CN215856071U - Centrifugal tube for separating extracellular vesicles and kit for extracting extracellular vesicles - Google Patents
Centrifugal tube for separating extracellular vesicles and kit for extracting extracellular vesicles Download PDFInfo
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- CN215856071U CN215856071U CN202121799956.4U CN202121799956U CN215856071U CN 215856071 U CN215856071 U CN 215856071U CN 202121799956 U CN202121799956 U CN 202121799956U CN 215856071 U CN215856071 U CN 215856071U
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 239000004005 microsphere Substances 0.000 claims abstract description 36
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 27
- 238000010828 elution Methods 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000007825 activation reagent Substances 0.000 claims description 4
- 230000000903 blocking effect Effects 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 239000006226 wash reagent Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 12
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 238000000605 extraction Methods 0.000 abstract description 7
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 5
- 210000001124 body fluid Anatomy 0.000 abstract description 4
- 239000010839 body fluid Substances 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 210000003463 organelle Anatomy 0.000 abstract description 4
- 150000003904 phospholipids Chemical class 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract 2
- 239000003480 eluent Substances 0.000 abstract 1
- 210000001808 exosome Anatomy 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 5
- 230000004913 activation Effects 0.000 description 3
- 238000001493 electron microscopy Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
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- 239000000090 biomarker Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 208000017169 kidney disease Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical class [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
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- 239000011534 wash buffer Substances 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract
The disclosure relates to a centrifuge tube for separating extracellular vesicles and a kit for extracting extracellular vesicles, and the method for extracting extracellular vesicles comprises the step of specifically enriching the extracellular vesicles in body fluid and tissues by means of titanium dioxide microspheres. Phosphate groups are contained in the phospholipid layer of the vesicle membrane of the extracellular vesicle, and the titanium dioxide microspheres can form a bidentate bond with the phosphate groups, so that the titanium dioxide microspheres can be specifically combined with the phosphate groups to achieve the enrichment effect, and then the target extracellular vesicle is eluted from the titanium dioxide microspheres through alkaline eluent. The vesicle extracted by the centrifugal tube has high purity and does not interfere with other proteins and organelles. The extraction method disclosed by the invention is simple, low in loss and low in requirements on production equipment, and is suitable for multi-sample production in a scientific research laboratory.
Description
Technical Field
The disclosure relates to the technical field of separation and extraction, in particular to a centrifuge tube for separating extracellular vesicles and a kit for extracting extracellular vesicles, which are particularly suitable for a method for extracting extracellular vesicles from body fluid or cell suspension at normal temperature and low speed.
Background
Extracellular Vesicles (EV) are a general term for vesicles having membrane structures released from cells, and are classified into different subtypes according to their particle sizes, and generally, exosomes refer to EVs having a particle size of less than 200nm, which are also called small extracellular vesicles (sEV), and mainly contain proteins, nucleic acids, lipids, and the like. Their shuttling between secretory cells and receptor cells mediates the transmission of biomolecules between cells, and is an important medium for information communication between cells of different tissues. A large number of documents show that the outer vesicle can promote tumorigenesis in vivo, control metastasis formation and tumor immune response; in the aspect of cardiovascular diseases, the compound can be used as a carrier of a cardiovascular system delivery system and has pathological effects on cardiac remodeling and blood vessel formation; in the case of renal disease, exosomes in urine can reflect the urinary system, from podocytes to the renal tubules, as potential biomarkers of renal disease; can also be used as a carrier of related disease proteins in liver diseases and neurodegenerative diseases, and becomes a potential biomarker for early diagnosis. Therefore, the research on the extracellular vesicles has huge market application value and demand in both basic research and clinical application.
