WO2020103192A1 - 外泌体的提取方法 - Google Patents

外泌体的提取方法

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Publication number
WO2020103192A1
WO2020103192A1 PCT/CN2018/119176 CN2018119176W WO2020103192A1 WO 2020103192 A1 WO2020103192 A1 WO 2020103192A1 CN 2018119176 W CN2018119176 W CN 2018119176W WO 2020103192 A1 WO2020103192 A1 WO 2020103192A1
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WIPO (PCT)
Prior art keywords
treatment
centrifugal
centrifugal force
extraction method
exosomes
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PCT/CN2018/119176
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English (en)
French (fr)
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廉翠红
朱艳霞
张远远
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深圳市第二人民医院
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Publication of WO2020103192A1 publication Critical patent/WO2020103192A1/zh

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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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for extracting exosomes.
  • hADSCs Human adipose-derived stem cells
  • hADSCs Human adipose-derived stem cells
  • fibroblasts and secretion By paracrine action on fibroblasts and secretion to promote the secretion of type 1 and type 2 collagen and fibronectin, promote collagen synthesis to exert anti-aging and antioxidant effects; in addition, hADSCs under the action of different inducing factors It can differentiate into adipocytes, osteoblasts, muscle cells, nerve cells, and can secrete a variety of cytokines that promote cell maturation and factors that promote angiogenesis, which shows that hADSCs have anti-inflammatory and damage repair effects.
  • hADSCs can be used to remove wrinkles, scars, and direct injections of scars; in addition, skin damage repair problems caused by various inflammations, ulcers, and burns are also expected to be solved by hADSCs.
  • Exosomes are tiny membrane vesicles secreted by most cells; in recent years, exosomes have shown superiority in many disease models with their advantages such as size and bimolecular membranes in many disease models. treatment effect. With the increase in research on exosomes, the research on exosomes derived from human fat-derived mesenchymal stem cells has also increased. However, the existing methods for extracting hADSCs and exosomes require a large amount of adipose tissue and cells, and have the disadvantages of being expensive and time-consuming.
  • the method of extracting adipose stem cells often uses mechanical equipment, so that the viability of the adipose stem cells is damaged, and the digestive juice will also damage the extracted adipose stem cells, and the steps are complicated. Therefore, in order to obtain high-quality and high-quality adipose stem cells, a simpler and more effective separation and cultivation method must be established.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, and to provide an extraction method for exosomes, which aims to solve the technical problems of complicated extraction steps, high cost, and unstable extraction effect of the existing exosomes.
  • An extraction method of exosomes includes the following steps:
  • the culture supernatant is subjected to the first centrifugal treatment, the supernatant is taken for the second centrifugal treatment, and then the supernatant is taken to obtain the centrifugal liquid;
  • the centrifugal liquid is subjected to ultrafiltration concentration treatment with a 100kd ultrafiltration tube to obtain ultrafiltrate;
  • a 0.22 ⁇ m filter is used for filtration and sterilization treatment to obtain a concentrated solution
  • the centrifugal force of the first centrifugal treatment is 600-1000g
  • the centrifugal force of the second centrifugal treatment is 2000-2800g
  • the centrifugal force of the third centrifugal treatment is 9000-11000g
  • the centrifugal force of the fourth centrifugal treatment is 90000-110000g.
  • the present invention provides a method for extracting exosomes.
  • a culture medium containing exosome-free serum is used for starvation culture of human fat-derived mesenchymal stem cells, so that the stem cells can be in a normal growth state without being inhibited from growth and proliferation.
  • the effective substances contained in the exosomes secreted are also closer to the exosomes in their natural state, and then the culture supernatant containing the exosomes is subjected to low-speed differential centrifugation (ie, first centrifugation first, then second Centrifugation) to remove cells and their debris, ultrafiltration concentration of the centrifuged liquid after low-speed differential centrifugation with a 100kd ultrafiltration tube to obtain ultrafiltrate with higher concentration of exosomes, and the ultrafiltrate is removed by third centrifugation After the impurities are directly filtered and sterilized with a 0.22 ⁇ m filter, the material with a particle size of more than 220nm is filtered out, and a concentrated liquid containing particles with a particle size of less than 220nm is further obtained.
  • low-speed differential centrifugation ie, first centrifugation first, then second Centrifugation
  • the liquid volume is concentrated due to the ultrafiltration concentration process and the third centrifugal process, so The filtration sterilization efficiency is greatly improved, and finally the concentrated solution is subjected to ultracentrifugation in the fourth centrifugal treatment to separate and extract it into exosomes.
  • the extraction method of exosomes combines both differential centrifugation, ultrafiltration and ultracentrifugation. Through this extraction process, it can be efficiently extracted from human fat source mesenchymal dry fine culture supernatant to high The concentration and high purity exosomes have the characteristics of simple operation, low cost and suitable for industrial production.
  • FIG. 1 is a schematic flowchart of the inventive method for extracting exosomes
  • Example 2 is a morphological diagram of the hADSCs separated in Example 1 of the present invention observed under a microscope;
  • Example 3 is a morphological view of the exosomes isolated in Example 2 of the present invention observed under a projection electron microscope;
  • Example 4 is a graph of DSL results of exosomes isolated in Example 5 of the present invention.
