CN112980795A - Separation and purification method for obtaining exosome with specified particle size by using flow cytometry sorting instrument - Google Patents
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Abstract
The invention discloses a separation and purification method for obtaining exosomes with specified particle sizes by using a flow cytometry sorter, which comprises the steps of sorting the exosomes by using the flow cytometry sorter, using a 50-micron air jet nozzle, wherein the pressure of sheath fluid is 75-80.5psi, and the pressure difference between a sample flow and the sheath fluid is 0.3-0.6 psi; the threshold is set to 0.01; calibrating positions of exosome particles with the particle sizes of 50nm, 100nm, 200nm and 500nm by using polystyrene beads; and separating and purifying the exosome particles with the particle size of 50-200nm by using a flow cytometer. The method can obtain exosome with the particle size of 50-200nm, has smaller particle size range, does not contain other particle size ranges, and has high purity.
Description
Technical Field
The invention belongs to the field of acquisition of nano biological particles, and particularly relates to a separation and purification method for acquiring exosomes with specified particle sizes by using a flow cytometry sorter.
Background
Cells release Extracellular Vesicles (EVs) of different sizes, and are increasingly paid more attention by researchers because signal molecules carried by the EVs and membrane contents play an important role in cell-cell communication. According to literature reports (Exosomes and Ectosomes in Intercellular Communication; 2019 cell-response of Exosome Composition), the role of outer vesicles of different sizes is also different, wherein Small Extracellular Vesicles (SEVS) with 50-150nm derived from endosomes can accurately partition RNA, DNA and protein out of the correct cells and determine their secretion mechanism, which is crucial for identifying biomarkers and designing future drug intervention measures.
In addition, physical parameters such as particle size and concentration are one of the main indexes for evaluating exosome separation, and with the continuous research of exosome in clinical application, higher requirements are provided for exosome separation methods and detection means.
The prior separation methods commonly used include ultracentrifugation, size exclusion, polymer precipitation and the like, but the particle size parameters of exosomes obtained by different separation and purification methods are different. The prior literature indicates that the exosome particles extracted by the ultracentrifugation method are mainly between 40 and 500nm, and the particle sizes are distributed; the exosome particles extracted by the size exclusion method are mainly between 40 and 120nm, and are mostly small-particle-size particles; the exosomes extracted by the polymer precipitation method are mainly distributed between 40 and 100nm, only a few exosomes are distributed above 100nm, and the number of particles with large particle size is small. The methods can not obtain the extracellular vesicles with the particle size of 50-200nm (including 50-150nm), and the development of downstream experiments is influenced to a certain extent, so that the purification and separation of the extracellular vesicles with specific particle size intervals are an urgent problem to be solved.
The flow cytometer can quantitatively characterize cells and biological particles with cell sizes, the incident laser beams irradiate the cells or the particles in the liquid flow system one by one, and after the scattered light and other fluorescence signals are received by the high-sensitivity detector, multi-parameter analysis is completed, such as the shape and the size of the particles. For particles with the same refractive index and suspension media thereof, the scattered light is generally related to the particle size, and the scattered light range of the existing sensitive flow cytometer is generally between 300-500 nm. However, exosomes, due to their small particle size, are usually within their background noise and cannot be analyzed and sorted by conventional methods.
Disclosure of Invention
In view of the above situation, the present invention provides a separation and purification method for obtaining exosomes with specified particle size by using a flow cytometer, in order to overcome the defects of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a separation and purification method for obtaining exosomes with specified particle sizes by using a flow cytometry sorter comprises the following steps:
(1) setting instrument parameters and selecting accessories: exosome sorting was performed using a MoFlo Astrios EQ flow cytometric sorter using a 50 μm air Jet nozzle (Jet-in-air), sheath fluid pressure was 75-80.5psi, and the difference between the sample stream pressure and sheath fluid pressure was 0.3-0.6 psi; the threshold is set to 0.01;
in flow sorting, different sheath fluid pressures are required for different sized air jet nozzles to maintain stable fluid flow, and with the present invention, a 50 μm nozzle can maintain stable fluid flow only at sheath fluid pressures greater than or equal to 80 psi.
