CN118256333A - Exosome acquisition device and exosome acquisition method - Google Patents
Exosome acquisition device and exosome acquisition method Download PDFInfo
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Abstract
The application provides an exosome collecting device and method, and relates to the field of biology. The device comprises: the cell filtering module is used for filtering the supernatant fluid of the exosomes to obtain the filtered supernatant fluid; the first centrifugal module comprises a first centrifugal rotor and a first centrifugal tube which are matched; centrifuging the supernatant subjected to the filtration treatment in the first centrifuge tube by using a first centrifugal rotor to obtain supernatant subjected to the first centrifugation; the second centrifugal module comprises a second centrifugal rotor and a second centrifugal tube which are matched; the supernatant after the first centrifugation is contained in the second centrifuge tube, and the supernatant in the second centrifuge tube is centrifuged by utilizing a second centrifugal rotor to obtain a precipitate after the second centrifugation; and the exosome treatment module is used for treating the precipitate after the second centrifugation to obtain an exosome. The application establishes a set of exosome standard preparation operation flow which is quick, efficient, economical and suitable for large-volume samples.
Description
Technical Field
The invention relates to the field of biology, in particular to an exosome acquisition device and method.
Background
Exosomes are 30-150nm extracellular vesicles released from a variety of cell types and perform a variety of cellular functions including intercellular communication, antigen presentation and tumorigenic proteins, mRNA and miRNA transfer. With the intensive research on exosomes, the application thereof is becoming more and more widespread. Meanwhile, exosomes can provide a variety of bioactive substances and components that are easily inactivated or degraded through a variety of pathways and safely transfer to target cells to participate in supervision, such as tissue repair, tumor diagnosis and treatment, and immunomodulation. Therefore, the exosome has various clinical application prospects: can be used as a drug delivery tool or a therapeutic agent for diseases, a novel biomarker for diagnosing diseases, or used for repairing and repairing the injuries related to regenerative medicine.
From the practical problems faced by the industrialization of exosomes, enterprises follow clinical demands, and finding the competitive advantage of exosomes in the fields of drug loading, medical science, tissue regeneration and the like compared with other products is a core factor that needs to be focused on in scientific research and clinical transformation. However, these studies are premised on the completion of stable, efficient, high purity exosome separation and extraction, which is also an important technical challenge currently limiting the progress in this field.
The optimal exosome extraction technical scheme is high-purity, harmless and low-cost, and can even meet the screening of different types of extracellular vesicles. Obviously, although there are numerous methods for separating exosomes at present, there is no exosome extraction device and method that can be performed quickly, efficiently, economically and suitable for large volumes of samples.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the method aims at the problems of lack of standardization, large-scale preparation difficulty and the like in the existing exosome preparation, adopts a classical ultracentrifugation method and combines filtration to stably extract and purify exosomes, and establishes a set of exosome standard preparation operation flow which is quick, efficient, economical and suitable for large-volume samples.
The first aspect of the present application discloses an exosome collection device, the device comprising:
The cell filtering module is used for filtering the supernatant fluid of the exosomes to obtain the filtered supernatant fluid;
The first centrifugal module comprises a first centrifugal rotor and a first centrifugal tube which are matched; centrifuging the supernatant subjected to the filtering treatment in the first centrifuge tube at a first rotating speed N1 by utilizing the first centrifugal rotor to obtain supernatant subjected to the first centrifugation;
The second centrifugal module comprises a second centrifugal rotor and a second centrifugal tube which are matched; the supernatant after the first centrifugation is contained in a second centrifuge tube, and the supernatant in the second centrifuge tube is centrifuged at a second rotating speed N2 by utilizing the second centrifuge rotor, so that a precipitate after the second centrifugation is obtained;
and the exosome treatment module is used for treating the precipitate after the second centrifugation to obtain an exosome.
In some embodiments, the cell filtration module comprises: a first filtration module and a second filtration module; sequentially processing the supernatant of the exosome by the first filtering module and the second filtering module to obtain filtered supernatant;
optionally, the first filtering module includes: a cell sieve of 100 μm;
Optionally, the second filtering module includes: cell sieves of 0.22 μm.
