods 0.000 claims abstract description 4
- 241000700605 Viruses Species 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 13
- 241000204031 Mycoplasma Species 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 238000010186 staining Methods 0.000 claims description 7
- 241000283690 Bos taurus Species 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 238000011109 contamination Methods 0.000 claims description 6
- 230000000120 cytopathologic effect Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 6
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 6
- 229920005644 polyethylene terephthalate glycol copolymer Polymers 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 102000001554 Hemoglobins Human genes 0.000 claims description 4
- 108010054147 Hemoglobins Proteins 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 230000003612 virological effect Effects 0.000 claims description 4
- 241000120506 Bluetongue virus Species 0.000 claims description 3
- 206010012735 Diarrhoea Diseases 0.000 claims description 3
- 241000239218 Limulus Species 0.000 claims description 3
- 208000007536 Thrombosis Diseases 0.000 claims description 3
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 claims description 3
- 229910052601 baryte Inorganic materials 0.000 claims description 3
- 239000010428 baryte Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 230000002542 deteriorative effect Effects 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 230000002458 infectious effect Effects 0.000 claims description 3
- 230000004660 morphological change Effects 0.000 claims description 3
- 230000003204 osmotic effect Effects 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000012372 quality testing Methods 0.000 claims description 3
- 241000701161 unidentified adenovirus Species 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 230000015271 coagulation Effects 0.000 claims description 2
- 238000005345 coagulation Methods 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000012757 fluorescence staining Methods 0.000 claims description 2
- 238000002798 spectrophotometry method Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 238000012009 microbiological test Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000204003 Mycoplasmatales Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for extracting fetal calf serum, which comprises the following steps: extracting serum, namely preparing a calf which is just born, directly extracting blood without feeding, and placing the extracted blood in a glass bottle for keeping; refrigerating and storing, namely preparing a temperature-controllable storage box, storing the glass bottles in the storage box, and controlling the temperature in the storage box within a range of-5 ℃ to-20 ℃; filtering, thawing raw serum under controlled conditions, performing sterile filtration, and performing final filtration step with multiple 0.1um filters, filling in laminar flow hood, which can maintain 100-grade conditions by certification, and filtering air with high efficiency air filter in filling room to maintain positive pressure. The serum of the present invention remains frozen throughout shipment and receipt, from the original processing site to our manufacturing facility. This rapid treatment ensures that endotoxin levels in serum are kept low and growth-promoting serum quality is kept at peak levels.
Description
Technical Field
The invention relates to the technical field of fetal calf serum, in particular to a method for extracting fetal calf serum.
Background
The fetal calf serum is a slightly viscous liquid with characteristics, light yellow and clear appearance, no hemolysis and no foreign matters. The fetal calf serum is obtained from fetal calf born by cesarean section; the new-born calf serum is taken from the newborn calf within 24 hours of birth; calf serum was obtained from calves born for 10-30 days. It is clear that fetal bovine serum is the highest quality because fetal bovine serum has not come into contact with the outside, and the serum contains the least amount of components harmful to cells, such as antibodies, complement, and the like.
The quality of serum is always reduced due to the problems of the method of storing the extracted fetal calf serum after extraction in the extraction process.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for extracting fetal calf serum.
The invention provides an extraction method of fetal calf serum, which comprises the following steps:
s1: extracting serum, namely preparing a calf which is just born, directly extracting blood without feeding, and placing the extracted blood in a glass bottle for keeping;
s2: refrigerating and storing, namely preparing a temperature-controllable storage box, storing the glass bottles in the storage box, and controlling the temperature in the storage box within a range of-5 ℃ to-20 ℃;
s3: filtering, thawing raw serum under controlled conditions, performing sterile filtration, and performing final filtration step with multiple 0.1um filters, filling in laminar flow hood, which can maintain 100-grade conditions by certification, and filtering air with high efficiency air filter in filling room to maintain positive pressure. Serum is aseptically dispensed into gamma-irradiated sterile PETG or PETE bottles, the filled containers are immediately labeled and frozen, and then maintained at a temperature below-5 ℃ to maintain product quality;
s4: centrifuging, centrifuging the filtered serum at a temperature below-5 ℃, standing, precipitating to obtain the required serum, controlling the temperature in the centrifuging process to prevent the influence on the centrifugation of the serum due to excessive temperature fluctuation, and carefully taking out the serum from a blood clot to avoid the pollution of the serum by red blood cells;
s5: quality testing, wherein the serum is tested by chemical analysis, microbial detection and virus detection;
s6 storage and transportation, FBS using gamma irradiation, sterile PETG or PETE bottle supply. The serum is stored at a temperature of between-5 ℃ and-20 ℃ in a frozen state, so that the product is prevented from deteriorating due to multiple freezing and thawing cycles of the serum, a smaller packaging size is used or the serum is divided into small portions suitable for one-time use, and when the serum and the equal portions are placed into a sterile container, a sterile technology is used.
Preferably, in S1, the blood extraction device and the glass bottle need to be sterilized for use.