The pressure difference between the sample flow and the sheath fluid determines the sorting sample loading speed, under the set condition of the invention (the pressure difference between the sample flow and the sheath fluid is 0.3-0.6psi), the liquid flow is stable, and the sorting efficiency can reach a higher level due to the sample loading speed. If other values are set, the above two aspects are affected.
(2) Calibrating positions of exosome particles with the particle sizes of 50nm, 100nm, 200nm and 500nm by using polystyrene beads;
(3) and separating and purifying the exosome particles with the particle size of 50-200nm by using a flow cytometer.
If the exosomes with other particle sizes are separated and purified, the set instrument parameters are the same as the parameters, such as the air injection nozzle, the pressure of sheath fluid, the pressure of sample flow and the like, and only the range of the gate is changed to circle out the required particle range.
Further, before the step (1), an exosome mixture with different particle sizes is prepared by an ultracentrifugation method.
Further, in step (1), instrument parameters are set, and the forward angle (FSC) and the lateral angle (SSC) use logarithmic (log) parameters.
Further, in the step (3), a diluent of polystyrene beads is used, wherein the dilution ratio is 1: 1000.
Further, in step (1), the sheath fluid pressure is set to 80 psi.
Further, before the step (1), a step of obtaining an exosome mixed liquor is also included.
Further, the step of obtaining an exosome mixture comprises: and obtaining an exosome mixed solution from the cell supernatant by an ultracentrifugation method.
Further, the step of obtaining an exosome mixture comprises:
when the fusion degree of the P4 generation PANC-1 cells is 75%, rinsing the PANC-1 cells by using a phosphate buffer solution with the concentration of 0.01M and the pH value of 7.2-7.4, adding a DMEM culture medium containing 10% by volume of fetal bovine serum without exosomes, culturing at 37 ℃ for 48 hours, collecting cell supernatant, and obtaining an exosome mixed solution from the cell supernatant by an ultracentrifugation method:
centrifuging at 4 deg.C and 300 Xg for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 70min, collecting precipitate, washing the precipitate with PBS buffer solution, centrifuging at 100000 Xg for 70min, collecting precipitate, and resuspending with PBS buffer solution to obtain exosome mixture.
The invention has the beneficial effects that:
(1) the invention utilizes the flow cytometry sorting instrument to rapidly separate and detect the exosome, and can prepare the exosome with the specified particle size. The method adopts a flow cytometry sorter to separate and purify exosomes with specified particle size, such as exosomes with particle size of 50-200 nm; the particle size of the separated and purified exosome is verified by a particle size meter, so that the bottleneck problem of research on obtaining exosomes with uniform particle size and specified particle size can be solved.
(2) The method can obtain exosome with the particle size of 50-200nm, has smaller particle size range, does not contain other particle size ranges, and has high purity.
(3) The minimum value of the particle size which can be detected by the fluorescent microspheres for the flow cytometer adopted by the invention reaches 70nm, and particles larger than 50nm can be obtained by using electronic noise as a boundary.
(4) The separation and purification method of the invention can not only obtain exosomes with the particle size of 50-200nm, but also obtain exosomes with other particle size ranges by applying the method; if the exosomes in other particle size ranges are separated and purified, the set instrument parameters are the same as the parameters (such as air injection nozzles, the pressure of sheath fluid, the pressure of sample flow and the like), and only the range of the trap is changed, so that the range of the required particles is trapped.
Drawings
FIG. 1 is a graph showing a sorting baseline of polystyrene beads with an electronic noise of 50nm, and polypropylene beads showing 100nm, 200nm and 300nm, respectively.
FIG. 2 is a diagram showing the sorting of mixed suspensions of exosomes of different particle sizes obtained by ultracentrifugation.
FIG. 3 is a graph showing the particle size distribution of exosomes obtained by ultracentrifugation (before sorting).
Figure 4 validation of exosome particle size after sorting.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments provided in the present application without any inventive step are within the scope of protection of the present application.
Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the specification. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of ordinary skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments without conflict.