In some embodiments, prior to the cell filtration module, further comprising: a third centrifugation module for removing cells from the cell culture broth; the third centrifugation module is used for centrifuging the cell culture solution at a third rotating speed N3 to obtain the supernatant of the exosome;
optionally, when the unit of the third rotation speed N3 is g, N3 is a positive number greater than or equal to 200, and preferably 300;
Optionally, when the unit of time of the third rotation speed N3 centrifugation is min, the centrifugation time is a positive number greater than or equal to 6.
In some embodiments, when the unit of the first rotation speed N1 is g, N1 is a positive number greater than or equal to 8000, preferably 10000;
optionally, centrifuging at the first rotational speed N1 for at least 30 minutes;
Optionally, when the unit of the second rotation speed N2 is g, N1 is a positive number greater than or equal to 80000, preferably 100000;
Optionally, the time of centrifugation at the second rotational speed N2 is at least 90min.
In some embodiments, after the cell filtration module, before the first centrifugation module, further comprises: a fourth centrifugation module that centrifigates the filtered supernatant at a fourth rotational speed N4;
optionally, when the unit of time of the fourth rotation speed N4 for centrifugation is min, the centrifugation time is a positive number greater than or equal to 15, and preferably 20.
In some embodiments, the method for processing the sediment after the second centrifugation comprises: re-suspending the precipitate by using PBS to obtain a mixed solution; centrifuging the mixture to obtain a separated supernatant and a precipitate; the separated precipitate is the exosome;
Optionally, the separated precipitate is resuspended by using PBS, and the mixture after resuspension is the exosome.
The second aspect of the application discloses an exosome collection method, which comprises the following steps:
s1: filtering the exosome supernatant to obtain a filtered supernatant;
s2: centrifuging the supernatant subjected to the filtration treatment in a first centrifuge tube at a first rotational speed N1 by using a first centrifugal rotor to obtain a supernatant subjected to the first centrifugation;
s3: centrifuging the supernatant after the first centrifugation in a second centrifuge tube at a second rotational speed N2 by using a second centrifugal rotor to obtain a precipitate after the second centrifugation;
s4: and treating the precipitate after the second centrifugation to obtain an exosome.
In some embodiments, the method of filtering exosome supernatant in S1 comprises: s11: obtaining exosome supernatant; s12: filtering the supernatant of the exosomes by using a 100 μm cell sieve to obtain a supernatant after the first filtration; s13: filtering the supernatant after the first filtering by using a cell sieve of 0.22 mu m to obtain the supernatant after the filtering treatment;
optionally, before the step S1, the method further includes: obtaining a cell culture solution; centrifuging the cell culture fluid at a third rotational speed N3 to obtain the exosome supernatant;
alternatively, the cell culture broth is derived from a culture medium.
In some embodiments, between S1 and S2, further comprising: centrifuging the supernatant after the filtration treatment at a fourth rotation speed N4.
In a third aspect, the application discloses an exosome collection device, the device comprising: a memory and a processor; the memory is used for storing program instructions; the processor is configured to invoke program instructions, which when executed, are configured to perform the steps of the method according to the second aspect of the application.
The application has the following beneficial effects:
1. The application creatively discloses an exosome collecting device and method, which are used for standardizing a unified operation flow, and the exosome obtained by the device and method is higher in yield, higher in purity, better in uniformity, simple in operation and suitable for large-scale production. Specifically, the supernatant is subjected to gradual gradient filtration (100 μm and 0.22 μm cell sieves) prior to ultracentrifugation to ensure removal of larger cell debris and broken organelles, and then subjected to large-scale extraction of exosomes according to the ultracentrifuge-adapted rotor, and finally enriched and purified by 100,000Xg.
