CN216513818U - Exosome separation system - Google Patents
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Abstract
The present invention provides an exosome-separating system comprising 1) a first dead-end filtration device for removing a cell component having a particle size larger than that of an exosome from a cell culture supernatant comprising the exosome; 2) a first vessel connected to the first dead-end filtration device, which contains filtrate filtered by the dead-end filtration device; and 3) a tangential flow filtration device for cyclically concentrating the filtrate in the first vessel while removing a cellular fraction having a particle size smaller than the particle size of the exosomes. The exosome separation system can be used for large-scale separation of exosomes, and solves the problems of complex exosome separation operation, small sample size and poor repeatability in the prior art.
Description
Technical Field
The present invention relates to exosome separation systems, in particular process systems for large-scale exosome separation.
Background
The Exosomes (Exosomes) are secreted by living cells, and have a size of 30-200nm and a density of 1.131.18g/ml of vesicular bodies with a bilayer membrane structure. The exosome carries various bioactive substances (such as protein, lipid, miRNA and the like), so the exosome secreted by cells can replace cells to play a role in repairing and treating various important organ and tissue injuries. Moreover, the application of exosomes avoids multiple potential risks of direct cell transplantation, has low immunogenicity, is convenient to store and transport, and shows unique advantages as a cell-free cell therapy technology. Because of their unique transport properties, exosomes have also been investigated as drug delivery vehicles, such as delivering RNA for gene therapy. As a delivery platform, exosomes are in great demand, and 10 is needed to perform experiments on only one animal (e.g., mouse)9-1011The large-scale production and the large-scale separation of the exosomes are important technologies for the clinical transformation of the exosomes.
The method is a precondition for wide application of exosome, and high-purity, high-yield and standardized exosome is obtained. However, there is no unified standard for the method for separating and purifying exosomes, which severely restricts the basic research and clinical application related to exosomes. At present, various methods for separating exosome, such as ultracentrifugation, density gradient centrifugation, immune separation, and polymerization precipitation, have been used, but all have certain limitations. Ultracentrifugation is the most commonly used method, considered as the "gold standard" for exosome extraction, but requires the provision of expensive ultracentrifuges, and is complex to operate, time-consuming, low in yield, difficult to process large-volume samples on a large scale; the immune separation method can only specifically enrich and express exosomes of one or more specific surface proteins, expensive reagents such as antibodies and the like are needed, and large-volume samples are difficult to process on a large scale; the exosome obtained by the polymerization precipitation method is usually mixed with impurities such as protein, polymer and the like, the purity is low, and the activity of the exosome cannot be guaranteed, so that the subsequent analysis is limited to a certain extent. Therefore, in order to accelerate the application of the exosome, it is important to develop a device for separating and purifying the exosome on a large scale.
Disclosure of Invention
In order to solve the above problems, the present invention provides an exosome-separating system comprising:
1) a first dead-end filtration device for removing a cell fraction having a particle size larger than the particle size of the exosomes from a cell culture supernatant comprising the exosomes;
2) a first vessel connected to the first dead-end filtration device, which contains filtrate filtered by the dead-end filtration device; and
3) tangential flow filtration means for cyclically concentrating said filtrate in said first vessel while removing cellular components having a particle size smaller than the particle size of said exosomes.
In some embodiments, the first dead-end filtration device comprises at least two dead-end filters connected in series.
In some embodiments, at least one of the dead-end filters is a capsule filter having a pore size of 0.22 μm.
In some embodiments, the tangential flow filtration device comprises a hollow fiber column with a molecular weight cut-off of 100 to 750 kD.
In some embodiments, the inlet and outlet of the tangential flow filtration device are each connected to a lower portion of the same side of the first vessel.
In some embodiments, the exosome separation system further comprises, a first peristaltic pump for inputting cell culture supernatant comprising the exosomes to the first dead-end filtration device; a second peristaltic pump for inputting the filtrate in the first container to the tangential flow filtration device.