The following three methods are currently used for separation and extraction. The first one is the most widely used ultracentrifugation method, which separates dead cells, cell debris, organelles, etc. from the sample by using centrifugal force of different intensities according to the different sedimentation coefficients of different components in the body fluid, and then obtains outer vesicles and partsSeparating proteins, resuspending with PBS, washing, and centrifuging to obtain the required protein. The method has the advantages that a large number of samples can be processed at one time, and the method is the most effective and reliable method for extracting the outer vesicles, but the method needs long time (8-10h), has high requirements on experimental equipment, and can interfere with foreign proteins. The second is a kit method, and currently, the most commonly used is EXOQuick kit developed by systembiospciens (sbi), and the series of reagents are based on polymer form Exosome extraction reagents, form a net-like result, capture microbubbles with certain diameters, and rapidly and effectively extract Exosome in various body fluid samples. The method is simple to operate, but the obtained purity is low. The third is TiO2Enrichment of microspheres by TiO2Forming a bidentate bond between the microsphere and a phosphate group contained in the phospholipid bilayer on the outer vesicle membrane to adsorb exosomes on TiO2And (4) enriching exosomes on the surface of the sphere by elution. The method is based on covalent bonding selective enrichment, has the advantages of high enrichment efficiency, less non-specific adsorption, short sample processing time and the like, is in accordance with the physicochemical properties of the outer vesicles, is simple and convenient, has low cost, and is suitable for scientific research requirements. Therefore, it is important to research the extraction method of the extracellular vesicles which have high purity, convenience and high efficiency and are free from interference of other proteins and organelles.
SUMMERY OF THE UTILITY MODEL
The object of the present disclosure is a centrifuge tube for isolating extracellular vesicles and a kit for extracting extracellular vesicles. The centrifugal tube is used for extracting the extracellular vesicles, so that the operation is simple, the requirement on experimental equipment is low, and the yield and the purity of the obtained extracellular vesicles are high.
In order to achieve the above object, a first aspect of the present disclosure provides a centrifuge tube for separating extracellular vesicles, the centrifuge tube including a tube body having a tube cavity, a sieve plate disposed in the tube body, the sieve plate dividing the tube cavity into an upper tube cavity and a lower tube cavity, the upper tube cavity containing titanium dioxide microspheres;
the lower tube cavity comprises a first lower tube cavity and a second lower tube cavity, the first lower tube cavity is provided with an upper opening, the sieve plate is closed, the lower opening of the first lower tube cavity extends into the second lower tube cavity, and the side wall of the second lower tube cavity is detachably connected to the centrifugal tube.
Optionally, the centrifuge tube has an outer diameter of 7.5-11mm, an inner diameter of 6-10mm, and a height of 28-35 mm.
Preferably, the centrifuge tube has an outer diameter of 9mm, an inner diameter of 7.1-7.3mm and a height of 31 mm.
Optionally, the total volume of the lumen is 0.6-0.8 mL; the volume ratio of the upper tube cavity to the lower tube cavity is 3-4: 1.
optionally, the sieve plate is used for blocking the titanium dioxide microspheres, and the distance between the sieve plate and the bottom of the centrifuge tube is 13-15 mm.
Optionally, the sieve plate has a pore size of 1-4 μm.
Optionally, the titanium dioxide microspheres have a size of 4.5-10 μm.
Preferably, the size of the titanium dioxide microspheres is 5 μm.
Optionally, the centrifuge tube further comprises a screw top cap, and the screw top cap and the tube body are detachably connected by a screw.
Optionally, the centrifuging tube outside still is provided with the sleeve pipe, the top edge of going up the lateral wall of lumen is provided with the locating part of outside extension, the locating part be used for with the centrifuging tube card is in on the sleeve pipe.
In a second aspect, the present disclosure provides a kit for extracting extracellular vesicles, the kit comprising the centrifuge tube of the first aspect and a carrier cassette for accommodating the centrifuge tube.
Optionally, the carrier cartridge further contains an activation reagent bottle, a wash reagent bottle, and an elution reagent bottle.
The centrifuge tube for extracting the extracellular vesicles is simple to operate, has low requirements on experimental equipment, and is high in yield and purity of the obtained extracellular vesicles.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
FIG. 1 is a schematic view of a centrifuge tube for separating extracellular vesicles without a cannula.