2. The application creatively optimizes the selection, culture condition, rotor selection, rotation speed and the like of the parent cells of the exosomes, solves the problem that the separation becomes difficult due to the different sizes, functions and sources of the exosomes, and the current situation that the parameters such as the centrifugal time, the centrifugal force, the rotor type and the like can influence the yield and the purity of the target exosomes; firstly, a human umbilical cord mesenchymal stem cell supernatant which is easy to obtain and stable is selected as a primary selection condition for the stable extraction of the exosome of the experiment, and the human umbilical cord mesenchymal stem cell supernatant has good immunoregulation characteristics, easily obtained parent tissue sources, high safety and good popularization value; secondly, when exosome is extracted, firstly, a serum-free culture medium is selected to enable human umbilical cord mesenchymal stem cells to fully release exosome, the culture medium does not contain any animal-derived component, recombinant or medical-grade human proteins are used, and the exosome has wide applicability and low cost and is suitable for large-scale preparation of the exosome of the laboratory; furthermore, the exosome is extracted by ultracentrifugation, and the supernatant of the exosome is filtered step by step before ultracentrifugation, so that large particulate matters are efficiently screened out; meanwhile, the selection and operation of a centrifugal rotor, centrifugal force and rotating speed of the ultracentrifugation are optimized, and then the exosome with high yield and high uniformity is obtained.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view of an apparatus according to a first aspect of an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method provided by a second aspect of an embodiment of the invention;
FIG. 3 is a schematic diagram of a computer device provided by an embodiment of the present invention;
Fig. 4 is a graph showing the result of the exosome form under the electron microscope according to the different extraction methods provided by the embodiment of the present application; wherein A is an exosome extracted by adopting the flow of the application, B is an exosome extracted by adopting a general ultracentrifugation method (without secondary filtration), and C is an exosome extracted by adopting a kit extraction method;
FIG. 5 is a graph showing the results of particle size analysis of exosomes obtained by different extraction methods according to the examples of the present application; wherein A is the average grain size and the exosome concentration of exosomes extracted by adopting the flow of the application, B is the average grain size and the exosomes concentration of exosomes extracted by adopting a general ultracentrifugation method (without secondary filtration), and C is the average grain size and the exosomes concentration of exosomes extracted by adopting a kit extraction method;
FIG. 6 is a BCA standard chart of different extraction methods provided by an embodiment of the present application; wherein A is an exosome standard curve extracted by adopting the flow of the application, the correlation is better, and the exosome protein concentration is high; b is an exosome standard curve extracted by adopting a general ultracentrifugation method (without secondary filtration), the concentration of exosome protein is reduced, C is an exosome standard curve extracted by adopting a kit extraction method, and the concentration of exosome protein is lower;
FIG. 7 is an expression diagram of markers Alix, TSG101 and CD63 in exosomes obtained by using a Western blot method to detect different extraction methods; wherein A is exosomes which are extracted by adopting the flow of the application and can express specific surface markers Alix, TSG101 and CD63; b is the exosome expressible specific surface markers Alix, TSG101 and CD63 extracted by general ultracentrifugation (without secondary filtration), C is the exosome expressible specific surface markers Alix, TSG101 and CD63 extracted by kit extraction.
Detailed Description
In order to enable those skilled in the art to better understand the present invention, the following description will make clear and complete descriptions of the technical solutions according to the embodiments of the present invention with reference to the accompanying drawings.
In some of the flows described in the specification and claims of the present invention and in the foregoing figures, a plurality of operations occurring in a particular order are included, but it should be understood that the operations may be performed out of order or performed in parallel, with the order of operations such as 101, 102, etc., being merely used to distinguish between the various operations, the order of the operations themselves not representing any order of execution. In addition, the flows may include more or fewer operations, and the operations may be performed sequentially or in parallel. It should be noted that, the descriptions of "first" and "second" herein are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, and are not limited to the "first" and the "second" being different types.
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments according to the invention without any creative effort, are within the protection scope of the invention.
The quality of separation and extraction of exosomes determines the stability and accuracy of subsequent experiments. The exosome extraction/purification method comprises the following steps: ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation, immunoaffinity separation (immunomagnetic beads, microfluidic techniques), and the like. Exosomes obtained by different extraction methods vary in quality and the results may not be reproducible. The current ultracentrifugation method is a gold standard for exosome separation, is the most common method for exosome extraction, is mature, is suitable for large-volume samples, and is also the exosome extraction method which is the basis for the patent selection. The ultracentrifugation method has the advantages of simple operation, no need of complex technical support, relatively low cost, suitability for large-volume samples, but time-consuming, and the number and quality of the obtained exosomes are affected by factors such as the type of the ultracentrifuge rotor, the size of the centrifugal force, the viscosity of the solution, and the like.
Almost all types of normal cells produce exosomes, such as human umbilical vein endothelial cells, mesenchymal stem cells, T cells, B cells, dendritic Cells (DCs), natural killer cells (NK), etc. Mesenchymal stem cells are multipotent stem cells with self-renewal and multipotent differentiation capacity. Umbilical cord-derived mesenchymal stem cells (HucMSCs) are readily available from human umbilical cord and can be isolated at low cost compared to other tissue-derived mesenchymal stem cells. Most importantly, hucMSCs rarely produce an in vitro immune response to allogeneic T cells. They secrete a large number of tolerance-related factors, including TGF-. Beta.1 and IL-10, indicating low immunogenicity and good immunomodulatory properties.