  • Example 5 is a graph showing the results of Western blotting of exosomes isolated in Example 5 of the present invention as a characteristic protein
  • Fig. 6 is a graph showing the results of BCA protein quantification of exosomes isolated in Example 5 of the present invention.
  • first”, “second”, “third”, “fourth”, etc. are for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as “first”, “second”, “third”, and “fourth” may explicitly or implicitly include one or more of the features.
  • An embodiment of the present invention provides a method for extracting exosomes, including the following steps:
  • S01 Cultivate human fat-derived mesenchymal stem cells in a medium containing exosome-free serum to obtain a culture supernatant, which contains exosomes;
  • the centrifugal force of the first centrifugal treatment is 600-1000g
  • the centrifugal force of the second centrifugal treatment is 2000-2800g
  • the centrifugal force of the third centrifugal treatment is 9000-11000g
  • the centrifugal force of the fourth centrifugal treatment is 90000-110000g.
  • a culture medium containing exosome-free serum is used to starve human adipose-derived mesenchymal stem cells, so that the stem cells can be in a normal growth state without being inhibited from growing.
  • the effective substances contained in the exosomes secreted by it are also closer to the exosomes in their natural state, and then the culture supernatant containing the exosomes is subjected to low-speed differential centrifugation (that is, first centrifugation, Second centrifugation) to remove cells and their debris, ultrafiltration concentration of the centrifuge after low speed differential centrifugation with a 100kd ultrafiltration tube to obtain ultrafiltrate with higher concentration of exosomes, the ultrafiltrate is passed through the third After centrifugation to remove impurities, directly filter and sterilize with a 0.22 ⁇ m filter, filter out the material with a particle size of more than 220nm, and further obtain a concentrated liquid containing particles with a particle size of less than 220nm.
  • low-speed differential centrifugation that is, first centrifugation, Second centrifugation
  • ultrafiltration concentration of the centrifuge after low speed differential centrifugation with a 100kd ultrafiltration tube to obtain ultrafiltrate with higher
  • the ultrafiltration concentration treatment and the third centrifugal treatment Due to the ultrafiltration concentration treatment and the third centrifugal treatment, the amount of liquid Concentration, so that the efficiency of filtration and sterilization is greatly improved, and finally the concentrated solution is subjected to ultracentrifugation in the fourth centrifugal treatment to separate and extract to exosomes.
  • the extraction method of exosomes combines both differential centrifugation, ultrafiltration and ultracentrifugation. Through this extraction process, it can be efficiently extracted from human fat source mesenchymal dry fine culture supernatant to high Concentrated, high-purity exosomes.
  • the method for extracting exosomes according to the embodiment of the present invention has high yield and high purity: because of the simple steps, the extraction method not only overcomes the shortcomings of the existing simple ultracentrifugation method and the defects of mixing impurities, but also solves To solve the problem of exosomal loss caused by adhesion of exosomes to the membrane after multiple concentrations of the existing simple ultrafiltration method, because a larger pore size 100KD ultrafiltration tube was selected to concentrate the supernatant, the 100KD ultrafiltration tube passed through the exocrine
  • the size of the body is a vesicle structure of 30-150nm. For example, the molecular weight of Alix protein is greater than 100KD, which can better separate exosomes.
  • the embodiment of the present invention is highly efficient in extracting exosomes, and can process a large amount of cell supernatant at one time.
  • the largest ultracentrifuge tube is only 30ml, and only 4ml at a time can only process 180ml (30ml * 6 According to one rotor, six such rotors can be placed), and after being concentrated in the embodiment of the present invention, the cell supernatant can be processed at a time 36 times that of the simple ultracentrifugation method.
  • exosomes extracted in this example showed a uniform shape of a round cup with a diameter of 30-150nm under electron microscope.
  • FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, ANXA5, TSG101 and other exosome-related proteins showed Positive expression indicates successful isolation of human adipose stem cell exosomes.
  • the 100KD ultrafiltration tube is a 15ml ultrafiltration tube.
  • the 15ml 100kd ultrafiltration tube can concentrate the 150ml supernatant to about 5ml, making the operation easier and the purity of the collected exosomes higher.
  • six 15mL 100kd ultrafiltration tubes (Millipore, UFC910096-1pk).
  • the centrifugal force of the first centrifugal process is 800 g
  • the centrifugal force of the second centrifugal process is 2500 g
  • the centrifugal force of the third centrifugal process is 10000 g
  • the centrifugal force of the fourth centrifugal process is 100000g.
  • the fourth centrifugation process may include two ultra-centrifugal separations of 100,000 g, and two ultra-ionizations to separate the exosomes in the concentrated solution, and a large number of exosomes with high purity and fewer impurities may be obtained.
  • the time for culturing human adipose-derived mesenchymal stem cells with a medium containing exosome-free serum is 24-48h, preferably 48h.
  • the extraction method of exosomes includes:
  • Conditional culture take 80-90% confluent cells grown in culture flask, and use 5ml of phosphate buffered saline (phosphate buffer) saline (abbreviated as PBS), shake up and down, wash 3 times to remove common exosomes from other sources to prevent other sources of exosomes from affecting the subsequent experimental analysis, and then add body serum without exocrine medium (Vivacell, C38010050)
  • PBS phosphate buffered saline
  • the prepared medium was treated for 48h, and the serum without exosomes was directly starved relative to the basic medium, which could make the cells in a normal state and would not inhibit the growth and proliferation of the cells, so the secreted exosomes contained effective substances It is also closer to the exosomes secreted in its natural state, which is more conducive to subsequent experiments;
  • Collect the culture supernatant collect the culture supernatant 150ml, shake the culture flask before collection to make it adhere to the cells And the exosome
  • the human adipose-derived mesenchymal stem cells are obtained by isolating and culturing hADSCs from adipose tissue to obtain fourth-generation cells, and then continuing to expand the cells to obtain cells with a fusion degree of 80-90%.