Preferably, in S5, the chemical analysis is performed by measuring the serum hemoglobin content spectrophotometrically using the osmotic pressure (vapor pressure method) and the pH value on the instrument; endotoxin content was determined by limulus reagent gel coagulation.
Preferably, in S5, the microbiological test is performed on each serum batch to confirm that there is no mycoplasma contamination within the limits of detection, the method used is to test culturable mycoplasma in culture medium using the bulk barite method and the nucleolar method, three different culture media are inoculated into serum samples and cultured under aerobic and anaerobic conditions, and non-culturable mycoplasma are tested by transferring the samples to indicator cell lines and staining with DNA fluorescence staining.
Preferably, in S5, the virus detection is performed by culturing a virus-sensitive cell culture previously proven to be free of virus contamination in a medium containing test serum, during which time the culture is microscopically examined to determine evidence of virus-induced morphological changes or cytopathic effects, after at least 21 days of multiple passages, detecting the presence of a particular viral agent in the culture by fluorescent antibody staining and detecting the presence of a cytopathic viral agent, such as infectious bovine diarrhea virus, bluetongue virus and bovine adenovirus, by fluorescent antibody staining.
The beneficial effects of the invention are as follows: whole blood is coagulated at refrigeration temperatures and then centrifuged at freezing temperatures, the raw serum product is immediately placed in bottles and transported after freezing to the manufacturing facility where the serum is kept frozen throughout shipping and receiving processes, from the original processing site to our manufacturing facility. This rapid treatment ensures that endotoxin levels in serum are kept low and growth-promoting serum quality is kept at peak levels.
Drawings
FIG. 1 is a flow chart of the method for extracting fetal calf serum according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1, a method for extracting fetal calf serum includes the following steps:
s1: extracting serum, namely preparing a calf which is just born, directly extracting blood without feeding, and placing the extracted blood in a glass bottle for keeping;
s2: refrigerating and storing, namely preparing a temperature-controllable storage box, storing the glass bottles in the storage box, and controlling the temperature in the storage box within a range of-5 ℃ to-20 ℃;
s3: filtering, thawing raw serum under controlled conditions, performing sterile filtration, and performing final filtration step with multiple 0.1um filters, filling in laminar flow hood, which can maintain 100-grade conditions by certification, and filtering air with high efficiency air filter in filling room to maintain positive pressure. Serum is aseptically dispensed into gamma-irradiated sterile PETG or PETE bottles, the filled containers are immediately labeled and frozen, and then maintained at a temperature below-5 ℃ to maintain product quality;
s4: centrifuging, centrifuging the filtered serum at a temperature below-5 ℃, standing, precipitating to obtain the required serum, controlling the temperature in the centrifuging process to prevent the influence on the centrifugation of the serum due to excessive temperature fluctuation, and carefully taking out the serum from a blood clot to avoid the pollution of the serum by red blood cells;
s5: quality testing, wherein the serum is tested by chemical analysis, microbial detection and virus detection;
s6 storage and transportation, FBS using gamma irradiation, sterile PETG or PETE bottle supply. The serum is stored at a temperature of between-5 ℃ and-20 ℃ in a frozen state, so that the product is prevented from deteriorating due to multiple freezing and thawing cycles of the serum, a smaller packaging size is used or the serum is divided into small portions suitable for one-time use, and when the serum and the equal portions are placed into a sterile container, a sterile technology is used.
In the present invention, the blood extraction device and the glass bottle need to be sterilized for use in S1, and the chemical analysis is performed using osmotic pressure (vapor pressure method) and pH value measured on the instrument, and the serum hemoglobin content is measured spectrophotometrically in S5; the endotoxin content is determined by the limulus reagent gel clotting method, S5, the microbiological test is carried out on each serum batch to determine the method used without mycoplasma contamination within the limits of detection, the mycoplasma culturable in the culture medium is detected by the bulk barite method and the nucleolar method, three different culture media are inoculated into the serum sample and cultured under aerobic and anaerobic conditions, the non-culturable mycoplasma is detected by transferring the sample to an indicator cell line and staining with DNA fluorescent staining, S5, the virus detection previously demonstrated the absence of virus contamination is cultured in a culture medium containing test serum, during which the culture is examined microscopically to determine the morphological changes caused by the virus or evidence of cytopathic effects, after a number of passages for at least 21 days, the presence of a specific virus preparation in the culture is detected by the fluorescent antibody staining method to determine the presence of a cytopathic virus preparation, such as infectious bovine diarrhea virus, bluetongue virus and bovine adenovirus.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. The method for extracting the fetal calf serum is characterized by comprising the following steps of:
s1: extracting serum, namely preparing a calf which is just born, directly extracting blood without feeding, and placing the extracted blood in a glass bottle for keeping;
s2: refrigerating and storing, namely preparing a temperature-controllable storage box, storing the glass bottles in the storage box, and controlling the temperature in the storage box within a range of-5 ℃ to-20 ℃;
s3: filtering, thawing raw serum under controlled conditions, performing sterile filtration, and performing final filtration step with multiple 0.1um filters, filling in laminar flow hood, which can maintain 100-grade conditions by certification, and filtering air with high efficiency air filter in filling room to maintain positive pressure. Serum is aseptically dispensed into gamma-irradiated sterile PETG or PETE bottles, the filled containers are immediately labeled and frozen, and then maintained at a temperature below-5 ℃ to maintain product quality;
s4: centrifuging, centrifuging the filtered serum at a temperature below-5 ℃, standing, precipitating to obtain the required serum, controlling the temperature in the centrifuging process to prevent the influence on the centrifugation of the serum due to excessive temperature fluctuation, and carefully taking out the serum from a blood clot to avoid the pollution of the serum by red blood cells;
s5: quality testing, wherein the serum is tested by chemical analysis, microbial detection and virus detection;
s6 storage and transportation, FBS using gamma irradiation, sterile PETG or PETE bottle supply. The serum is stored at a temperature of between-5 ℃ and-20 ℃ in a frozen state, so that the product is prevented from deteriorating due to multiple freezing and thawing cycles of the serum, a smaller packaging size is used or the serum is divided into small portions suitable for one-time use, and when the serum and the equal portions are placed into a sterile container, a sterile technology is used.