The exosome (HucMSCs-EXo) derived from the human umbilical cord mesenchymal stem cells can simulate part of functions of the umbilical cord mesenchymal stem cells, play roles in promoting tissue repair and immunoregulation, and the exosome has no cell nucleus structure, cannot be amplified in a host body, has better safety in a clinical application process, and therefore has better popularization value. The application selects the easily obtained and stable human umbilical cord mesenchymal stem cell supernatant as the primary selection condition for the stable extraction of the exosome of the experiment.
Currently, over 80% of researchers choose ultracentrifugation to extract exosomes. The ultracentrifugation method is to circulate with different centrifugal forces or centrifugal times, phase out larger particles, finally obtain exosomes precipitated at the bottom, and is mainly divided into 2 steps, firstly, dead cells, cell fragments and precipitates of large vesicles are removed by medium-low speed centrifugation, and then exosomes precipitates are obtained by centrifugal separation with 100,000Xg. The ultracentrifugation method is favored because the method is not polluted by separating reagents, the exosome is large in acquisition amount, simple to operate, mature in technology and relatively mature in method, and is the first choice for large-scale exosome preparation. However, exosomes vary in size, function and origin, which makes separation difficult. It was found that the parameters such as centrifugation time, centrifugal force, rotor type, etc. all affect the yield and purity of the target exosomes.
Fig. 1 is a schematic diagram of an exosome collection device according to an embodiment of the present invention, specifically, the device includes:
101: a cell filtration module; the cell filtering module is used for filtering the supernatant fluid of the exosome to obtain the filtered supernatant fluid;
in some embodiments, the cell filtration module comprises: a first filtration module and a second filtration module; sequentially processing the supernatant of the exosome by the first filtering module and the second filtering module to obtain filtered supernatant;
in some more specific embodiments, the first filtration module comprises: a cell sieve of 100 μm;
in some more specific embodiments, the second filtration module comprises: cell sieves of 0.22 μm.
In some embodiments, eradication of fragments and vesicles greater than 200nm is important, and if more stringent filtration is required, 100nm filters may be used, but it is noted that if viscous fluids are used, these filters are easily blocked, resulting in loss of exosome material. Furthermore, 100nm filtration may affect the morphology of exosomes, and if the exosomes are large in scale they may be lost. Therefore, the application firstly selects a 100 mu m cell sieve to filter the supernatant of the human umbilical cord mesenchymal stem cells, and continuously adopts a 0.22 mu m filter sieve to filter the supernatant after reaching a new 50mL centrifuge tube, so as to gradually filter large particles. After filtration, the exosomes were pelleted using an ultracentrifugation method of 100,000Xg and harvested.
In some embodiments, after the cell filtration module, before the first centrifugation module, further comprises: a fourth centrifugation module that centrifigates the filtered supernatant at a fourth rotational speed N4;
In some more specific embodiments, when the fourth rotation speed N4 is centrifuged for a time unit of min, the centrifugation time is a positive number greater than or equal to 15, preferably 20.
102: A first centrifugal module; the first centrifugal module comprises a first centrifugal rotor and a first centrifugal tube which are matched; centrifuging the supernatant subjected to the filtering treatment in the first centrifuge tube at a first rotating speed N1 by utilizing the first centrifugal rotor to obtain supernatant subjected to the first centrifugation;
in a more specific embodiment, when the unit of the first rotation speed N1 is g, N1 is a positive number greater than or equal to 8000, preferably 10000, so as to remove larger cell fragments and broken organelles;
In more specific embodiments, centrifuging at the first rotational speed N1 is for at least 30 minutes;
103: a second centrifugal module; the second centrifugal module comprises a second centrifugal rotor and a second centrifugal tube which are matched; the supernatant after the first centrifugation is contained in a second centrifuge tube, and the supernatant in the second centrifuge tube is centrifuged at a second rotating speed N2 by utilizing the second centrifuge rotor, so that a precipitate after the second centrifugation is obtained;
in more specific embodiments, when the unit of the second rotation speed N2 is g, N1 is a positive number greater than or equal to 80000, preferably 100000;
In a more specific embodiment, the centrifugation time is at least 90 minutes at the second rotational speed N2.