In some embodiments, the exosome-separation system further comprises a second vessel connected to the permeate port of the tangential flow filtration device for receiving permeate from the tangential flow filtration device.
In some embodiments, the exosome-separation system further comprises a third vessel connected to the first vessel for containing the concentrated filtrate comprising the exosomes.
In some embodiments, the exosome-separation system further comprises a second dead-end filtration device for performing sterile filtration, intermediate the first container and the third container.
In some embodiments, the exosomes have an average particle size of 30-200 nm.
The process system for large-scale separation of exosomes provided by the utility model solves the problems of complex operation, small sample size, poor repeatability and incapability of being used for large-scale preparation of exosomes in the prior art.
Drawings
FIG. 1 is a schematic structural diagram of an exosome-isolating system of the present invention.
FIG. 2 is a structural diagram of one embodiment of the exosome-isolation system of the present invention.
FIG. 3 is a graph showing the proliferation of cultured cells over time.
FIG. 4 is an electron micrograph of isolated exosomes. (A) Electron micrographs of exosomes isolated from example 2Day4 cell supernatant; (B) example 3 electron micrograph of isolated exosomes; (C) example 4 electron micrograph of isolated exosomes; (D) example 5 electron micrograph of isolated exosomes; (E) example 6 electron micrograph of isolated exosomes; (F) example 7 electron micrograph of isolated exosomes by ultracentrifugation.
Figure 5 is a schematic of the particle size distribution of isolated exosomes. (A) Figure of particle size distribution of exosomes isolated on different days of culture in example 2; (B) example 3 schematic of the particle size distribution of isolated exosomes; (C) example 4 schematic of the particle size distribution of isolated exosomes; (D) example 5 schematic of the particle size distribution of isolated exosomes; (E) example 6 schematic of the particle size distribution of isolated exosomes; (F) example 7 schematic of the particle size distribution of isolated exosomes. The particle size (nm) is plotted on the abscissa, and the concentration (number of particles/mL) is plotted on the ordinate.
FIG. 6 is an electrophoresis diagram of protein marker expression of exosomes isolated in example 4.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
"dead-end filtration," also known as "vertical filtration" or "full-scale filtration," refers to the process in which a solvent and a solute (e.g., small particulate matter) smaller than the filtration pores are driven under pressure through the filtration pores of a filtration device (the direction of flow being generally perpendicular to the surface of the filtration membrane), while particles larger than the filtration pores are retained and typically retained in the filtration device, e.g., deposited on the surface of the filtration membrane. The pressurization or vacuum can provide the required pressure for filtration. In the filtration process, as the number of particles accumulated on the surface of the filtration membrane increases, the filtration resistance increases and the filtration rate decreases. Thus, dead-end filtration is intermittent and typically requires periodic stopping to clean the fouling layer on the membrane surface or to replace the filtration membrane.
"tangential flow filtration," also known as "cross-flow filtration," refers to filtration in which the liquid flow direction is at a substantially perpendicular angle to the filtration direction, i.e., the liquid flow direction is substantially parallel to the surface of the filter membrane (i.e., tangential flow), and the filtrate flow direction is substantially perpendicular to the surface of the filter membrane. The tangential flow can effectively clean the surface of the filtering membrane, and the intercepted substances on the surface are washed away, so that the filtering membrane can be restored to maintain the filtering capacity. The tangential flow filtration has flexible operation mode, and can be operated intermittently or continuously.
Where particle size is mentioned, for example, reference to a cell component having a particle size greater than the particle size of the exosomes means that the mean particle size of the cell component is greater than the mean particle size of the exosomes, for example, the mean particle size of the cell component may be 1.5 times, 2 times, 3 times, 4 times or more greater than the mean particle size of the exosomes, or that the particle size of the majority of the cell component is greater than the mean particle size of the exosomes, for example, at least 60%, 70%, 80% or at least 90% of the cell component has a particle size greater than the mean particle size of the exosomes. Similarly, the meaning of a cellular component or particulate matter having a particle size smaller than the particle size of the exosomes can be readily understood by the person skilled in the art. Since the particle size of the exosomes to be isolated and their distribution range may vary from one secretory cell to another (or from cell state to cell state), the pore size of the filtration device used herein may also be adjusted accordingly.