2. The method of claim 1, wherein the blood extraction device and the glass bottle are sterilized for use in step S1.
3. The method for extracting fetal calf serum as claimed in claim 1, wherein in the step S5, the chemical analysis is performed by measuring the content of serum hemoglobin by using an osmotic pressure (vapor pressure method) and pH value on an instrument and measuring the content of serum hemoglobin by using a spectrophotometric method; endotoxin content was determined by limulus reagent gel coagulation.
4. The method of claim 1, wherein in step S5, the microbial detection is performed on each serum batch to confirm that the method used is free from mycoplasma contamination within the detection limit, the method uses a large-volume barite method and a nucleolus method to detect the culturable mycoplasma in the culture medium, three different culture mediums are inoculated on the serum sample and cultured under aerobic and anaerobic conditions, and the non-culturable mycoplasma is detected by transferring the sample to the indicator cell line and staining by DNA fluorescence staining.
5. The method of claim 1, wherein in step S5, the virus detection is performed by culturing a virus-sensitive cell culture previously proven to be free of virus contamination in a culture medium containing test serum, during which the culture is examined microscopically to determine the presence of morphological changes or evidence of cytopathic effects caused by the virus, and after at least 21 days of multiple passages, the culture is examined for the presence of specific viral agents by fluorescent antibody staining, and the presence of cytopathic viral agents, such as infectious bovine diarrhea virus, bluetongue virus and bovine adenovirus, is examined by fluorescent antibody staining.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010169061.6A CN111304143A (en) | 2020-03-12 | 2020-03-12 | Method for extracting fetal calf serum |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010169061.6A CN111304143A (en) | 2020-03-12 | 2020-03-12 | Method for extracting fetal calf serum |
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| CN111304143A true CN111304143A (en) | 2020-06-19 |
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| CN202010169061.6A Pending CN111304143A (en) | 2020-03-12 | 2020-03-12 | Method for extracting fetal calf serum |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898080A (en) * | 2019-11-01 | 2020-03-24 | 广西中医药大学 | Application of horseshoe crab plasma in promoting growth and development |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101112334A (en) * | 2006-07-28 | 2008-01-30 | 兰州民科生物技术中心 | Fetal cattle blood serum production technology |
| CN102268401A (en) * | 2011-06-08 | 2011-12-07 | 庞观龙 | Production method of newborn calf serum with immunoglobulin removed |
| CN108192855A (en) * | 2018-03-21 | 2018-06-22 | 天津博奥科生物科技有限公司 | A kind of extracting method of fetal calf serum |
-
2020
- 2020-03-12 CN CN202010169061.6A patent/CN111304143A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101112334A (en) * | 2006-07-28 | 2008-01-30 | 兰州民科生物技术中心 | Fetal cattle blood serum production technology |
| CN102268401A (en) * | 2011-06-08 | 2011-12-07 | 庞观龙 | Production method of newborn calf serum with immunoglobulin removed |
| CN108192855A (en) * | 2018-03-21 | 2018-06-22 | 天津博奥科生物科技有限公司 | A kind of extracting method of fetal calf serum |
Non-Patent Citations (2)
| Title |
|---|
| 刘文静: "保证牛血清质量促进生物制品品质的提高", vol. 11, no. 5, pages 59 * |
| 山东省学生体质健康调查研究组: "生物制品及其原辅材料质量标准控制实用手册", 山东科学技术出版社, pages: 369 - 370 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898080A (en) * | 2019-11-01 | 2020-03-24 | 广西中医药大学 | Application of horseshoe crab plasma in promoting growth and development |
| CN110898080B (en) * | 2019-11-01 | 2021-07-06 | 广西中医药大学 | Application of horseshoe crab plasma in promoting growth and development |
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Application publication date: 20200619 |