CN109666622A - A kind of method that the extraction of cell excretion body is isolated - Google Patents
A kind of method that the extraction of cell excretion body is isolated Download PDFInfo
- Publication number
- CN109666622A CN109666622A CN201910070512.8A CN201910070512A CN109666622A CN 109666622 A CN109666622 A CN 109666622A CN 201910070512 A CN201910070512 A CN 201910070512A CN 109666622 A CN109666622 A CN 109666622A
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- supernatant
- cell
- excretion body
- culture medium
- cell culture
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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
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
Isolated method is extracted the invention discloses a kind of cell excretion body, including following operating procedure: target cell being put into selected culture medium, 72h is harvested after cell culture, and centrifugation obtains cell culture medium supernatant；The cell culture medium supernatant of harvest is carried out 4 DEG C using refrigerated centrifuge, and 6000xg is centrifuged 30min, supernatant in centrifuge tube is transferred to after centrifugation and newly manages spare, precipitating discarding；With disposable syringe Aspirate supernatant, supernatant is slowly dropped to the cell filter membrane that specification is 0.22um and is filtered；Cell culture medium is put into ultracentrifuge and carries out 4 DEG C, 100000xg ultracentrifugation 70min, supernatant is removed after centrifugation and retains sediment fraction；Cleaning resuspension is carried out to precipitating using PBS buffer solution, liquid is placed again into ultracentrifuge and carries out 4 DEG C, 100000xg ultracentrifugation 70min, supernatant is carefully sucked out after centrifugation and retains sediment fraction；It is resuspended and is precipitated using PBS buffer solution, as extract the excretion body product of separation.
Description
Technical field
The present invention relates to excretion body extraction and separation technology field, specially a kind of cell excretion body extracts isolated method.
Background technique
Cell excretion body extracting method mainly has a supercentrifugation at present, density-gradient centrifugation method, the high polymer precipitation method with
And immunomagnetic beads method etc..Existing excretion body extracting method is in operability, cost, is difficult in extraction efficiency and product purity
Balance, usually easy to operate, the time-consuming less extracted excretion body purity of method is difficult to ensure.And it can obtain outside high-purity
Secreting the extracting method of body, often time-consuming, complicated for operation and expensive device is needed to support.
Summary of the invention
Isolated method is extracted, the purpose of the present invention is to provide a kind of cell excretion body to solve in above-mentioned background technique
The problem of proposition.
To achieve the above object, the invention provides the following technical scheme: a kind of cell excretion body extracts isolated method, packet
Include following operating procedure:
S1: cell culture medium supernatant: target cell being put into selected culture medium, and 72h is harvested after cell culture, centrifugation
Obtain cell culture medium supernatant；
S2: low-speed centrifugal: carrying out 4 DEG C using refrigerated centrifuge for the cell culture medium supernatant harvested in S1), 6000xg from
Supernatant in centrifuge tube is transferred to after centrifugation and newly manages spare, precipitating discarding by heart 30min；
S3: membrane filtration: the supernatant that will be obtained in S2) is drawn with disposable syringe, supernatant is slowly dropped to rule
Lattice are that the cell filter membrane of 0.22um is filtered；
S4: ultracentrifugation: being put into ultracentrifuge for the cell culture medium obtained in S3) and carry out 4 DEG C, 100000xg hypervelocity
It is centrifuged 70min, supernatant is removed after centrifugation and retains sediment fraction；
S5: cleaning is resuspended；The precipitating obtained in S4) is subjected to cleaning resuspension using PBS buffer solution；
S6: ultracentrifugation again: the liquid obtained in S5) is placed again into ultracentrifuge and carries out 4 DEG C, 100000xg is super
Speed centrifugation 70min, is carefully sucked out supernatant and retains sediment fraction after centrifugation；
S7: precipitating is resuspended: the precipitating obtained in S6) is resuspended using PBS buffer solution, as extracts the outer of separation
Secrete body product.