  • the method for separating and culturing hADSCs from adipose tissue includes:
  • the primary human fat-derived mesenchymal stem cells are subcultured.
  • the above method for separating and cultivating hADSCs from adipose tissue is simple and easy to operate, efficient and stable, and saves time and cost. It can be used to extract primary cells with good vitality and fast proliferation from a small amount of adipose tissue for subsequent extraction of exosomes Lay a solid foundation. Further, the adipose tissue sample is digested with a digestion solution containing 0.25% trypsin and 0.1% type I collagenase, so that the viable and fast-promoting raw material can be extracted from the trace adipose tissue by the double enzyme method Generation cells. More preferably, the complete culture includes: 10% fetal bovine serum, high glucose DMEM, and 1% penicillin / streptomycin.
  • the method of separating and culturing hADSCs from adipose tissue includes: (1) Under sterile conditions, a surgeon takes approximately 500 mg of normal adipose tissue mass from the omentum of a patient undergoing laparotomy and places it in a 1X In a 50ml centrifuge tube of PBS solution, transport on ice, and transport the adipose tissue in vitro for no more than 1h; (2) Isolate fat-derived mesenchymal stem cells: rinse the adipose tissue 3 times with PBS solution to make the washing solution colorless, use The ophthalmic scissors try to cut the small blood vessels visible in the fat tissue as much as possible.
  • Cultivate purified fat-derived mesenchymal stem cells digest the primary fat stem cells obtained by inoculation, and wait for the cells to grow to 100% fusion at 37 ° C , Can be digested with 0.25% pancreatin and 0.04% EDTA (volume ratio 1: 1), and inoculated into a new culture flask according to the area ratio of 1: 3. After multiple passages, purified mesenchymal stem cells can be obtained.
  • the maximum time for digesting the isolated primary cells is no more than 30 minutes, preferably 20 minutes.
  • Exosome-free serum medium is: 10% exosome-free serum (Vivacell, C38010050) + high-sugar DMEM (gibco, C11995500BT) + 1% penicillin / streptomycin (gibco, 15140163);
  • the ultracentrifuge is Optima MAX-XP desktop ultracentrifuge (Beckman Coulter);
  • the ultra-isolated tube required in the ultracentrifuge is the sealed tube 8.9ml (Beckman Coulter, 361623);
  • the 0.22 ⁇ m filter is a sterile syringe filter (Millipore, SLGP033RB);
  • hADSCs stem cell isolation and culture method
  • Fig. 2 is a morphological view of hADSCs isolated in this example observed under a microscope.
  • the hADSCs of the fourth-generation cryopreservation culture were continuously cultured, and exosomes were extracted from the cell supernatant.
  • Ultracentrifugation collect the concentrated solution by ultracentrifugation at 100000g, 2h, 4 °C. After washing with PBS, it was centrifuged again at 100,000g for 2h at 4 ° C.
  • Figure 3 is the morphology of the exosomes isolated from this example observed under a projection electron microscope; the results of electron microscopy can be found that the exosomes extracted in this example have a cleaner background and have a typical exosome
  • the boundary of the membrane is a typical cup-shaped structure), and the particle size is mostly around 80nm; most of the exosomes obtained are typical in morphological structure, indicating that the damage is small.
  • the hADSCs of the fourth-generation cryopreservation culture were continuously cultured, and exosomes were extracted from the cell supernatant.
  • Ultracentrifugation collect the concentrated solution by ultracentrifugation at 100000g, 2h, 4 °C. After washing with PBS, it was centrifuged again at 100,000g for 2h at 4 ° C.
  • the hADSCs of the fourth-generation cryopreservation culture were continuously cultured, and exosomes were extracted from the cell supernatant.
  • Centrifugation collect 100,000g of concentrated liquid by ultracentrifugation, 2h, 4 °C. After washing with PBS, it was centrifuged again at 100,000g for 2h at 4 ° C.
  • the 4th-generation cryopreserved ADSCS was continuously cultured, and exosomes were extracted from the cell supernatant.
  • Conditional culture 80-90% confluent cells were grown in T175cm 2 culture flasks, starved with exosome-free serum (Vivacell, C38010050) prepared medium for 48 hours, and 150 ml of supernatant of cultured cells was collected.
  • Ultrafiltration concentration use six 15ml 100kd ultrafiltration tubes (Millipore, UFC910096-1pk) for ultrafiltration concentration under the following conditions: 4 °C, 4000g, 25min;
  • Figures 4-6 are graphs of the identification results of the extracted exosomes in this example.