"removing" cellular components from a stock solution (e.g., cell culture supernatant) by filtration refers to separating these cellular components from the exosomes to be isolated. One skilled in the art will appreciate that removing certain cellular components by filtration means that these cellular components are completely removed, but are reduced in their presence, for example by an amount of at least 50%, at least 80%, at least 90%, or at least 95%, although a 100% reduction may be desirable.
The exosome separation methods and process systems provided herein combine dead-end filtration with tangential flow filtration to achieve large-scale exosome separation.
Fig. 1 shows a schematic structure of an exosome-separation system provided herein, whose main components include multiple dead-end filters, one tangential flow filter, multiple containers, and multiple peristaltic pumps, connecting tubes and tube clamps, etc.
As shown in fig. 1, a first dead-end filter 1 is connected in series with a second dead-end filter 2, through which a stock solution including exosomes, such as cell culture supernatant, is sequentially filtered under the action of a peristaltic pump 3 to remove larger cell debris and large cell vesicles. The filtered raw liquor enters the intermediate product container 5 through an opening 5a in the intermediate product container 5. The outlet 5b of the intermediate product container 5 is connected to the inlet 4a of the tangential flow filter 4 and the liquid in the intermediate product container 5 is pumped into the tangential flow filter 4 by means of the peristaltic pump 8. The tangential flow filter 4 may be a hollow fiber column (PUR) having a filter pore size smaller than the exosome particle size, capable of removing water and other small molecule impurities. The outlet 4b of the tangential flow filter 4 is connected to a further inlet 5c of the intermediate product container 5, so that liquid flowing in a direction substantially parallel to the tangential flow filter membrane surface is returned to the intermediate product container 5 via the outlet 4b of the tangential flow filter 4, while permeate flowing through the pores of the tangential flow filter membrane flows as waste liquid via the permeate port 4c into the waste liquid container 6. The liquid returning to the intermediate product container 5 is re-introduced into the tangential flow filter 4 by means of the peristaltic pump 8, circulating for tangential flow filtration. Due to the concentration effect of tangential flow filtration, the liquid volume in the intermediate product container 5 decreases and the exosome concentration increases, stopping tangential flow filtration after the desired volume is reached. The intermediate product container 5 is provided with a further outlet 5d connected to the product container 7. The product container 7 is intended to contain a prepared liquid comprising exosomes, i.e. an exosome product.
The containers and the filter can be connected by connecting pipes (such as flexible pipes, for example, silicone pipes). The hose may be equipped with a hose clamp (e.g., a robert clamp) to close or open the fluid flow, and the hose and the inlet of the container may be connected by a luer fitting.
When the exosome separation system provided herein is used for exosome preparation, impurities (whole cells, larger cell fragments, organelles, particulate matter, etc.) having a particle size larger than that of exosomes are first removed from a stock solution (e.g., cell culture supernatant including exosomes) by a first dead-end filter 1 and a second dead-end filter 2, and then impurities (e.g., small cell membrane fragments, proteins, etc.) having a particle size smaller than that of exosomes and a part of water are removed by circulation filtration through a tangential flow filter 4, allowing the exosomes to be concentrated (e.g., 30-50 times concentrated), thereby obtaining a high-concentration exosome product.