Preferably, the cell culture medium supernatant volume is no less than 30ml, and serum is free of in cell culture medium supernatant or is made
With the serum for being free of excretion body.
Preferably, S2) in precipitating include cell culture medium in dead cell and big cell fragment ingredient, S4) in sink
It forms sediment and partially contains excretion body.
Preferably, S4) in carry out ultracentrifugation before to used ultracentrifugation pipe carry out sterilization treatment.
Preferably, S5) and S7) in PBS buffer solution used be sterile neutral solution, and do not contain insoluble particles.
Preferably, S7) in PBS buffer solution used the use of volume is 50ul, gained excretion body is placed in -80 DEG C of refrigerators
Long-term preservation.
Compared with prior art, the beneficial effects of the present invention are: cell excretion body extracting method proposed by the invention, knot
The method for having closed ultracentrifugation filter centrifugation, it is easy to operate, it is time saving and energy saving, and do not need special device.It is extracted outer
It secretes that body content is more, and purity is higher, can be used for a series of biological experiments.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
A kind of method that the extraction of cell excretion body is isolated, including following operating procedure:
S1: cell culture medium supernatant: target cell being put into selected culture medium, and 72h is harvested after cell culture, centrifugation
Obtain cell culture medium supernatant；
S2: low-speed centrifugal: carrying out 4 DEG C using refrigerated centrifuge for the cell culture medium supernatant harvested in S1), 6000xg from
Supernatant in centrifuge tube is transferred to after centrifugation and newly manages spare, precipitating discarding by heart 30min；
S3: membrane filtration: the supernatant that will be obtained in S2) is drawn with disposable syringe, supernatant is slowly dropped to rule
Lattice are that the cell filter membrane of 0.22um is filtered；
S4: ultracentrifugation: being put into ultracentrifuge for the cell culture medium obtained in S3) and carry out 4 DEG C, 100000xg hypervelocity
It is centrifuged 70min, supernatant is removed after centrifugation and retains sediment fraction；
S5: cleaning is resuspended；The precipitating obtained in S4) is subjected to cleaning resuspension using PBS buffer solution；
S6: ultracentrifugation again: the liquid obtained in S5) is placed again into ultracentrifuge and carries out 4 DEG C, 100000xg is super
Speed centrifugation 70min, is carefully sucked out supernatant and retains sediment fraction after centrifugation；
S7: precipitating is resuspended: the precipitating obtained in S6) is resuspended using PBS buffer solution, as extracts the outer of separation
Secrete body product.
Further, the cell culture medium supernatant volume is no less than 30ml, in cell culture medium supernatant without serum or
Use the serum for being free of excretion body.
Further, S2) in precipitating include cell culture medium in dead cell and big cell fragment ingredient, S4) in
Sediment fraction contains excretion body.
Further, S4) in carry out ultracentrifugation before to used ultracentrifugation pipe carry out sterilization treatment.
Further, S5) and S7) in PBS buffer solution used be sterile neutral solution, and do not contain insoluble particles.
Further, S7) in PBS buffer solution used the use of volume is 50ul, gained excretion body is placed in -80 DEG C of refrigerators
Interior long-term preservation.
Embodiment: Morphology observation is carried out to extracted excretion body using transmission electron microscope:
It is added dropwise on 10 microlitres of excretion body suspensions to copper mesh, is stored at room temperature 20 minutes.Then 2% uranium acetate negative staining is added dropwise
2 minutes, suck residual liquid.Then 2% uranium acetate negative staining is added dropwise again 5 minutes, sucks residual liquid, using saturating after drying
Radio sem observation, it can be observed that the imitated vesicle structure of duplicature.