Unless defined otherwise, technical or scientific terms referred to herein shall have the ordinary meaning as understood by those of ordinary skill in the art to which this application belongs. Reference to "a," "an," "the," and similar words throughout this application are not to be construed as limiting in number, and may refer to the singular or the plural. The present application is directed to the use of the terms "including," "comprising," "having," and any variations thereof, which are intended to cover non-exclusive inclusions; reference to "connected," "coupled," and the like in this application is not intended to be limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. Reference herein to "a plurality" means greater than or equal to two. "and/or" describes an association relationship of associated objects, meaning that three relationships may exist, for example, "A and/or B" may mean: a exists alone, A and B exist simultaneously, and B exists alone. Reference herein to the terms "first," "second," "third," and the like, are merely to distinguish similar objects and do not denote a particular ordering for the objects.
Reagents, instruments, and the like used in the following examples are commercially available. The PBS refers to: phosphoric acid buffer solution with concentration of 0.01M and pH of 7.2-7.4.
Example 1 culture of human pancreatic cancer cells PANC-1
Human pancreatic cancer cells PANC-1 were derived from pancreatic cancer ductal cells, purchased from cell bank of Chinese academy of sciences (catalog number: TCTU 98), cultured adherent to DMEM medium (brand: Corning Cellgro, catalog number: 10-013-CV) containing 90% volume fraction fetal bovine serum, at 37 ℃ with a volume fraction of 5% carbon dioxide.
Example 2 isolation and particle size distribution detection of exosomes derived from human pancreatic cancer cell PANC-1
When the fusion degree of the P4 generation PANC-1 cells was about 75%, the PANC-1 cells were rinsed 2 times with PBS, and DMEM medium containing 10% by volume of exosome-free fetal bovine serum (brand: SURR, catalog number: SURR 050QY) (specifically, 75cm was added2The cell culture flask of (3) was filled with 30ml of DMEM medium, brand: Corning Cellgro, catalog No.: 10-013-CV), culturing at 37 ℃ for 48h, collecting 80mL of cell supernatant, and obtaining an exosome mixed solution from the cell supernatant by an ultracentrifugation method:
centrifuging at 4 deg.C and 300 Xg for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 70min, collecting precipitate, washing the precipitate with 10mL PBS, centrifuging at 100000 Xg for 70min, collecting precipitate, and resuspending with 200mL PBS to obtain exosome suspension.
The exosome weight suspension was put into a particle sizer for particle size distribution testing (brand: Malvern, model: Zetasizer Nano ZS 90) and found to have a particle size range between 0-400nm (as shown in figure 3).
Example 3 flow sorting and particle size distribution detection of exosomes derived from human pancreatic cancer cells PANC-1
In this example, the exosome heavy suspension prepared in example 2 was subjected to flow sorting (using a flow cytometric sorter, brand: Beckman Coulter, model: Moflo Astrios EQ). In some embodiments, other methods may be used to prepare the exosome mixture, which is then separated and purified using a flow sorter. In other embodiments, the cell supernatant is collected and the separation and purification process is used to obtain exosomes of a specified particle size using a flow cytometric sorter.
In this embodiment, the separation and purification method for obtaining exosomes with specified particle size by using a flow cytometer includes the following steps:
(1) debugging and setting a sorting scheme of the flow cytometer: the droplet delay was manually adjusted using a 50 μm air jet nozzle, sheath fluid pressure 80psi, sample stream pressure 80.3psi, and held constant throughout the experiment.
In this embodiment, the frequency and amplitude of droplet oscillation are adjusted to stabilize the liquid flow, obtain a certain droplet delay (droplet delay value is 25 ± 5), and maintain the droplet delay for a long time to perform sorting.
The 50 μm air jet nozzle was chosen to minimize sorting droplets, so that sorted exosomes achieved a certain concentration requirement, which was beneficial for downstream experiments.
The threshold is set at 0.01 and the Forward (FSC) and lateral (SSC) angles use logarithmic (log) parameters. In the experiment of utilizing flow cytometer separation purification exosome, the setting of threshold is equivalent to setting detectable exosome particle size, and the bigger the threshold is, the bigger the detectable exosome particle is, and the exosome that the particle is little can be cut off by the threshold, unable detection, sets up the threshold to 0.01 in this embodiment, can make the particle that is greater than electronic noise all detected.
(2) A baseline plot of the different particle sizes (50nm, 100nm, 200nm, 300nm) was drawn using a polystyrene bead Photon Correlation Spectroscopy (PCS) control mixing kit (cat # 6602336, Beckman Coulter) (as shown in FIG. 1). The polystyrene beads were diluted 1000 times with ultrapure water.