FIG. 2 is a schematic diagram of a centrifuge tube for separating extracellular vesicles.
FIG. 3 is the result of electron microscopy of outer vesicles in serum, from which the outer vesicle particles of a typical saucer-like sample can be seen.
FIG. 4 is a graph showing the particle size-concentration distribution of outer vesicles in serum; the results show that the Exosome Diameter (nm) 131.0, fits the outer vesicle particle size (< 200 nm).
FIG. 5 is a schematic diagram of the principle of titania-assisted extraction of vesicles.
Description of the reference numerals
1 spiral upper cover
2 pipe body
3 Upper pipe cavity
4 sieve plate
5 lower tubular cavity
6 first lower lumen
7 second lower pipe cavity
8 locating part
9 casing pipe
Detailed Description
The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
Referring to fig. 1, 2 and 4, a first aspect of the present disclosure provides a centrifuge tube for separating extracellular vesicles, the centrifuge tube including a tube body 2 having a tube cavity, a screen plate 4 disposed in the tube body, the screen plate 4 dividing the tube cavity into an upper tube cavity 3 and a lower tube cavity 5, the upper tube cavity 3 containing titanium dioxide microspheres;
When the device is used, the titanium dioxide microspheres contained in the upper tube cavity are combined with the extracellular vesicles in the sample, so that the extracellular vesicles cannot pass through the sieve plate, and other substances can enter the lower tube cavity through the sieve plate and are discarded.
Optionally, the centrifuge tube has an outer diameter of 7.5-11mm, an inner diameter of 6-10mm, and a height of 28-35 mm.
Preferably, the centrifuge tube has an outer diameter of 9mm, an inner diameter of 7.1-7.3mm and a height of 31 mm.
Optionally, the total volume of the lumen is 0.6-0.8 mL; the volume ratio of the upper tube cavity 3 to the lower tube cavity 5 is 3-4: 1.
according to the present disclosure, the centrifugal tube with the size as described above is used for extracting extracellular vesicles, the using method is simple, the loss is low, and the extraction of extracellular vesicles is facilitated.
Optionally, the sieve plate 4 is used for blocking the titanium dioxide microspheres, and the distance between the sieve plate 4 and the bottom of the centrifuge tube is 13-15 mm.
According to the present disclosure, the sieve plate can block the titanium dioxide microspheres, improving the purity of extracellular vesicles extracted using the centrifuge tube; sieve apart from centrifuge tube bottom have as above the distance of height is favorable to discarding the outflow of liquid, the outer vesicle of the cell through elution of being more convenient for enters into lower lumen.
Optionally, the sieve plate 4 has a pore size of 1-4 μm.
Optionally, the titanium dioxide microspheres have a size of 4.5-10 μm.
Preferably, the size of the titanium dioxide microspheres is 5 μm.
According to the present disclosure, the phospholipid layer of the vesicle membrane of the extracellular vesicle contains phosphate groups, and the titanium dioxide microspheres can form bidentate bonds with the phosphate groups, so as to be specifically bound to the extracellular vesicle.
Optionally, the centrifuge tube further comprises a spiral upper cover 1, and a detachable spiral connection is arranged between the spiral upper cover 1 and the tube body 2.
Optionally, the centrifuge tube outside still is provided with sleeve 9, the upper edge of going up the lateral wall of lumen 2 is provided with outside extension locating part 8, locating part 8 be used for with the centrifuge tube card is in on sleeve 9.
According to the present disclosure, the centrifuge tube needs to slightly unscrew the spiral upper cover 1 during the centrifugation process, so as to facilitate the liquid outflow.
In a second aspect, the present disclosure provides a kit for extracting extracellular vesicles, the kit comprising a first portion of the centrifuge tube and a carrier cassette for holding the centrifuge tube.
Optionally, the carrier cartridge further contains an activation reagent bottle, a wash reagent bottle, and an elution reagent bottle.