In some embodiments, the first dead-end filter 1 and the second dead-end filter 2 have different filter pore sizes, forming a multi-stage filtration, e.g. the first dead-end filter 1 has a filter pore size of 1 μm for removing larger cell debris and large cell vesicles; the second dead-end filter 2 has a filter pore size of 0.22 μm and is used to remove microvesicles of 200nm or more. Depending on the total amount of the dope to be treated and the amount of impurities, in some embodiments, only one dead-end filter may be used; in other embodiments, three or more dead-end filters connected in series may be used. In some embodiments, at least one of the dead-end filters is a bag filter (surface area, e.g., 0.2-0.4 m)2) (ii) a In other embodiments, the dead-end filters are all capsule filters. For venting or pressure control purposes, the first dead-end filter 1 and/or the first dead-end filter 2 can be provided with a closable vent, respectively, which can be connected to the environment via the disposable filter membranes 10 and 11, respectively.
In some embodiments, the inlet 5c and the outlet 5b of the intermediate product container 5 are located on the same side of the intermediate product container 5. More preferably, the inlet 5c and the outlet 5b of the intermediate product container 5 are located on the same side of the intermediate product container 5 and are arranged in a lower position (i.e. lower, e.g. close to the bottom surface of the container) of the intermediate product container 5. This arrangement keeps both the inlet 5c and the outlet 5b of the intermediate product container 5 below the liquid level therein to prevent the generation of a large amount of bubbles.
In some embodiments, three- way valves 12 and 13 may be provided between the connecting line between the outlet 5b of the intermediate product container 5 and the inlet 4a of the tangential flow filter 4 and between the outlet 4b of the tangential flow filter 4 and the inlet 5c of the intermediate product container 5 to facilitate cleaning or sterilization of the intermediate product container 5 and the tangential flow filter 4 with a cleaning or sterilizing agent (e.g., NaOH). In some embodiments, a pressure sensor 14 may be provided on the connecting line between the outlet 5b of the intermediate product container 5 and the inlet 4a of the tangential flow filter 4 to monitor the pressure conditions within the tangential flow filter 4. In some embodiments, a pressure regulating valve 15 is provided on the connecting line between the outlet 4b of the tangential flow filter 4 and the inlet 5c of the intermediate product container 5 for regulating the inlet pressure of the tangential flow filter 4.
In some embodiments, the tangential flow filter 4 comprises a hollow fiber column. In one embodiment, the molecular weight cut-off is 100-750kD, and the surface area is 0.4-1m2Polyether sulfone PES material is adopted. In one embodiment, peristaltic pump 8 rotates at 450rpm to deliver liquid from intermediate product container 5 to tangential flow filter 4 at a corresponding flow rate of 1520mL/min, maintaining the pressure indicated by the pressure sensor ≈ 0.2 bar.
In some embodiments, the intermediate product container 5 may be a pouch container, i.e. an intermediate product pouch. In the preparation of the exosome product, the intermediate product bag is placed obliquely with the outlet 5b and inlet 5c to one side of the intermediate product bag and the lower position keeping it below the liquid level prevents the generation of a large amount of air bubbles. Similarly, the waste liquid container 6 and the product container 7 may be pouch containers.
In some embodiments, a further dead-end filter 9 is provided on the connection of the outlet 5d of the intermediate product container 5 to the product container 7 for sterile filtration by means of a peristaltic pump.
Fig. 2 is a specific example of an exosome-isolation system provided herein. As shown in fig. 2, the first and second dead-end filters 1 and 2 connected in series, the tangential flow filter 4, the peristaltic pumps 3 and 8, and the like are contained in one tank 16. The cabinet 16 is also provided with a door 17, a cabinet drawer 18 and a touch screen 19. In this particular example, the intermediate product container 5, the waste liquid container 6, and the product container 7 are each a pouch container, wherein the intermediate product container 5 and the product container 7 are located inside the cabinet drawer 18, and the waste liquid container 6 is located outside the cabinet. The weighing device 20 may be used to weigh the total weight of the waste container to determine the concentration or concentration of exosomes in the intermediate product container 5. The touch screen 19 can display the operating status of the process system, control process parameters such as the speed of the peristaltic pump, etc.