Cell excretion body extracting method proposed by the invention, the method for combining ultracentrifugation filter centrifugation, behaviour
Make simply, it is time saving and energy saving, and do not need special device.Extracted excretion body content is more, and purity is higher, can be used for a system
Column biological experiment.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of cell excretion body extracts isolated method, it is characterised in that: including following operating procedure:
S1: cell culture medium supernatant: target cell being put into selected culture medium, and 72h is harvested after cell culture, and centrifugation obtains
Cell culture medium supernatant；
S2: low-speed centrifugal: the cell culture medium supernatant harvested in S1) is carried out 4 DEG C using refrigerated centrifuge, 6000xg centrifugation
Supernatant in centrifuge tube is transferred to after centrifugation and newly manages spare, precipitating discarding by 30min；
S3: membrane filtration: the supernatant that will be obtained in S2) is drawn with disposable syringe, supernatant is slowly dropped to specification is
The cell filter membrane of 0.22um is filtered；
S4: ultracentrifugation: being put into ultracentrifuge for the cell culture medium obtained in S3) and carry out 4 DEG C, 100000xg ultracentrifugation
70min removes supernatant and retains sediment fraction after centrifugation；
S5: cleaning is resuspended；The precipitating obtained in S4) is subjected to cleaning resuspension using PBS buffer solution；
S6: ultracentrifugation again: being placed again into ultracentrifuge for the liquid obtained in S5) and carry out 4 DEG C, 100000xg exceed the speed limit from
Heart 70min is carefully sucked out supernatant and retains sediment fraction after centrifugation；
S7: precipitating is resuspended: the precipitating obtained in S6) is resuspended using PBS buffer solution, as extracts the excretion body of separation
Product.
2. a kind of cell excretion body according to claim 1 extracts isolated method, it is characterised in that: the cell culture
Base supernatant volume is no less than 30ml, serum is free of in cell culture medium supernatant or using the serum for being free of excretion body.
3. a kind of cell excretion body according to claim 1 extracts isolated method, it is characterised in that: S2) in precipitating wrap
Include the dead cell and big cell fragment ingredient in cell culture medium, S4) in sediment fraction contain excretion body.
4. a kind of cell excretion body according to claim 1 extracts isolated method, it is characterised in that: S4) in surpassed
Sterilization treatment is carried out to used ultracentrifugation pipe before speed centrifugation.
5. a kind of cell excretion body according to claim 1 extracts isolated method, it is characterised in that: S5) and S7) in institute
It is sterile neutral solution with PBS buffer solution, and does not contain insoluble particles.
6. a kind of cell excretion body according to claim 1 extracts isolated method, it is characterised in that: S7) in PBS used
Buffer solution is 50ul using volume, and gained excretion body is placed in long-term preservation in -80 DEG C of refrigerators.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910070512.8A CN109666622A (en) | 2019-01-25 | 2019-01-25 | A kind of method that the extraction of cell excretion body is isolated |
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| CN201910070512.8A CN109666622A (en) | 2019-01-25 | 2019-01-25 | A kind of method that the extraction of cell excretion body is isolated |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110496138A (en) * | 2019-06-11 | 2019-11-26 | 中国农业大学 | A kind of extracting method and its application of yak milk excretion body |
| CN110669723A (en) * | 2019-11-08 | 2020-01-10 | 赵凯 | Differential centrifugation method-based cell exosome extraction process |
| CN110964694A (en) * | 2019-11-11 | 2020-04-07 | 浙江卫未生物医药科技有限公司 | Method for extracting exosome based on density gradient centrifugation and ultracentrifugation |