  • Figures 4 and 6 above were used to measure the concentration and particle size of exosomes with a nanoparticle analyzer (DSL) combined with standard protein quantification (BCA): the exosomal DSL results show the measured pbs after ultracentrifugation Nanoparticles with an average particle size of 110.4nm accounted for 98.6% in the resuspended solution;
  • Figure 6 is the linear relationship between the OD value and the standard curve of the standard protein concentration after protein quantification in the exosomes: R 2 is 0.9954, which is in accordance with the BCA kit error Within the range, according to this linear formula, the protein concentration of exosomes is calculated to be about 730.15 (ug / ml);
  • Figure 5 is the Western blot detection of the characteristic protein on the surface of exosomes, including: Rich in the four transmembrane protein family involved in exosome transport (CD63,

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Abstract

一种外泌体的提取方法，包括如下步骤：用含无外泌体血清的培养基培养人脂肪来源间充质干细胞，得到培养上清液，所述培养上清液中含有外泌体；将所述培养上清液进行第一离心处理后，取上清进行第二离心处理，然后取上清得到离心液；将所述离心液用100KD超滤管进行超滤浓缩处理，得到超滤液；将所述超滤液进行第三离心处理后，用0.22μm过滤器进行过滤除菌处理，得到浓缩液；将所述浓缩液进行第四离心处理，弃上清得到所述外泌体。

Description

外泌体的提取方法 技术领域
本本发明属于生物技术领域，具体涉及一种外泌体的提取方法。
 背景技术
皮肤作为人体最大的器官总是会受到各种物理性、机械性、化学性和病原微生物性的侵袭。皮肤的抗氧化、抗衰老、抗炎及损伤修复是临床医师非常关心的研究问题。
人脂肪来源间充质干细胞（human adipose-derived stem cells，hADSCs）是一类存在于脂肪组织中，具有自我更新和多向分化潜能的干细胞。通过旁分泌作用于成纤维细胞以及分泌促进其分泌1、2型的胶原和纤连蛋白，促进胶原合成，以发挥抗衰老、抗氧化作用；除此之外，hADSCs在不同的诱导因子作用下可以分化成脂肪细胞、成骨细胞、肌细胞、神经细胞，而且还可以能分泌多种促进细胞成熟的细胞因子和促进血管生成的因子，这些表明hADSCs具有抗炎、损伤修复的作用。因此，实际应用中，比如整形美容上，可以用hADSCs来除皱、瘢痕和疤痕的直接注射；另外，因为各种炎症、溃疡、烧伤等造成的皮肤损伤修复问题也有望通过hADSCs来解决。
外泌体（Exosomes）是一种被大多数细胞分泌的微小膜泡；近年来，外泌体凭借其大小及双分子膜等优势在多种疾病模型中呈现出了较细胞水平更为卓越的治疗效果。随着外泌体研究增多，人脂肪来源间充质干细胞外泌体的相关研究也日益增多。然而，现有提取hADSCs及外泌体方法需要大量脂肪组织和细胞，存在价格昂贵、费时费力的缺点。比如提取脂肪干细胞方法常使用机械设备，使得脂肪干细胞提取的细胞活力收到损伤，消化液也会损伤提取出来的脂肪干细胞，而且步骤冗杂。因此，要想获得高质、高量的脂肪干细胞，必须建立一套更简便有效的分离培养方法。
而外泌体领域的研究时间尚短，存在多种问题，如外泌体的提取方法学规范、统一定量及鉴定等。关于外泌体的提取有超速离心、试剂盒、超滤法、蔗糖密度梯度离心等，然而各种方法均有其利弊。超速离心法是目前外泌体相关文章中的主流方法，由于离心步骤繁琐，费事费力，而且步骤多导致实验中容易污染，且损耗量大，使得最终回收的外泌体不稳定。而且对于抽提细胞上清来说，更是极为不请便，试想用提取300ml的上清需要6个50ml离心管，无论是过滤还是后续的每一步的离心去沉淀，都具有操作极其不便的缺点，总之非常麻烦。而超滤法存在外泌体会堵塞膜孔，造成浓缩效率低，浓缩管重复利用差，甚至堵塞在膜孔的外泌体还可能会粘连成团，造成损失及最后的数据有误差，对于后续实验也有影响；最后浓缩液中外泌体的浓度不稳定，为后续实验操作和分析来带困扰等缺点。