In some embodiments, ultra-high speed centrifugation is based on density and size between exosomes in one sample, which typically requires a series of centrifugal forces at different rotational speeds (low and high speed centrifugation) and for a certain time to separate exosomes. Second, by extending the centrifugation time, significantly higher vesicle yields can be obtained, indicating that a conventional centrifugation protocol of 70min is essentially inadequate for isolation of exosomes. Thus, the present application first applies a relatively low centrifugal force (300 Xg) to remove cells from the cell culture broth, then gradient filters the supernatant and then applies a high centrifugal force (10,000 Xg) to remove large cell debris and broken organelles. Finally, high-speed centrifugation (100,000Xg) was performed to increase the centrifugation time to 90min, thereby collecting the exosomes in the supernatant.
104: An exosome treatment module; and the exosome treatment module is used for treating the precipitate after the second centrifugation to obtain an exosome.
In some embodiments, the method for processing the sediment after the second centrifugation comprises: re-suspending the precipitate by using PBS to obtain a mixed solution; centrifuging the mixture to obtain a separated supernatant and a precipitate; the separated precipitate is the exosome;
in some embodiments, the isolated pellet is resuspended in PBS and the mixture after resuspension is the exosome.
In some embodiments, prior to the cell filtration module, further comprising: a third centrifugation module for removing cells from the cell culture broth; the third centrifugation module is used for centrifuging the cell culture solution at a third rotating speed N3 to obtain the supernatant of the exosome;
In some more specific embodiments, when the third rotational speed N3 is in g, N3 is a positive number of 200 or more, preferably 300, to remove cells from the cell culture broth for lower centrifugal force;
in some more specific embodiments, when the third rotation speed N3 is centrifuged for a time unit of min, the centrifugation time is a positive number greater than or equal to 6.
In some embodiments, the selection of extraction rotors is as follows: two rotor types commonly used in exosome research are the swinging bucket rotor (SW) and the fixed angle rotor (FA). These are functionally very different, since the rotor is caused to protrude horizontally from the axis of rotation during rotation, the FA rotor being maintained at a constant angle throughout centrifugation. The results indicate that SW is generally longer than FA rotor settling path length, which may result in lower pelletization efficiency. Therefore, the laboratory purchased a Thermo ultracentrifuge Sorvall WX80, with the chosen rotor being a fixed angle rotor adapted to the centrifuge, F37L-8X 100, F50L-8X 39 and F50-24X 1.5, for centrifugation and purification of exosomes 10,000 Xg and 100,000 Xg, respectively.
Fig. 2 is a schematic flow chart of an exosome collection method according to an embodiment of the present invention, where the method includes:
s1: filtering the exosome supernatant to obtain a filtered supernatant;
s2: centrifuging the supernatant subjected to the filtration treatment in a first centrifuge tube at a first rotational speed N1 by using a first centrifugal rotor to obtain a supernatant subjected to the first centrifugation;
s3: centrifuging the supernatant after the first centrifugation in a second centrifuge tube at a second rotational speed N2 by using a second centrifugal rotor to obtain a precipitate after the second centrifugation;
s4: and treating the precipitate after the second centrifugation to obtain an exosome.
In some embodiments, the method of filtering exosome supernatant in S1 comprises: s11: obtaining exosome supernatant; s12: filtering the supernatant of the exosomes by using a 100 μm cell sieve to obtain a supernatant after the first filtration; s13: filtering the supernatant after the first filtering by using a cell sieve of 0.22 mu m to obtain the supernatant after the filtering treatment;
In some embodiments, before the step S1, the method further includes: obtaining a cell culture solution; centrifuging the cell culture fluid at a third rotational speed N3 to obtain the exosome supernatant;
In some embodiments, the cell culture broth is obtained from a culture medium. The culture medium comprises one or more of the following components: serum starvation culture, exosome-removing serum culture, serum-free system culture, and other sample sources; serum-free system culture is preferred. Among them, serum culture without exosomes and serum-free system culture are common means for exosome culture. Exosome-removing serum culture is used for reducing or avoiding the influence of serum starvation culture on cell secretion exosomes, and the exosome-removing serum can be suitable for most cell culture, can basically maintain the same growth rate and morphology as those in normal fetal bovine serum, and is a mode selected by most students. Serum-free system culture is from starvation culture to exosome-free serum culture to serum-free system culture. This is for the culture medium improvement and optimization made by exosome studies. The culture medium of the existing serum-free system can be divided into a serum-free culture medium containing platelet lysate (containing exosomes), a serum-free culture medium with a definite formula and the like, wherein the serum-free system with the definite formula enables the result of researching the cell exosomes to be more accurate, but the serum-free system is high in price and high in large-scale extraction cost for the exosomes. Therefore, the culture medium selected by the application is a serum-free exosome, and is suitable for culturing primary and passage mesenchymal stem cells from different sources; and does not contain any animal-derived components, using recombinant or medical grade human proteins; and the method has wide applicability, and the addition of various brand additives shows better performance, so that the method is suitable for large-scale preparation of the laboratory external genitalia.