Provided herein are a method and a separation system (multi-stage filtration system) for large-scale preparation of exosomes, in order to solve the problems of complex operation, small sample size, poor reproducibility, and inability to be used for large-scale preparation of exosomes in the prior art. In some embodiments, a microcarrier is used for three-dimensional cell culture, cell supernatant is harvested, two-stage filtration is performed to remove large-particle impurities and proteins in the cell supernatant, exosome is circularly concentrated through a hollow fiber membrane with a proper pore size and material, small-molecule protein impurities are removed, and finally exosome solution is obtained. In general, the technical solution provided herein can be divided into two parts: large-scale preparation of exosomes and large-scale isolation of exosomes.
The separation methods and separation systems provided herein are further illustrated by the following specific examples.
Example 1:
the purpose is as follows: obtaining a large-volume culture supernatant through three-dimensional culture of MSCs so as to prepare exosome on a large scale subsequently.
The method comprises the following brief operation steps: umbilical cord mesenchymal stem cells are added at a ratio of 1.5X 108Inoculation into 7g of 3D
In the microtubes, day D0 of inoculationSupplementing to 3L with serum-free medium, inoculating D1 supplemented medium to 5L on day1, three-dimensionally culturing for 5 days, sampling and counting cells every day, collecting supernatant of the culture medium, and placing in four-degree refrigerator at 4L. The proliferation of cells obtained by cell technology is shown in FIG. 3. The cell proliferation number is increased along with the increase of the culture days, and the total cell number reaches 1.98 multiplied by 10 on the 5 th day of culture9And (4) respectively.
Example 2:
the purpose is as follows: evaluation MSCs three-dimensional culture increased with the number of culture days, and exosomes were harvested consecutively for analysis of secreted exosome changes.
The method comprises the following brief operation steps:
a. preparing a microcarrier: gently grasp 20 micro-slides using flat-head forcepsTM(20 mg/piece) was put into a 500mL air-permeable culture flask with built-in impeller (hereinafter referred to as "flask"), 40mL of complete medium was taken and added to the flask along the side arm port of the flask, and the flask was shaken to place microslipsTMDispersing in culture medium for use;
b. cell preparation: every 20 micro-slidesTM10mL of cell suspension was prepared at a density of 1.2X 108Each cell/mL for standby;
c. cell inoculation: uniformly mixing the cell suspension, adding the cell suspension (total 10mL) into the culture along a side arm sample port of a culture bottle, supplementing 150mL of complete culture medium, ensuring the final volume to be 200mL, and screwing a bottle cap;
d. cell culture: transfer of culture flasks to pre-installed 3D FloTrixTMSetting the stirring speed to 35rpm by a touch screen controller at the bottle position center of a stirring table of the miniSpin bioreactor for culture;
e. supplementing 100ml of complete culture medium 24h (Day1) after inoculation, wherein the final volume is 300ml, and the rotating speed is adjusted to 40 rpm;
f. pumping 250ml of cell supernatant by using a peristaltic pump at Day4, 6 and 8 respectively, preserving at-80 ℃, and simultaneously supplementing 250ml of fresh complete culture medium;
day10 Total cell supernatants were collected and stored at-80 ℃.
h. Before exosome separation, cell supernatants harvested by Day4, 6, 8 and 10 were re-lysed at 4 ℃ and exosomes were extracted by ultracentrifugation, briefly as follows: taking a proper amount of cell culture supernatant, centrifuging at 300g and 4 ℃ for 10min, and removing the precipitate; centrifuging the supernatant at 2000g and 4 ℃ for 10min, and discarding the precipitate again; centrifuging the supernatant at 4 ℃ for 30min at 10000g, and transferring the supernatant into an ultracentrifuge tube; centrifuging the supernatant 100000g at 4 deg.C for 90min, and discarding the supernatant; adding 1ml PBS for heavy suspension precipitation to prepare an exosome premix; transferring the exosome premix into a centrifuge tube, performing ultracentrifugation for 90min at 100000g and 4 ℃, discarding the supernatant, and adding a proper amount of PBS (injection: the whole process is performed at 4 ℃) to re-suspend to obtain an exosome extract.