因此，现有技术有待改进。
 技术问题
本发明的目的在于克服现有技术的上述不足，提供一种外泌体的提取方法，旨在解决现有外泌体提取步骤繁琐、成本高，以及提取效果不稳定的技术问题。
 技术解决方案
为实现上述发明目的，本发明采用的技术方案如下：
一种外泌体的提取方法，包括如下步骤：
用含无外泌体血清的培养基培养人脂肪来源间充质干细胞，得到培养上清液，所述培养上清液中含有外泌体；
将所述培养上清液进行第一离心处理后，取上清进行第二离心处理，然后取上清得到离心液；
将所述离心液用100kd超滤管进行超滤浓缩处理，得到超滤液；
将所述超滤液进行第三离心处理后，用0.22μm过滤器进行过滤除菌处理，得到浓缩液；
将所述浓缩液进行第四离心处理，弃上清得到所述外泌体；
其中，所述第一离心处理的离心力为600-1000g，所述第二离心处理的离心力为2000-2800g，所述第三离心处理的离心力为9000-11000g，所述第四离心处理的离心力为90000-110000g。
 有益效果
本发明提供外泌体的提取方法，先用含无外泌体血清的培养基对人脂肪来源间充质干细胞进行饥饿培养，这样可使干细胞处于正常生长状态，不会被抑制生长增殖，其所分泌的外泌体所包含的有效物质也更贴近其自然状态下的外泌体，然后将含有外泌体的培养上清液进行低速差速离心（即先第一离心处理、再第二离心处理）以去除细胞及其碎片，用100kd超滤管对低速差速离心后的离心液进行超滤浓缩得到外泌体浓度更高的超滤液，将超滤液经过第三离心处理去除杂质后直接用0.22μm过滤器过滤除菌，过滤掉粒径为220nm以上的物质，进一步得到含颗粒粒径小于220nm的浓缩液，因超滤浓缩处理和第三离心处理使得液体量浓缩，这样过滤除菌效率得到大大提高，最后将浓缩液进行超速离心的第四离心处理分离提取到外泌体。该外泌体的提取方法，既结合了差速离心，又结合了超滤和超速离心，通过该提取流程，最终可高效地从人脂肪来源间充质干细培养上清液中提取到高浓度、高纯度的外泌体，具有操作简单、成本低，适合产业化生产的特点。
 附图说明
图1为发明的外泌体提取方法的流程示意图；
图2为本发明实施例1中分离得到的hADSCs在显微镜下观察到的形态图；
图3为本发明实施例2中分离得到的外泌体在投射电镜下观察到的形态图；
图4为本发明实施例5中分离得到的外泌体在DSL结果图；
图5为本发明实施例5中分离得到的外泌体做特征蛋白质免疫印迹结果图；
图6为本发明实施例5中分离得到的外泌体做BCA蛋白定量结果图。
 本发明的实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图和实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”、“第四”的特征可以明示或者隐含地包括一个或者更多个该特征。
本发明实施例提供了一种外泌体的提取方法，包括如下步骤：
S01：用含无外泌体血清的培养基培养人脂肪来源间充质干细胞，得到培养上清液，所述培养上清液中含有外泌体；
S02：将所述培养上清液进行第一离心处理后，取上清进行第二离心处理，然后取上清得到离心液；
S03：将所述离心液用100kd超滤管进行超滤浓缩处理，得到超滤液；
S04：将所述超滤液进行第三离心处理后，用0.22μm过滤器进行过滤除菌处理，得到浓缩液；
S05：将所述浓缩液进行第四离心处理，弃上清得到所述外泌体；
其中，所述第一离心处理的离心力为600-1000g，所述第二离心处理的离心力为2000-2800g，所述第三离心处理的离心力为9000-11000g，所述第四离心处理的离心力为90000-110000g。
本发明实施例提供的外泌体的提取方法，先用含无外泌体血清的培养基对人脂肪来源间充质干细胞进行饥饿培养，这样可使干细胞处于正常生长状态，不会被抑制生长增殖，其所分泌的外泌体所包含的有效物质也更贴近其自然状态下的外泌体，然后将含有外泌体的培养上清液进行低速差速离心（即先第一离心处理、再第二离心处理）以去除细胞及其碎片，用100kd超滤管对低速差速离心后的离心液进行超滤浓缩得到外泌体浓度更高的超滤液，将超滤液经过第三离心处理去除杂质后直接用0.22μm过滤器过滤除菌，过滤掉粒径为220nm以上的物质，进一步得到含颗粒粒径小于220nm的浓缩液，因超滤浓缩处理和第三离心处理使得液体量浓缩，这样过滤除菌效率得到大大提高，最后将浓缩液进行超速离心的第四离心处理分离提取到外泌体。
该外泌体的提取方法，既结合了差速离心，又结合了超滤和超速离心，通过该提取流程，最终可高效地从人脂肪来源间充质干细培养上清液中提取到高浓度、高纯度的外泌体。具体地，本发明实施例的外泌体提取方法获得率高、纯度高：该提取方法因为步骤简单，不仅克服了现有单纯超速离心法步骤多来带的失误和混入杂质的缺点，而且解决了现有单纯超滤法多次浓缩后外泌体粘连在膜上造成的外泌体流失的问题，因为选择了更大孔径的100KD超滤管来浓缩上清，100KD超滤管通过外泌体的大小是30-150nm的囊泡结构，如Alix蛋白的分子量就大于100KD，这样可使外泌体更好地分离，太小的分子量容易发生滤孔堵塞；而且该提取方法价格低廉：超滤管和超离管均可重复利用3-5次，分离方法简单，综合起来成本低，节省人力和物力，适合产业化生产外泌体；操作简单，无需其他辅助工具或者仪器。