According to the present disclosure, the titanium dioxide (TiO)2) The microspheres are activated by Wash Buffer in an activation reagent bottle, according to the disclosure, the activation of the titanium dioxide microspheres is to weigh the titanium dioxide microspheres into a centrifuge tube, suspend the titanium dioxide microspheres by precooled Wash Buffer (abbreviated as 'WB'), centrifuge for 1min at 5000g after short vortex, discard the activation solution, replace new cold WB, and repeat the operation for 2-3 times.
According to the disclosure, the incubation is to add the pretreated sample into a centrifuge tube filled with activated titanium dioxide microspheres, uniformly vortex the sample, and incubate the sample for 5min at 4 ℃; the purification is to repeatedly blow and beat the incubated titanium dioxide microspheres in a washing reagent bottle by using precooled WB, and the step is repeated for 2-3 times; the Elution is to add an Elution Buffer (EB for short) into an Elution reagent bottle, incubate for 10min at 4 ℃, then centrifuge for 1min at 1000g, collect the effluent liquid to obtain an outer vesicle suspension, and store at-80 ℃.
According to the disclosure, the pretreatment of the sample is to remove impurities or cell debris after high-speed centrifugation in a low-temperature environment or after concentration for a certain time; the centrifugal speed of the high-speed centrifugation is more than 10000g, and the processing time of a certain time is more than 10 min.
According to the disclosure, when the activated titanium dioxide microspheres are mixed with the pretreated sample, the sample and the titanium dioxide microspheres are fully mixed, that is, the titanium dioxide microspheres are resuspended, and the temperature of the sample is 4 ℃, and the incubation time is 5 min.
According to the disclosure, the use of pre-cooled WB is aimed at washing away the impure proteins which are not completely adsorbed and are not firmly adsorbed on the surface of the titanium dioxide microspheres.
According to the disclosure, the ratio of the required amount of the titanium dioxide microspheres to the sample volume is 40-60 [ mu ] g:1 [ mu ] L, wherein the volume of WB can be 200-; the EB volume during elution may be 20-50. mu.L.
According to the disclosure, the extracellular vesicles extracted by the centrifugal tube have the characteristics of high purity, convenience, high efficiency and no interference of other proteins and organelles.
Example 1
By means of a TiO compound2A method for microsphere-specific enrichment of extracellular vesicles, comprising the steps of:
required sample 50. mu.L serum (human)
1)TiO2And (3) activation: 2.5mg TiO are weighed2Microspheres (5 μm in diameter) were placed in a spin column and 200 μ L of pre-cooled WB was used to suspend the TiO2Centrifuging the microspheres for 1min at 5000g after the brief vortex, discarding the activating solution, replacing with new cold WB, and repeating the operation for 3 times;
2) sample pretreatment: transferring 60 μ L of untreated serum into a new centrifuge tube, centrifuging at 20000g at 4 deg.C for 10 min;
3)TiO2adding the mixture into a sample for incubation: pipette 50 μ L of pretreated serum (note do not aspirate pellet) into the activated TiO loading chamber2Uniformly swirling in a centrifugal column of the microspheres, and incubating for 5min at 4 ℃;
4) and (3) purification: sucking 500 microliter of precooled WB into a centrifugal column, and repeatedly blowing the TiO obtained after the step 3 by using a pipette2Microspheres were centrifuged at 500g for 30s and the effluent discarded. This step was repeated 3 times;
5) and (3) elution: adding EB, incubating at 4 deg.C for 10min, centrifuging at 1000g for 1min, collecting eluate as outer vesicle suspension, and storing at-80 deg.C.
Test example 1
The outer vesicle suspensions obtained in the examples were tested and the results were obtained as shown in fig. 2 and fig. 3. FIG. 2 is the result of electron microscopy of outer vesicles in serum, and typical saucer-like outer vesicle particles can be seen under the electron microscopy. FIG. 3 is a graph showing the particle size-concentration distribution of outer vesicles in serum; the results show that the Exosome Diameter (nm) 131.0, fits the outer vesicle particle size (< 200 nm).
