Method for evaluating exosome by counting exosome nanoparticles

Abstract

The invention relates to the technical field of biological medicines, in particular to a method for evaluating exosomes by counting exosome nanoparticles. According to the nanoparticle counting scheme for evaluating the exosome, exosome samples of different batches in the same preparation method or exosome samples of exosome products of the same batch treated by different methods can be directly evaluated through a membrane rupture particle ratio according to parameters of nanoparticle number change caused by exosome vesicle rupture; besides, the particle number of the exosome sample before and after treatment and before and after membrane rupture is measured, the preservation rate of the treated exosome is calculated according to the measured result, and finally, the treatment effect can be quantitatively evaluated according to the preservation rate of the treated exosome, so that the treated exosome can be more effectively evaluated; therefore, quality detection and quality control are carried out.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for evaluating exosomes by counting exosome nanoparticles. Background Art

[0002] Extracellular vesicles refer to particles released by cells, wrapped in a lipid bilayer, and unable to self-replicate (without a functional nucleus). In the description of extracellular vesicle subpopulations, there is a class of terms based on assumed biogenesis processes, such as exosomes and ectosomes. Exosomes refer to extracellular vesicles released from the inside of cells through multivesicular bodies (MVBs), and ectosomes refer to extracellular vesicles formed by budding from the cell membrane. Current separation technologies make it difficult to enrich extracellular vesicles produced by different mechanisms, and the characterization of the corresponding extracellular vesicle subpopulations is also difficult to achieve.

[0003] Exosomes, commonly referred to as small extracellular vesicles, are very small, with a diameter generally less than 200nm. They are mainly composed of lipids and proteins. In addition to proteins, a variety of nucleic acids are found in their contents, including DNA, mRNA, microRNA and ncRNA. Almost all cells can secrete exosomes, and exosomes exist naturally in body fluids, including blood, saliva, urine, cerebrospinal fluid and breast milk. Exosomes can be secreted by a variety of cells and body fluids in the human body, including endothelial cells, immune cells, platelets, smooth muscle cells, etc. When they are secreted from host cells into receptor cells, exosomes can regulate the biological activity of receptor cells through the proteins, nucleic acids, lipids, etc. they carry.

[0004] Exosomes have very important biological functions and are a hot topic in life science research, especially the research and application of stem cell exosomes. Exosome nanoparticle counting is the most commonly used method to evaluate exosomes, which is generally obtained by detecting and calculating the number of particles with a diameter of 30-200nm (or 30-150nm). However, after the exosomes are frozen or freeze-dried, the exosome particle count is found to increase instead of decrease. This is because after preservation or other treatments, some exosome vesicles rupture and release the contents, which increases the particle count of 30-200nm (or 30-150nm), resulting in errors in exosome counting. The current counting or evaluation method cannot solve the changes of exosomes before and after certain treatments (such as different storage conditions, freeze-drying or preparations), nor is it conducive to quantitative horizontal comparison after different storage conditions or different treatments. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for evaluating exosomes by counting exosome nanoparticles. The method provided by the present invention can more effectively evaluate the treated exosomes, thereby performing quality detection and quality control.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0008] The exosome sample to be evaluated is perforated, and the number of particles in the exosome sample before and after perforation is measured, which are recorded as N t and N tr ; The particle size of the particles is 30-200nm or 30-150nm;

[0009] The membrane-broken particle ratio of the exosome sample to be evaluated was calculated according to formula I:

[0010] R t =N tr / N t Formula I;

[0011] Among them, R t is the ratio of membrane-broken particles, N tr N is the number of particles in the exosome sample to be evaluated after membrane rupture. t is the number of particles in the exosome sample to be evaluated before membrane rupture;

[0012] The exosome samples to be evaluated include any one of 1) to 3): 1) exosome samples from different batches using the same preparation method, 2) exosome samples from the same batch of exosome products treated by different methods, 3) treated exosome samples that need to be quantitatively evaluated;

[0013] When the exosome sample to be evaluated is 1), the larger the membrane-broken particle ratio is, the higher the purity of the exosomes is or the better the integrity is;