Example 3:
after umbilical cord mesenchymal stem cells were cultured in three dimensions for 4d, cell supernatants (prepared according to example 1) were collected for use in the isolation of purified exosomes in this example as well as in the examples below.
The purpose is as follows: using the exosome separation system provided herein, the effect of cobert deep paperboard in exosome separation was evaluated
The method comprises the following brief operation steps:
a. filtering 500ml of three-dimensional cell supernatant with 0.04-9.0 μm deep paperboard (CDFCDCSD 0140PCP) at flow rate of 25 ml/min;
b. c, further filtering the cell supernatant obtained after filtration in the step a by using a 0.04-0.4 mu m (Corbert, CDFCDCSD0102PCP) deep paperboard at the flow rate of 25 ml/min;
c. the cell supernatant from step b was then concentrated to 20-30ml by Tangential Flow Filtration (TFF) at a flow rate of 100ml/min, a shear force of 3psi, and a treatment time of about 3.5 h. Wherein the hollow fiber membrane is made of Polyethersulfone (PES) and has a surface area of 75cm2The inner diameter of the fiber is 1mm, the molecular weight cut-off is 500kd, and the obtained solution is the exosome extracting solution.
Example 4:
the purpose is as follows: using the exosome separation system provided herein, the effect of a cobott-capsule filter in exosome separation was evaluated.
The method comprises the following brief operation steps:
a. 500ml of three-dimensional cell supernatant is firstly processed by 1 mu m PES capsuleFilter (surface area 180 cm)2Koppet, C01BBPAFPA1P) at a flow rate of 25 ml/min;
b. the cell supernatant filtered in step a was passed through a 0.22 μm PES capsule filter (surface area 180 cm)2Kobaite, C01BBSLENA1P) was further filtered at a flow rate of 25 ml/min;
c. the cell supernatant filtered in step b was then concentrated to 20-30ml by TFF at a flow rate of 100ml/min, a shear force of 3psi and a treatment time of about 3.5 h. Wherein the hollow fiber membrane (REPLIGEN, D02-E500-10-N) is PES with a surface area of 75cm2The inner diameter of the fiber is 1mm, the molecular weight cut-off is 500kd, and the obtained solution is the exosome extracting solution.
Example 5:
the purpose is as follows: using the exosome separation system provided herein, the effect of shanghai coronary eurocyst filters in exosome separation was evaluated.
The method comprises the following brief operation steps:
a. three-dimensional cell supernatant 3.75L was first passed through a1 μm PES bag filter (surface area 2000 cm)2Shanghai Guanghai Europe purification science and technology Limited, KSL-2H2H-5-S1) at a flow rate of 200 ml/min;
b. the cell supernatant filtered in step a was passed through a 0.22 μm PES capsule filter (surface area 2000 cm)2Shanghai Guanghai Europe purification science and technology Limited, KSL-2H2H-5-S2Y) at a flow rate of 200 ml/min;
c. the cell supernatant filtered in step b was then concentrated to 130ml by TFF at a flow rate of 650ml/min and a shear force of 3psi for about 2 h. Wherein the hollow fiber membrane (REPLIGEN, S04-E300-10-N) is PES with a surface area of 0.1m2The inner diameter of the fiber is 1mm, the molecular weight cutoff is 300kd, and the obtained solution is the exosome extracting solution.
Example 6:
the purpose is as follows: using the exosome separation system provided herein, the effect of Shandong Bora biological hollow fiber membranes in exosome separation was evaluated
The method comprises the following brief operation steps:
a. 3.75L of the three-dimensional cell supernatant passes through a1 μm PES bag filter(surface area 2000cm2Shanghai Guanghai Europe purification science and technology Limited, KSL-2H2H-5-S1) at a flow rate of 200 ml/min;
b. the cell supernatant filtered in step a was passed through a 0.22 μm PES capsule filter (surface area 2000 cm)2Shanghai Guanghai Europe purification science and technology Limited, KSL-2H2H-5-S2Y) at a flow rate of 200 ml/min;
c. the cell supernatant filtered in step b was then concentrated to 130ml by TFF, with a shear of 3psi and a treatment time of about 0.5 h. Wherein the hollow fiber membrane (US-300-500K, Bona Biotechnology group, Inc. in Shandong) is PES with a surface area of 0.4m2The inner diameter of the fiber is 1mm, the molecular weight cut-off is 500kd, and the obtained solution is the exosome extracting solution.