| CN111269872A (en) * | 2020-01-21 | 2020-06-12 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN111979089A (en) * | 2019-05-23 | 2020-11-24 | 比欧泰克生物技术服务(北京)有限公司 | Large-volume stem cell exosome separation and concentration device and concentration process |
| CN112011498A (en) * | 2019-05-31 | 2020-12-01 | 广州北斗生物科技有限公司 | Purification and concentration preparation method of stem cell exocrine body |
| CN112094809A (en) * | 2020-10-19 | 2020-12-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method for extracting exosome from serum or plasma |
| CN112458045A (en) * | 2020-11-30 | 2021-03-09 | 南京医科大学附属口腔医院 | Method for culturing odontogenic stem cells and separating exosomes |
| CN112779208A (en) * | 2019-11-11 | 2021-05-11 | 李传浩 | Method for extracting and separating extracellular culture medium |
| CN113234677A (en) * | 2021-04-27 | 2021-08-10 | 西南医科大学附属医院 | Method for extracting exosome from in-vitro tumor tissue |
| CN113502256A (en) * | 2021-06-11 | 2021-10-15 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113980903A (en) * | 2021-11-04 | 2022-01-28 | 中南大学湘雅医院 | Colorectal cancer cell exosome and separation and extraction method and application thereof |
| CN114317402A (en) * | 2021-12-14 | 2022-04-12 | 思我特(杭州)食品科技有限公司 | Method for extracting and identifying goat milk exosome |
| CN115197827A (en) * | 2022-07-29 | 2022-10-18 | 深圳技术大学 | Cell exosome filtering and extracting device and extracting method |
| CN115322954A (en) * | 2022-08-30 | 2022-11-11 | 柳州市人民医院 | Method for separating and purifying influenza virus infected epithelial cell exosomes |
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2019
- 2019-01-25 CN CN201910070512.8A patent/CN109666622A/en active Pending
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111979089A (en) * | 2019-05-23 | 2020-11-24 | 比欧泰克生物技术服务(北京)有限公司 | Large-volume stem cell exosome separation and concentration device and concentration process |
| CN112011498A (en) * | 2019-05-31 | 2020-12-01 | 广州北斗生物科技有限公司 | Purification and concentration preparation method of stem cell exocrine body |
| CN110496138B (en) * | 2019-06-11 | 2021-08-27 | 中国农业大学 | Extraction method and application of yak milk exosome |
| CN110496138A (en) * | 2019-06-11 | 2019-11-26 | 中国农业大学 | A kind of extracting method and its application of yak milk excretion body |
| CN110669723A (en) * | 2019-11-08 | 2020-01-10 | 赵凯 | Differential centrifugation method-based cell exosome extraction process |
| CN112779208A (en) * | 2019-11-11 | 2021-05-11 | 李传浩 | Method for extracting and separating extracellular culture medium |
| CN110964694A (en) * | 2019-11-11 | 2020-04-07 | 浙江卫未生物医药科技有限公司 | Method for extracting exosome based on density gradient centrifugation and ultracentrifugation |
| CN111269872A (en) * | 2020-01-21 | 2020-06-12 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN111269872B (en) * | 2020-01-21 | 2021-10-29 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN112094809A (en) * | 2020-10-19 | 2020-12-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method for extracting exosome from serum or plasma |
| CN112458045A (en) * | 2020-11-30 | 2021-03-09 | 南京医科大学附属口腔医院 | Method for culturing odontogenic stem cells and separating exosomes |
| CN113234677A (en) * | 2021-04-27 | 2021-08-10 | 西南医科大学附属医院 | Method for extracting exosome from in-vitro tumor tissue |
| CN113502256A (en) * | 2021-06-11 | 2021-10-15 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113502256B (en) * | 2021-06-11 | 2023-09-19 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113980903A (en) * | 2021-11-04 | 2022-01-28 | 中南大学湘雅医院 | Colorectal cancer cell exosome and separation and extraction method and application thereof |
| CN114317402A (en) * | 2021-12-14 | 2022-04-12 | 思我特(杭州)食品科技有限公司 | Method for extracting and identifying goat milk exosome |
| CN115197827A (en) * | 2022-07-29 | 2022-10-18 | 深圳技术大学 | Cell exosome filtering and extracting device and extracting method |
| CN115322954A (en) * | 2022-08-30 | 2022-11-11 | 柳州市人民医院 | Method for separating and purifying influenza virus infected epithelial cell exosomes |
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Application publication date: 20190423 |