[0014] When the exosome sample to be evaluated is 2), the larger the membrane-breaking particle ratio is, the lower the loss rate of exosomes after treatment is, and the better the treatment effect is;

[0015] When the exosome sample to be evaluated is 3), the following steps are also included:

[0016] The exosome sample before treatment is subjected to membrane rupture, and the number of particles in the exosome sample before treatment and after membrane rupture is determined; the particle size of the particles is 30-200 nm or 30-150 nm; the exosome sample before treatment is an exosome sample from the same batch as the exosome sample to be evaluated before treatment;

[0017] The preservation rate of the treated exosome samples was calculated according to Formula II and Formula III:

[0018] P=(N t-cal -N r ) / (NN r )×100% Formula II;

[0019] N t-cal =N t ×(N tr / N r ) Formula III;

[0020] Among them, P is the preservation rate of the processed exosome sample, N t-cal is the calibrated N t Value, N t is the number of particles in the exosome sample to be evaluated before membrane rupture, N is the number of particles in the exosome sample before treatment before membrane rupture, and N r N is the number of particles after the membrane of the exosome sample was ruptured before treatment. tr is the number of particles after membrane rupture of the exosome sample to be evaluated;

[0021] The protective effect of the treatment on exosomes was quantitatively evaluated based on the preservation rate of the treated exosome samples. The greater the preservation rate of the treated exosomes, the lower the loss rate of the treated exosomes, and the better the protective effect of the treatment on exosomes.

[0022] Preferably, the treatment comprises one or more of storage treatment under different conditions, freeze-drying treatment and purification treatment.

[0023] Preferably, the determination method comprises an exosome nanoparticle counting method.

[0024] Preferably, the method for counting exosome nanoparticles is NTA nanoparticle counting.

[0025] Preferably, the reagent used for membrane disruption includes: tissue and / or cell lysis solution.

[0026] Preferably, the tissue and / or cell lysate is RIPA lysate.

[0027] Preferably, the membrane rupture time is ≥25 min.

[0028] Preferably, when the treatment methods are ≥2, the preservation rates of exosomes after different treatments are calculated using Formula II and Formula III, respectively.

[0029] Preferably, the loss rate = 100% - P.

[0030] The present invention provides an application of the method described in the above technical solution in exosome quality detection and / or quality control.

[0031] Beneficial effects:

[0032] The present invention proposes a scheme for evaluating the counting of nanoparticles of exosomes. By using the parameter of the change in the number of nanoparticles caused by the rupture of exosome vesicles, the exosome samples from different batches of the same preparation method, or the exosome samples from the same batch of exosome products treated by different methods can be directly evaluated through the ruptured particle ratio; in addition, by measuring the number of particles of the exosome samples before and after the treatment and before and after the rupture of the membrane, and calculating the preservation rate of the treated exosomes according to the measurement results, the treatment effect can be quantitatively evaluated according to the preservation rate of the treated exosomes, so that the treated exosomes can be evaluated more effectively, thereby performing quality inspection and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0034] Figure 1 It is the particle size and concentration distribution result of exosome NSCE240612F1 in Example 1;

[0035] Figure 2 The ruptured particle size and concentration distribution results of exosome NSCE240612F1 in Example 1;

[0036] Figure 3 It is the particle size and concentration distribution result of exosome NSCE240612F1DG in Example 1;

[0037] Figure 4 The ruptured particle size and concentration distribution results of exosome NSCE240612F1DG in Example 1. DETAILED DESCRIPTION

[0038] The present invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0039] The exosome sample to be evaluated is perforated, and the number of particles in the exosome sample before and after perforation is measured, which are recorded as N t and N tr ; The particle size of the particles is 30-200nm or 30-150nm;

[0040] The membrane-broken particle ratio of the exosome sample to be evaluated was calculated according to formula I:

[0041] R t =N tr / N t Formula I;

[0042] Among them, R t is the ratio of membrane-broken particles, Ntr N is the number of particles in the exosome sample to be evaluated after membrane rupture. t is the number of particles in the exosome sample to be evaluated before membrane rupture;