本发明实施例提取外泌体高效，能一次处理大量的细胞上清，而对于现有单纯的超速离心法，最大的超离管也才30ml，一次4h也单纯只能处理180ml（30ml*6，按照一个转头可以放6个这样的转子），而本发明实施例浓缩后超离，一次能处理的细胞上清是单纯超速离心法的36倍。
本实施例提取到的外泌体电镜下看直径在30-150nm之间，呈均一大小的圆杯形态，FLOT1、ICAM、ALIX、CD81、CD63、EpCAM、ANXA5、TSG101等外泌体相关蛋白呈阳性表达，说明成功分离到人脂肪干细胞外泌体。
进一步地，所述100KD超滤管为15ml的超滤管。15ml的100kd超滤管可使150ml的上清液浓缩至5ml左右，使得操作更加简单，收集到的外泌体纯度更高，具体地，每次可用6个15mL的100kd超滤管（Millipore,UFC910096-1pk）。进一步地，上述提取方法中，所述第一离心处理的离心力为800g，所述第二离心处理的离心力为2500g，所述第三离心处理的离心力为10000g，所述第四离心处理的离心力为100000g。进一步地，第四离心处理可以包括两次100000g超速离心分离，两次超离对浓缩液中的外泌体进行分离，可得到了大量的纯度高且杂质更少的外泌体。更进一步地，所述用含无外泌体血清的培养基培养人脂肪来源间充质干细胞的时间为24-48h，优选48h。
本发明一优选实施例中，如图1所示，外泌体的提取方法包括：
（1）条件培养：取培养瓶中生长80-90%融合的细胞，用5ml的磷酸缓冲盐溶液（phosphate buffer saline，简称PBS）上下晃动，清洗3遍，以去除普通中的其他来源的外泌体以防其他来源外泌体影响后续实验分析，然后再加用无外泌培养基体血清（Vivacell，C38010050）配制培养基处理48h，无外泌体的血清相对于基础培养基直接饥饿处理，可以使得细胞处于正常状态，并不会抑制细胞的生长增殖，所以其所分泌的外泌体所包含的有效物质也更贴近其自然状态下所分泌的外泌体，更利于进行后续的实验；（2）收集培养上清：收集培养上清液150ml，收集前需要晃动下培养瓶，使得附着于细胞上的及在底部的外泌体充分收集，收集完培养上清后可再用5ml的PBS依次冲洗一下培养瓶并收集冲洗液。（3）离心：离心4℃，800g，10mins；去上清（用移液管轻吸，以防沉淀混入上清，影响后续的超滤），弃死亡细胞，再离心2500g，4℃，10mins，去上清，弃细胞碎片；（4）过滤：用15ml的100kd超滤管，4℃，4000g，25min（注意滤筒斜面和转轴垂直，平行转头和成角转头所能加的过滤液最大量不同）；（5）离心：收集超滤液超速离心10000g，4℃，1h，去除杂质。（6）除去细菌：用0.22μm过滤器过滤超滤液，最后用2mlPBS洗一下，超滤和高速离心后，液体量已经浓缩，这时候过滤可以大大的提高工作效率。（7）超速离心：收集超滤液置于8.9ml超离管，超速离心100000g，4℃，2h。小心移去上清液，留下0.5ml，再用PBS重悬至8.9ml，再次离心100000g，4℃，2h，用1ml长枪头轻轻弃去上清，然后用200ul PBS重悬外泌体，收集后-80℃保存。
进一步地，上述步骤S01中，所述人脂肪来源间充质干细胞是：从脂肪组织中分离培养hADSCs得到第4代细胞后继续扩大培养得到融合度达80~90%的细胞。
优选地，所述从脂肪组织中分离培养hADSCs的方法包括：
收集脂肪组织样本，然后进行消化处理，得到消化液；
从所述消化液中收集含单个核细胞的液体后，移入完全培养基中终止消化，然后分离出原代人脂肪来源间充质干细胞；
将所述原代人脂肪来源间充质干细胞进行传代培养。
上述从脂肪组织中分离培养hADSCs的方法简单易操作，高效稳定，省时花费低，可从微量脂肪组织中用双酶法提取活力好、增殖速度快的原代细胞，为后续提取外泌体奠定坚实基础。进一步地，用含有0.25%胰蛋白酶和0.1%I型胶原酶的消化液对所述脂肪组织样品中进行消化处理，这样可以从微量脂肪组织中用双酶法提取活力好，增殖速度快的原代细胞。更优选地，所述完全培养包括：10％胎牛血清、高糖DMEM和1%青霉素/链霉素。
在一优选实施例中，从脂肪组织中分离培养hADSCs的方法包括：（1）无菌条件下，由外科医生自开腹手术患者大网膜取正常脂肪组织块约500mg，置于装有1X PBS溶液的50ml离心管中，冰上运输，运输离体脂肪组织时间不超过1h；（2）分离脂肪来源的间充质干细胞：用PBS液冲洗脂肪组织3遍，使得冲洗液无色，用眼科剪尽量剪破脂肪组织中肉眼可见的细小血管，冲洗血液后，尽量剪碎脂肪组织，移入50ml离心管中，加入0.25％胰蛋白酶（无EDTA，gibco）和0.1％胶原酶（I型，gibco）混合消化液（体积比1：1），混合消化液和组织体积比1：2-3。37℃恒温摇床振荡消化5 min，移液管吹打消化10min．静置待液面分为3层：上层为黄色油状脂肪细胞层，中层为脂肪组织层，下层为含单个核细胞的液体．吸出下层液体移入含完全培养基（10％胎牛血清(gibco)+高糖DMEM(gibco)+1%青霉素/链霉素）的离心管中按照1：2体积终止消化。在剩余脂肪中加入新的胰酶和胶原酶，重复以上步骤继续消化，重复3次，每次消化时间小于30mins。将收集到的液体1 500 r／min离心15 min，去除悬浮的脂肪细胞和脂滴，去上清，向细胞沉淀中加入含10％胎牛血清DMEM H+重悬细胞，移入T25培养瓶中，37℃、5％C0 2培养箱孵育，24h后观察换液；（3）培养纯化脂肪来源的间充质干细胞：消化接种得到的原代脂肪干细胞，待细胞在37℃生长至100%融合后，可以用0.25％胰酶和0.04％EDTA（体积比1：1）消化传代消化，按照1：3的面积比例接种到新的培养瓶。经过多次传代后，可以得到纯化的间充质干细胞。所述分离原代细胞消化最长时间为不超过30min，以20min为宜。