In some embodiments, between S1 and S2, further comprising: centrifuging the supernatant after the filtration treatment at a fourth rotation speed N4.
Fig. 3 is a computer device according to an embodiment of the present invention, where the device includes: a memory and a processor; the memory is used for storing program instructions; the processor is configured to invoke program instructions, which when executed, are configured to perform the steps of the method described above.
Examples
1. Experimental materials
1.1 Main instruments and consumables:
| Name of the name | Production area | Manufacturer' s | Product model |
| High-speed refrigerated centrifuge | Germany | Thermo | X3R |
| Ultracentrifuge | Germany | Thermo | SorvallWX80 |
| F37L-8×100 fixed angle rotor | Germany | Thermo | 096-087056 |
| F50-8×39 fixed angle rotor | Germany | Thermo | 096-087051 |
| F50-24×1.5 fixed angle rotor | Germany | Thermo | 096-247028 |
1.2 Main reagents:
1.3 main consumables:
| Name of the name | Production area | Manufacturer' s | Specification of specification | Lot number | Product goods number |
| EP pipe | Germany | Thermo | 1.5mL | 21003035 | 314352 |
2. Experimental method
(1) Collecting and removing conditioned media
1) When the cells reach 70% -80%, the complete culture medium is taken out, and the cells are washed three times with sterile PBS. Replaced with a similar volume of exosome-specific medium (BC-T4 medium).
2) The cells were returned to the incubator for 24-48 hours.
3) Removal of conditioned media from adherent cells: cell supernatants from exosomes were collected by pipette and transferred to 50mL centrifuge tubes. The cells were centrifuged for 10min at 300 Xg, 4 ℃.
4) After centrifugation, the supernatant was filtered through a 100 μm cell sieve and then filtered through a 0.22 μm filter, and the filtered supernatant was stored at 4℃for 1 week or-80℃for a long period of time.
(2) Removal of cells, dead cells and cell debris
1) If not stored, the mixture can be centrifuged directly for 20min at 2000 Xg at 4 ℃.
2) The supernatant was removed and transferred to an ultracentrifuge rotor (F37L-8X 100).
3) One side of each ultracentrifuge tube was labeled with a waterproof label and centrifuged at 10,000Xg for 30min at 4 ℃.
(3) Collecting exosome fraction
1) The supernatant was transferred to a centrifuge tube fitted with an F50-8X 39 rotor.
2) Centrifuge to 90min,100,000Xg, 4 ℃. (particle = exosome + contaminating protein)
3) The supernatant was removed completely and discarded.
(4) Cleaning exosomes
1) Particles in the tube were resuspended in 1mL PBS using a micropipette.
2) Centrifuge for 1h,100,000Xg, 4 ℃.
3) The supernatant was removed as completely as possible without touching the bottom of the tube where the particles were located. For a fixed angle rotor, the supernatant was decanted, the holding tube was inverted, and the remaining liquid on the sides and mouth was pipetted with micropipettes.
4) To resuspend the particles (i.e. exosomes): 50. Mu.L/tube of PBS was added and then resuspended.
5) The exosomes were placed in 50 μl tubes and stored for 1 year at-80 ℃. Avoiding repeated freezing and thawing.
3. Experimental results
3.1 Electron microscope identification results
The exosomes derived from the human umbilical cord mesenchymal stem cells have a circular or oval vesicle-shaped structure with a double-layer membrane structure, are granular, have a diameter of about 30-150 nm and a relatively complete membrane structure, are typical morphological characteristics of the exosomes (fig. 4), wherein fig. 4A is the exosomes obtained by the method of the application, fig. 4B is the exosomes obtained by a general ultracentrifugation method (without secondary filtration), and fig. 4C is the exosomes obtained by a kit extraction method, and the results show that the exosomes extracted by the method of the application are high in yield and high in uniformity.