In FIG. 1, R represents the gate of the flow chart, i.e., the range of the circle, R5 represents the position of the fluorescent microsphere with 200nm, R4 represents the position of the fluorescent microsphere with 100nm, R6 represents the position of the fluorescent microsphere with 300nm, R7 represents the electronic noise, and R8 represents the position of the fluorescent microsphere with 50-200 nm.
(3) Exosome solutions of the specified particle size (50-200nm) were sorted from the exosome mixture according to a baseline plot (as shown in R8 in figure 2). In FIG. 2, R represents the gate of the flow chart, i.e., the range of the circle, R5 represents the position of the fluorescent microsphere with 200nm, R4 represents the position of the fluorescent microsphere with 100nm, R6 represents the position of the fluorescent microsphere with 300nm, R7 represents the electronic noise, and R8 represents the position of the fluorescent microsphere with 50-200 nm.
(4) And (5) verifying the particle size range of the exosomes obtained by sorting by using a particle size analyzer.
The exosomes obtained after the flow sorting were put into a particle sizer for particle size distribution detection (laser particle sizer, brand: Malvern, model: Zetasizer Nano ZS 90), and the particle size range of the exosomes was found to be between 50-200nm (as shown in fig. 4).
It should be understood by those skilled in the art that various features of the above-described embodiments can be combined in any combination, and for the sake of brevity, all possible combinations of features in the above-described embodiments are not described in detail, but rather, all combinations of features which are not inconsistent with each other should be construed as being within the scope of the present disclosure.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application.
Claims (7)
1. A separation and purification method for obtaining exosomes with specified particle sizes by using a flow cytometry sorter is characterized by comprising the following steps of:
(1) setting instrument parameters and selecting accessories: using a flow cytometric sorter for exosome sorting, using a 50 μm air jet nozzle, the pressure of the sheath fluid being 75-80.5psi, the pressure difference between the sample stream pressure and the sheath fluid pressure being 0.3-0.6 psi; the threshold is set to 0.01;
(2) calibrating positions of exosome particles with the particle sizes of 50nm, 100nm, 200nm and 500nm by using polystyrene beads;
(3) and separating and purifying the exosome particles with the particle size of 50-200nm by using a flow cytometer.
2. The method according to claim 1, wherein logarithmic parameters are used for the forward angle and the lateral angle when setting the instrument parameters in step (1).
3. The method for separating and purifying exosomes with specified particle size according to claim 1, wherein in the step (3), a diluent of polystyrene beads is used, wherein the dilution factor is 1: 1000.
4. The method according to claim 1, wherein in step (1), the pressure of the sheath fluid is set to 80 psi.
5. The method for separating and purifying exosomes with specified particle size according to claim 1, which is characterized by further comprising the step of obtaining exosome mixed liquor before the step (1).
6. The method for separating and purifying exosomes with specified particle size according to claim 5, wherein the step of obtaining exosome mixed liquor comprises the following steps: and obtaining an exosome mixed solution from the cell supernatant by an ultracentrifugation method.
7. The method for separating and purifying exosomes with specified particle size according to claim 6, wherein the step of obtaining exosome mixed liquor comprises the following steps:
when the fusion degree of the P4 generation PANC-1 cells is 75%, rinsing the PANC-1 cells by using a phosphate buffer solution with the concentration of 0.01M and the pH value of 7.2-7.4, adding a DMEM culture medium containing 10% by volume of fetal bovine serum without exosomes, culturing at 37 ℃ for 48 hours, collecting cell supernatant, and obtaining an exosome mixed solution from the cell supernatant by an ultracentrifugation method:
centrifuging at 4 deg.C and 300 Xg for 10min, and collecting supernatant; centrifuging at 2000 Xg for 10min, and collecting supernatant; centrifuging at 10000 Xg for 30min, and collecting supernatant; centrifuging at 100000 Xg for 70min, collecting precipitate, washing the precipitate with PBS buffer solution, centrifuging at 100000 Xg for 70min, collecting precipitate, and resuspending with PBS buffer solution to obtain exosome mixture.
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