[0043] The exosome samples to be evaluated include any one of 1) to 3): 1) exosome samples from different batches using the same preparation method, 2) exosome samples from the same batch of exosome products treated by different methods, 3) treated exosome samples that need to be quantitatively evaluated;

[0044] When the exosome sample to be evaluated is 1), the larger the membrane-broken particle ratio is, the higher the purity of the exosomes is or the better the integrity is;

[0045] When the exosome sample to be evaluated is 2), the larger the membrane-breaking particle ratio is, the lower the loss rate of exosomes after treatment is, and the better the treatment effect is;

[0046] When the exosome sample to be evaluated is 3), the following steps are also included:

[0047] The exosome sample before treatment is subjected to membrane rupture, and the number of particles in the exosome sample before treatment and after membrane rupture is determined; the particle size of the particles is 30-200 nm or 30-150 nm; the exosome sample before treatment is an exosome sample from the same batch as the exosome sample to be evaluated before treatment;

[0048] The preservation rate of the treated exosome samples was calculated according to Formula II and Formula III:

[0049] P=(N t-cal -N r ) / (NN r )×100% Formula II;

[0050] N t-cal =N t ×(N tr / N r ) Formula III;

[0051] Among them, P is the preservation rate of the processed exosome sample, N t-cal is the calibrated N t Value, N t is the number of particles in the exosome sample to be evaluated before membrane rupture, N is the number of particles in the exosome sample before treatment before membrane rupture, and N r N is the number of particles after the membrane of the exosome sample was ruptured before treatment. tr is the number of particles after membrane rupture of the exosome sample to be evaluated;

[0052] The protective effect of the treatment on exosomes was quantitatively evaluated based on the preservation rate of the treated exosome samples. The greater the preservation rate of the treated exosomes, the lower the loss rate of the treated exosomes, and the better the protective effect of the treatment on exosomes.

[0053] In the present invention, the number of exosome nanoparticles is N, and the average number of nanoparticles of each exosome after membrane rupture is kN, where k is the membrane rupture coefficient. Generally, exosomes from the same batch have the same k value, and the same number of exosomes from the same batch have the same number of nanoparticles after membrane rupture.

[0054] As an embodiment, when the exosome samples to be evaluated are exosome samples from different batches prepared by the same method, the present invention performs membrane rupture treatment on the exosome samples to be evaluated, thereby destroying the membrane structure of the exosomes and releasing the contents, and counting the nanoparticles before and after membrane rupture, and calculating the membrane rupture particle ratio. The same number of exosomes from the same batch have the same number of nanoparticles after membrane rupture. If some exosome vesicles have ruptured before membrane rupture, and the more ruptured exosomes are, the more nanoparticles are released, and the membrane rupture particle ratio is smaller, so that the treatment effect of the exosomes can be evaluated according to the membrane rupture coefficient.

[0055] As an embodiment, the reagent used for membrane disruption includes: tissue and / or cell lysis solution.

[0056] As an embodiment, the tissue and / or cell lysate is RIPA lysate.

[0057] As an embodiment, the membrane rupture time is ≥25 min.

[0058] As an embodiment, the determination method may be an exosome nanoparticle counting method; as another embodiment, the exosome nanoparticle counting method is NTA nanoparticle counting. As an embodiment, a nanoparticle tracking analyzer NanosightNS300 may be used to count nanoparticles to obtain a particle concentration of exosomes with a diameter within a specified range (30-200nm or 30-150nm), and the total exosome count result is the nanoparticle concentration × exosome sample volume.

[0059] As an implementation mode, when the exosome samples to be evaluated are exosome samples treated by different methods from the same batch of exosome products, since the exosomes from the same batch have the same membrane rupture coefficient, after different treatments, the more exosomes are lost and the more nanoparticles are released, then the higher the nanoparticle count of the exosome sample before membrane rupture after treatment, the smaller the membrane rupture particle ratio (according to formula I, at this time the membrane rupture particle ratio is equal to the ratio of the nanoparticle count after membrane rupture after treatment to the nanoparticle count before membrane rupture after treatment); therefore, the smaller the membrane rupture particle ratio of the exosome sample, the higher the loss rate of exosomes after treatment, and the worse the protective effect of the treatment method on exosomes, so that the protective effect of different treatment methods on exosomes can be evaluated only based on the membrane rupture coefficient.