Example 7 (comparative):
the purpose is as follows: exosomes were isolated by conventional ultracentrifugation methods for comparison to exosomes obtained by the isolation methods provided herein.
The method comprises the following brief operation steps: taking 40ml MSCs three-dimensional culture supernatant, extracting exosome by ultracentrifugation (see example 2 for ultracentrifugation)
Example 8:
exosomes extracted by the different methods in the above examples were identified and the results are as follows.
8.1 Electron microscopy of exosomes
Exosomes were observed by negative staining. The brief method is as follows: the sealing film is cut and paved on a fume hood experiment table, a drop of sample, pure water and 2% uranyl acetate are respectively dripped on the sealing film, then a copper sheet is covered on the sample for 2min, the copper sheet is sucked dry on filter paper, the filter paper is washed on the pure water, the filter paper is sucked dry, the copper sheet is placed on the uranium drop for 2min, the uranium is sucked dry by the filter paper, after the copper mesh is dried, the copper mesh is placed in a sample box, and an electron microscope picture is taken under 80 kV. The transmission electron microscope model used is as follows: h-7650 (Hitachi).
FIGS. 4A-4F are electron micrographs of exosomes prepared in examples 2-7 (where the example 2 exosome was the Day4 exosome), respectively. The classical structure of the exosome can be observed by the exosome separated in each embodiment under an electron microscope.
8.2 exosome particle size distribution and concentration analysis
Exosomes isolated in examples 3, 4, 5 and 6 were diluted 50-fold, exosomes isolated in example 2 and example 7 (comparative) were diluted 400-fold, and the particle concentration was 1 × 108-5×109particles/mL range, detected using a nanoparticle tracking analyzer (Nanosight LM10, Malvern Panalytical). The results are shown in FIGS. 5A-5F.
FIG. 5A is a graph showing the particle size distribution of exosomes isolated from cell supernatants after culturing MSC4, 6, 8, and 10 days in example 2, in which the total number of particles isolated from 500ml of the supernatant was 4.13X 10, respectively12，3.4×1012，2.0×1012，2.16×1012The average particle diameters were 170.5, 208.3, 186.0, and 177.8nm, respectively.
FIG. 5B is a schematic diagram showing the particle size distribution of the exosomes isolated in example 3, the total number of particles being 4.37X 1011The average particle diameter was 181 nm.
FIG. 5C is a schematic diagram showing the particle size distribution of the exosomes isolated in example 4, with the total number of particles being 1.32X 1012The average particle size was 132.2 nm.
FIG. 5D is a schematic diagram showing the particle size distribution of the exosomes isolated in example 5, wherein the total number of particles obtained from 500ml supernatant isolation is 1.28X 1012The average particle diameter was 113.5 nm.
FIG. 5E is a schematic diagram of the particle size distribution of the exosomes isolated in example 6, wherein the total number of particles obtained from 500ml supernatant isolation is 9.36X 1011The average particle size was 123.8 nm.
FIG. 5F is a graph showing the particle size distribution of exosomes isolated in example 7, wherein the total number of particles obtained by 500ml supernatant (converted) isolation is 1.37X 1012The average particle size was 197.5 nm.
Comparing the number of exosome particles and the particle size of exosomes in each culture day of MSCs in example 2 (fig. 5A), it was found that the number of exosome particles was large and the average particle size was small in the supernatant of day4, so that MSCs were cultured for 4 days, and it was excellent to collect the supernatant and extract exosomes.