As can be seen from FIGS. 2 and 3, the operation of extracting the extracellular vesicles by using the centrifugal tube is simple, the requirement on experimental equipment is low, and the yield and the purity of the obtained extracellular vesicles are high.
The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. The centrifugal tube for separating the extracellular vesicles is characterized by comprising a tube body (2) with a tube cavity, wherein a sieve plate (4) is arranged in the tube body, the sieve plate (4) divides the tube cavity into an upper tube cavity (3) and a lower tube cavity (5), and the upper tube cavity (3) contains titanium dioxide microspheres;
lower lumen (5) include first lumen (6) and second lower lumen (7) that top-down set up, the upper shed of first lumen (6) is by sieve (4) are closed, the lower shed of first lumen (6) extends into lumen (7) under the second, the lateral wall detachably of lumen (7) is connected under the second on the centrifuging tube.
2. The centrifuge tube of claim 1, wherein the centrifuge tube has an outer diameter of 7.5-11mm, an inner diameter of 6-10mm, and a height of 28-35 mm.
3. The centrifuge tube of claim 1, wherein the total volume of the lumen is 0.6-0.8 mL; the volume ratio of the upper tube cavity (3) to the lower tube cavity (5) is 3-4: 1.
4. the centrifuge tube according to claim 1, wherein the sieve plate (4) is used for blocking the titanium dioxide microspheres, and the distance between the sieve plate (4) and the bottom of the centrifuge tube is 13-15 mm.
5. The centrifuge tube according to claim 1 or 3, wherein the sieve plate (4) has a pore size of 1-4 μm.
6. The centrifuge tube of claim 5, wherein the titanium dioxide microspheres are 4.5-10 μ ι η in size.
7. The centrifuge tube according to claim 1, wherein the centrifuge tube further comprises a screw top cap (1), the screw top cap (1) and the tube body (2) having a detachable screw connection therebetween.
8. The centrifuge tube according to claim 7, wherein a sleeve (9) is further arranged outside the centrifuge tube, an upper edge of a side wall of the upper tube cavity (3) is provided with a limiting member (8) extending outwards, and the limiting member (8) is used for clamping the centrifuge tube on the sleeve (9).
9. A kit for extracting extracellular vesicles, comprising the centrifuge tube according to any one of claims 1 to 8 and a carrier case for accommodating the centrifuge tube.
10. The kit of claim 9, wherein the carrier cartridge further contains an activation reagent bottle, a wash reagent bottle, and an elution reagent bottle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164203A (en) * | 2022-02-11 | 2022-03-11 | 北京同创正业生物科技有限公司 | Extracellular vesicle purification material and purification method |
| CN115420886A (en) * | 2022-09-13 | 2022-12-02 | 中国人民解放军军事科学院军事医学研究院 | A method for extracting liposomes in plasma based on titanium dioxide microspheres and determining the content of free drug, encapsulated drug and total drug |
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2021
- 2021-08-03 CN CN202121799956.4U patent/CN215856071U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164203A (en) * | 2022-02-11 | 2022-03-11 | 北京同创正业生物科技有限公司 | Extracellular vesicle purification material and purification method |
| CN114164203B (en) * | 2022-02-11 | 2022-05-10 | 北京同创正业生物科技有限公司 | Extracellular vesicle purification material and purification method |
| CN115420886A (en) * | 2022-09-13 | 2022-12-02 | 中国人民解放军军事科学院军事医学研究院 | A method for extracting liposomes in plasma based on titanium dioxide microspheres and determining the content of free drug, encapsulated drug and total drug |
| CN115420886B (en) * | 2022-09-13 | 2024-01-12 | 中国人民解放军军事科学院军事医学研究院 | Method for extracting liposome in blood plasma based on titanium dioxide microspheres and measuring free drug, encapsulated drug and total drug content |
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