本发明先后进行过多次试验，现举一部分试验结果作为参考对发明进行进一步详细描述，下面结合具体实施例进行详细说明。
混合消化液：0.25％胰蛋白酶（无EDTA，gibco，15050-065）和0.1％胶原酶（I型，gibco，17100-017）；
完全培养基，10％胎牛血清（gibco，10099-141）+高DMEM（gibco）+1%青霉素/链霉素（gibco）；
无外泌体血清培养基为：10%无外泌体血清（Vivacell，C38010050）+高糖DMEM(gibco，C11995500BT)+1%青霉素/链霉素（gibco，15140163）；
100kd超滤管(Millipore,UFC910096-1pk)；
超速离心机的为Optima MAX-XP台式超速离心机（Beckman Coulter）；
超速离心机中所需超离管为指封管8.9ml（Beckman Coulter，361623）；
0.22μm过滤器为无菌针头式过滤器(Millipore,SLGP033RB)；
实施例1
hADSCs干细胞分离培养方法：
取500mg人脂肪组织，用PBS清洗2-3遍，并用手术剪/镊子去除血管、结缔组织。尽量只保存脂肪细胞，因为红细胞凋亡后产物、脂滴，会影响脂肪干细胞生长。将剪碎的匀浆状脂肪，移入50ml离心管，按照1：2体积比加入0.25%胰酶（无EDTA）+0.1%的I型胶原酶（1：1）混合消化液，反复吹打消化充分。静置后待50ml离心管分层后，倒掉上层液，用移液枪小心将底层的细胞悬液吸入15ml离心管，并加入完全培养基（DMEM H+、10%FBS、1%青霉素/链霉素），终止消化，离心机1500r、10mins。倒弃上清，留下面的脂肪细胞，补培养基，吹打混匀后，接种于T25上。反复消化大约3-4次，直至脂肪组织消化完全。2天后换液，弃去未贴壁的细胞，以后每3天换液。当细胞融合达100%时，用0.25%胰蛋白酶进行常规消化，并将细胞按照1∶3的比例进行传代。
图2为本实施例分离得到的hADSCs在显微镜下观察到的形态图。
实施例2
将第4代冻存培养的hADSCs继续培养，从细胞上清液中提取外泌体。
（1）收集培养：在T175cm 2培养瓶中生长细胞，养到80-90%融合时，用无外泌体血清的培养基中饥饿处理24h，收集培养细胞的上清液。
（2）差速离心：800g离心，5mins；弃细胞及其碎片，再2000g离心，10mins；弃细胞及其碎片；
（3）超滤：用100KD超滤管进行超滤浓缩处理；
（4）除菌：收集超滤液10000g离心，4℃，1h，去除杂质；再用0.22μm过滤器 (Millipore,SLGP033RB)过滤。
（5）超速离心：收集浓缩液100000g超速离心，2h，4℃。用PBS清洗后，再100000g超速离心，2h，4℃。
图3为本实施例分离得到的外泌体在投射电镜下观察到的形态图；电镜结果图可以发现该实施例提取的外泌体具有背景更干净，且拥有外泌体典型（具有明显的膜边界，呈典型的杯状结构）的结构，粒径大小也大多在80nm左右；所获的外泌体大多形态结构典型，说明损伤小。
实施例3
将第4代冻存培养的hADSCs继续培养，从细胞上清液中提取外泌体。
（1）收集培养：在T175cm 2培养瓶中生长细胞，养到80-90%融合时，用无外泌体血清的培养基中饥饿处理48h，收集培养细胞的上清液。
（2）差速离心：800g离心，5mins；弃细胞及其碎片，再2000g离心，10mins；弃细胞及其碎片；
（3）超滤：用100KD超滤管进行超滤浓缩处理；
（4）除菌：收集超滤液10000g离心，4℃，1h，去除杂质；再用0.22μm过滤器 (Millipore,SLGP033RB)过滤。
（5）超速离心：收集浓缩液100000g超速离心，2h，4℃。用PBS清洗后，再100000g超速离心，2h，4℃。
实施例4
将第4代冻存培养的hADSCs继续培养，从细胞上清液中提取外泌体。
（1）收集培养：在T175cm 2培养瓶中生长细胞，养到80-90%融合时，用无外泌体血清的培养基中饥饿处理24h，收集培养细胞的上清液。
（2）离心：800g离心，5mins；弃细胞及其碎片，再2000g离心，10mins；弃细胞及其碎片；
（3）超滤：用6个15mL的100kd超滤管（Millipore,UFC910096-1pk），4℃，4000g，25min；
（4）除菌：收集超滤液10000g离心，4℃，1h，去除杂质；再用0.22μm过滤器 (Millipore,SLGP033RB)过滤。
（5）离心：收集浓缩液100000g超速离心，2h，4℃。用PBS清洗后，再100000g超速离心，2h，4℃。
实施例5
将第4代冻存培养的ADSCS继续培养，从细胞上清液中提取外泌体。