3.2NTA detection results
Particle size analysis showed that: compared with other methods, the exosome obtained by the method has smaller particle size and larger concentration, the average particle size is 126.0nm, and the exosome concentration can reach 2.8x10 12/mL (figure 5A). The average particle size of the exosomes extracted by general ultracentrifugation was 144.0nm, and the exosomes concentration was 8.2×10 11/mL (fig. 5B). The average particle size of exosomes obtained by the kit extraction method is 120.0nm, the exosomes concentration is 1.4×10 11/mL (figure 5C), and the dispersity is large and the uniformity is poor. Although all three methods described above are consistent with the physical characteristics of the exosome particle size, it is apparent that the exosome obtained by the method of the present application is of higher quality and purity (FIG. 5).
3.3BCA protein concentration assay
The results show that the method adopted by the application has larger concentration of the exosome protein compared with other methods. The exosome standard curve of the method adopted by the application is y=0.0008× -0.00119, and R 2 =0.9987, so that the correlation is good; the dilution concentration was 125.333. Mu.g/mL at 50 times, and the original concentration was 6.267. Mu.g/. Mu.L (FIG. 6A). Typical ultracentrifugation standard curves are y=0.0014× -0.0552, r 2 = 0.9823; the dilution was performed 50 times at 98.643. Mu.g/mL and the original concentration was 4.932. Mu.g/. Mu.L (FIG. 6B), resulting in a decrease in the concentration of the exosome protein. The kit extraction calibration curve is y=0.0013×+0.0087, and r 2 = 0.9902; the dilution was 50-fold at 41.333. Mu.g/mL, the original concentration was 2.067. Mu.g/. Mu.L (FIG. 6C), and the exosome protein concentration was low. Therefore, the exosome extraction method adopted by the application has higher yield.
3.4Western Blot detection results
The results show that exosomes extracted by the extraction method of the application (fig. 7A), general ultracentrifugation method (fig. 7B) and kit method (fig. 7C) all expressed specific surface markers Alix, TSG101 and CD63.
Aiming at the defects of low purity and small acquisition amount during exosome extraction, the application optimizes the selection and culture conditions of exosome parent cells, the filtration, rotor selection, rotating speed and the like during exosome super-separation on the basis of adopting an ultracentrifugation classical extraction method.
The results of the verification of the present verification embodiment show that assigning an inherent weight to an indication may moderately improve the performance of the present method relative to the default settings.
It will be clear to those skilled in the art that, for convenience and brevity of description, specific working procedures of the above-described systems, apparatuses and units may refer to corresponding procedures in the foregoing method embodiments, which are not repeated herein.
In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices, and methods may be implemented in other manners. For example, the apparatus embodiments described above are merely illustrative, e.g., the division of the units is merely a logical function division, and there may be additional divisions when actually implemented, e.g., multiple units or components may be combined or integrated into another system, or some features may be omitted or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed with each other may be an indirect coupling or communication connection via some interfaces, devices or units, which may be in electrical, mechanical or other form.
The units described as separate units may or may not be physically separate, and units shown as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In addition, each functional unit in the embodiments of the present invention may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit. The integrated units may be implemented in hardware or in software functional units.
Those of ordinary skill in the art will appreciate that all or part of the steps in the various methods of the above embodiments may be implemented by a program to instruct related hardware, the program may be stored in a computer readable storage medium, and the storage medium may include: read Only Memory (ROM), random access Memory (RAM, random Access Memory), magnetic or optical disk, and the like.
Those of ordinary skill in the art will appreciate that all or a portion of the steps in implementing the methods of the above embodiments may be implemented by a program to instruct related hardware, where the program may be stored in a computer readable storage medium, where the storage medium may be a read only memory, a magnetic disk or optical disk, etc.
While the foregoing describes a computer device provided by the present invention in detail, those skilled in the art will appreciate that the foregoing description is not meant to limit the invention thereto, as long as the scope of the invention is defined by the claims appended hereto.