Example 3 (fig. 5B) compared to example 4 (fig. 5C), example 4 yielded a higher number of exosome particles, a smaller average particle size, and better example 4.
Example 4 (fig. 5C) compared to example 5 (fig. 5D), the exosome particle number did not differ much from the average particle size.
Example 4 (fig. 5C) compared to example 6 (fig. 5E), example 4 obtained a slightly larger number of exosome particles, with a comparable particle size.
Example 4 (fig. 5C) compared to example 7 (fig. 5F), the average exosome particle size was smaller and better in example 4.
8.3 exosome western blot detection
An appropriate amount of the exosome solution isolated in example 4 was taken, added with a loading buffer, and heated in a boiling water bath for 5min to fully denature the protein. Protein samples were added to wells of SDS-PAGE gels, and the protein samples were separated by 80V electrophoresis and subjected to membrane transfer. After the membrane conversion is finished, the membrane is soaked in a sealing solution for sealing for 1h with the protein surface facing upwards, then the membrane is incubated overnight at the temperature of 4 ℃ for one time, then an enzyme-labeled secondary antibody is used for incubation for 1h at room temperature, finally the protein membrane is placed in an ECL luminescent solution for dark reaction for 5min, and then the ECL luminescent solution is placed in a chemiluminescence imaging system for color development imaging (as shown in figure 6).
As can be seen from the above description of the examples, the capsule filtration-TFF method is a more preferable mode of the exosome separation and purification method, and the obtained exosome has clear structure, smaller particle size and higher concentration. In addition, the filter and the hollow fiber membrane used in the exosome separation system can be amplified in equal proportion, and are used for separating and purifying exosomes in cell supernatants with larger volumes.
Claims (10)
1. An exosome-separation system, comprising:
1) a first dead-end filtration device for removing a cell fraction having a particle size larger than the particle size of the exosomes from a cell culture supernatant comprising the exosomes;
2) a first vessel connected to the first dead-end filtration device, which contains filtrate filtered by the dead-end filtration device; and
3) tangential flow filtration means for cyclically concentrating said filtrate in said first vessel while removing cellular components having a particle size smaller than the particle size of said exosomes.
2. An exosome-separating system according to claim 1, in which the first dead-end filtering means comprises at least two dead-end filters connected in series.
3. An exosome-separating system according to claim 2, wherein at least one said dead-end filter is a capsule filter having a pore size of 0.22 μm.
4. The exosome separation system according to claim 1, wherein the tangential flow filtration device comprises a hollow fiber column with a molecular weight cut-off of 100-750 KD.
5. An exosome-separating system according to claim 1, wherein the inlet and outlet of the tangential flow filtration device are each connected to a lower portion of the same side of the first vessel.
6. An exosome-separating system according to claim 1, further comprising a first peristaltic pump for inputting cell culture supernatant comprising the exosomes to the first dead-end filtration device; a second peristaltic pump for inputting the filtrate in the first container to the tangential flow filtration device.
7. An exosome separating system according to claim 1, further comprising a second vessel connected to the permeate port of said tangential flow filtration device for containing permeate from said tangential flow filtration device.
8. An exosome separation system according to claim 1, further comprising a third vessel connected to the first vessel for containing the concentrated filtrate comprising the exosomes.
9. An exosome separation system according to claim 8, further comprising a second dead-end filtration device for performing sterile filtration intermediate the first vessel and the third vessel.
10. An exosome-separating system according to claim 1, in which the exosomes have an average particle size of 30-200 nm.
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| CN116555006A (en) * | 2023-06-14 | 2023-08-08 | 中山大学附属第一医院 | Continuous concentration, purification and extraction system and method for extracellular vesicles including exosomes |
| CN116555006B (en) * | 2023-06-14 | 2024-02-09 | 中山大学附属第一医院 | Continuous concentration, purification and extraction system and method for extracellular vesicles including exosomes |
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