（1）条件培养：在T175cm 2培养瓶中生长80-90%融合的细胞，用无外泌体血清（Vivacell，C38010050）的配制的培养基饥饿处理48h，收集培养细胞的上清液150ml。
（2）差速离心：800g离心，4℃，10mins；去上清，弃死亡细胞，再2500g离心，4℃，10mins，去上清，弃细胞碎片；
（3）超滤浓缩：用6个15ml的100kd超滤管（Millipore,UFC910096-1pk）超滤浓缩，条件为：4℃，4000g，25min；
（4）除菌：收集超滤液10000g离心，4℃，1h，去除杂质；再用0.22μm过滤器过滤超滤液；
（5）超速离心：收集浓缩液置于8.9ml超离管，100000g超速离心4℃，2h。小心移去上清液，留下0.5ml，再用PBS重悬至8.9ml，再来一次100000g超速离心，4℃，2h。
图4-图6为本实施例中进一步对提取的外泌体的鉴定结果图。具体地，上述图4和图6分别用纳米颗粒分析仪（DSL）结合标准蛋白定量（BCA）进行外泌体浓度和粒径的测量：外泌体DSL结果图显示所测量的超速离心后pbs重悬液里平均粒径为110.4nm的纳米颗粒占98.6%；图6是对外泌体进行了蛋白定量后OD值和标准蛋白浓度标准曲线的线性关系：R 2为0.9954符合BCA试剂盒的误差范围之内，根据此线性公式，计算得到外泌体的蛋白浓度约为730.15（ug/ml）；图5为外泌体表面的普遍存在的特征蛋白免疫印迹检测，包括：外泌体膜上富含参与外泌体运输的四跨膜蛋白家族（CD63，CD81和CD9），反映膜受体结合能力的ESCRT复合体相关的蛋白（TSG101）以及参与切割脱离质膜形成独立膜结构的过程的蛋白（ALIX）等：图中CD63对应面积为34770；TGS101对应面积为72850，ALIX对应面积为674343；面积的大小代表单位量样品蛋白表达量，该免疫印迹结果表明：提取到了稳定的外泌体。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (15)

  1. 一种外泌体的提取方法，其特征在于，包括如下步骤：
    用含无外泌体血清的培养基培养人脂肪来源间充质干细胞，得到培养上清液，所述培养上清液中含有外泌体；
    将所述培养上清液进行第一离心处理后，取上清进行第二离心处理，然后取上清得到离心液；
    将所述离心液用100KD超滤管进行超滤浓缩处理，得到超滤液；
    将所述超滤液进行第三离心处理后，用0.22μm过滤器进行过滤除菌处理，得到浓缩液；
    将所述浓缩液进行第四离心处理，弃上清得到所述外泌体；
    其中，所述第一离心处理的离心力为600-1000g，所述第二离心处理的离心力为2000-2800g，所述第三离心处理的离心力为9000-11000g，所述第四离心处理的离心力为90000-110000g。
  2. 如权利要求1所述的提取方法，其特征在于，所述第一离心处理的离心力为800g。
  3. 如权利要求1所述的提取方法，其特征在于，所述第二离心处理的离心力为2500g.
  4. 如权利要求1所述的提取方法，其特征在于，所述第三离心处理的离心力为10000g。
  5. 如权利要求1所述的提取方法，其特征在于，所述第四离心处理的离心力为100000g。
  6. 如权利要求1所述的提取方法，其特征在于，所述第一离心处理的离心力为800g，所述第二离心处理的离心力为2500g，所述第三离心处理的离心力为10000g，所述第四离心处理的离心力为100000g。
  7. 如权利要求1所述的提取方法，其特征在于，所述100KD超滤管为15ml的超滤管。
  8. 如权利要求1所述的提取方法，其特征在于，所述超滤浓缩处理的条件为：离心力4000g，时间25min。
  9. 如权利要求1所述的提取方法，其特征在于，所述含无外泌体血清的培养基包括：10%无外泌体血清、高糖DMEM和1%青霉素/链霉素。
  10. 如权利要求1所述的提取方法，其特征在于，所述用含无外泌体血清的培养基培养人脂肪来源间充质干细胞的时间为24-48h。
  11. 如权利要求10所述的提取方法，其特征在于，所述用含无外泌体血清的培养基培养人脂肪来源间充质干细胞的时间为48h。
  12. 如权利要求1-11任一项所述的提取方法，其特征在于，所述人脂肪来源间充质干细胞是：从脂肪组织中分离培养hADSCs得到第4代细胞后继续扩大培养得到融合度达80~90%的细胞。
  13. 如权利要求12所述的提取方法，其特征在于，所述从脂肪组织中分离培养hADSCs的方法包括：
    收集脂肪组织样本，然后进行消化处理，得到消化液；
    从所述消化液中收集含单个核细胞的液体后，移入完全培养基中终止消化，然后分离出原代人脂肪来源间充质干细胞；
    将所述原代人脂肪来源间充质干细胞进行传代培养。
  14. 如权利要求13所述的制备方法，其特征在于，用含有0.25%胰蛋白酶和0.1%I型胶原酶的消化液对所述脂肪组织样品中进行消化处理。
  15. 如权利要求13所述的制备方法，其特征在于，所述完全培养包括：10％胎牛血清、高糖DMEM和1%青霉素/链霉素。
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