Claims (10)
1. An exosome collection device, the device comprising:
The cell filtering module is used for filtering the supernatant fluid of the exosomes to obtain the filtered supernatant fluid;
The first centrifugal module comprises a first centrifugal rotor and a first centrifugal tube which are matched; centrifuging the supernatant subjected to the filtering treatment in the first centrifuge tube at a first rotating speed N1 by utilizing the first centrifugal rotor to obtain supernatant subjected to the first centrifugation;
The second centrifugal module comprises a second centrifugal rotor and a second centrifugal tube which are matched; the supernatant after the first centrifugation is contained in a second centrifuge tube, and the supernatant in the second centrifuge tube is centrifuged at a second rotating speed N2 by utilizing the second centrifuge rotor, so that a precipitate after the second centrifugation is obtained;
and the exosome treatment module is used for treating the precipitate after the second centrifugation to obtain an exosome.
2. The exosome collection apparatus according to claim 1, wherein the cell filtration module comprises: a first filtration module and a second filtration module; sequentially processing the supernatant of the exosome by the first filtering module and the second filtering module to obtain filtered supernatant;
optionally, the first filtering module includes: a cell sieve of 100 μm;
Optionally, the second filtering module includes: cell sieves of 0.22 μm.
3. The exosome collection apparatus according to claim 1, further comprising, prior to the cell filtration module: a third centrifugation module for removing cells from the cell culture broth; the third centrifugation module is used for centrifuging the cell culture solution at a third rotating speed N3 to obtain the supernatant of the exosome;
optionally, when the unit of the third rotation speed N3 is g, N3 is a positive number greater than or equal to 200, and preferably 300;
Optionally, when the unit of time of the third rotation speed N3 centrifugation is min, the centrifugation time is a positive number greater than or equal to 6.
4. The exosome collection device according to claim 1, wherein when the unit of the first rotational speed N1 is g, N1 is a positive number greater than or equal to 8000, preferably 10000;
optionally, centrifuging at the first rotational speed N1 for at least 30 minutes;
Optionally, when the unit of the second rotation speed N2 is g, N1 is a positive number greater than or equal to 80000, preferably 100000;
Optionally, the time of centrifugation at the second rotational speed N2 is at least 90min.
5. The exosome collection apparatus according to claim 1, further comprising, after the cell filtration module and before the first centrifugation module: a fourth centrifugation module that centrifigates the filtered supernatant at a fourth rotational speed N4;
optionally, when the unit of time of the fourth rotation speed N4 for centrifugation is min, the centrifugation time is a positive number greater than or equal to 15, and preferably 20.
6. The exosome collection apparatus according to claim 1, wherein the processing method for processing the sediment after the second centrifugation comprises: re-suspending the precipitate by using PBS to obtain a mixed solution; centrifuging the mixture to obtain a separated supernatant and a precipitate; the separated precipitate is the exosome;
Optionally, the separated precipitate is resuspended by using PBS, and the mixture after resuspension is the exosome.
7. A method of exosome collection, the method comprising:
s1: filtering the exosome supernatant to obtain a filtered supernatant;
s2: centrifuging the supernatant subjected to the filtration treatment in a first centrifuge tube at a first rotational speed N1 by using a first centrifugal rotor to obtain a supernatant subjected to the first centrifugation;
s3: centrifuging the supernatant after the first centrifugation in a second centrifuge tube at a second rotational speed N2 by using a second centrifugal rotor to obtain a precipitate after the second centrifugation;
s4: and treating the precipitate after the second centrifugation to obtain an exosome.
8. The exosome collection method according to claim 1, wherein the method of filtering the exosome supernatant in S1 comprises: s11: obtaining exosome supernatant; s12: filtering the supernatant of the exosomes by using a 100 μm cell sieve to obtain a supernatant after the first filtration; s13: filtering the supernatant after the first filtering by using a cell sieve of 0.22 mu m to obtain the supernatant after the filtering treatment;
optionally, before the step S1, the method further includes: obtaining a cell culture solution; centrifuging the cell culture fluid at a third rotational speed N3 to obtain the exosome supernatant;
alternatively, the cell culture broth is derived from a culture medium.
9. The exosome collection method according to claim 1, further comprising, between S1 and S2: centrifuging the supernatant after the filtration treatment at a fourth rotation speed N4.
10. An exosome collection device, the device comprising: a memory and a processor; the memory is used for storing program instructions; the processor being adapted to invoke program instructions, which when executed, are adapted to carry out the steps of the method according to any of claims 7-9.
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