[0060] As an implementation method, the loss rate = 100% - P.

[0061] As an embodiment, the treatment includes one or more of storage treatment under different conditions, freeze-drying treatment and purification treatment.

[0062] As an embodiment, the present invention can also quantitatively evaluate the processed exosome samples, that is, quantitatively evaluate the preservation rate or loss rate of the processed exosome samples, such as evaluating the effect of freezing the same exosome product at -80°C for a certain period of time. Freezing at -80°C may cause the rupture of exosome vesicles and the loss of exosomes. At this time, only counting the nanoparticles of the exosomes cannot evaluate the preservation effect of the exosomes at this temperature.

[0063] Calculate the preservation rate or loss rate of exosomes under this treatment condition: Assume that the number of exosome nanoparticles before treatment (cryopreservation) is N, and the number of exosome nanoparticles after exosome membrane rupture is N r ;

[0064] After processing (freezing), the number of exosome nanoparticles is N t , after exosome membrane rupture, N tr ;

[0065] Assuming the preservation rate of exosomes is P, then according to the definition of membrane rupture coefficient k, k is equal to the ratio of the total number of nanoparticles before and after membrane rupture in the same exosome, that is, k = N r / N;

[0066] After processing (cryopreservation), the total number of exosome particles preserved without rupture of the vesicles is N × P;

[0067] After treatment (freezing), the exosomes with ruptured vesicles, i.e., the lost exosomes, are N(1-P);

[0068] After treatment (freezing), the total number of nanoparticles in the exosomes with ruptured vesicles is N(1-P)×k; then after treatment (freezing), the total number of exosome nanoparticles N t=N×P+N(1-P)×k;

[0069] After calculation, we get: P = (N t -N r ) / (N-kN)=(N t -N r ) / (NN r )×100%.

[0070] In general, the results of nanoparticle counting for the same batch, the same volume or the same number of exosomes before and after treatment should be consistent, that is, N tr =N r , then there is no need to obtain N tr ; However, due to the existence of errors, it is possible that N tr ≠N r , then we can make an approximate evaluation as needed (ignore N tr Results), you can also use N tr To calibrate N t The value of N after calibration t The value is N t-cal , considering factors such as exosome volume error or different volumes or amounts of exosomes from the same batch used in different treatments, N t-cal =N t ×(N tr / N r ), after calculation, we get the formula: P = (N t-cal -N r ) / (N-kN)=(N t-cal -N r ) / (NN r )×100%.

[0071] Then the loss rate of exosomes is 100%-P.

[0072] The method provided by the present invention is mainly applicable to the evaluation of the loss effect or protective effect of different treatments (such as storage, freeze-drying or purification, etc.) on exosomes.

[0073] As an embodiment, when the treatment methods are ≥2, Formula II and Formula III can also be used to calculate the preservation rate of exosomes after different treatments, so as to quantitatively evaluate the effects of different treatments.

[0074] Based on the above advantages, the present invention provides an application of the method described in the above technical solution in exosome quality detection and / or quality control.

[0075] In order to further illustrate the present invention, a method for evaluating exosomes by counting exosome nanoparticles provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0076] Example 1

[0077] The present invention provides a method for evaluating exosomes by counting exosome nanoparticles, comprising the following steps:

[0078] processing the exosomes to obtain processed exosomes;

[0079] The exosomes before treatment and the exosomes after treatment are subjected to membrane permeation operations respectively, to obtain four samples, namely, the exosome sample before treatment, the exosome sample after treatment, the exosome membrane permeation sample before treatment, and the exosome membrane permeation sample after treatment;

[0080] The number of particles in the four samples was determined by using an exosome nanoparticle counting method, and the particle size of the particles was 30-200 nm or 30-150 nm;

[0081] The preservation rate of exosomes after treatment was calculated according to formula II and formula III:

[0082] P=(N t-cal -N r ) / (NN r )×100% Formula II;

[0083] N t-cal =N t ×(N tr / N r ) Formula III;

[0084] Among them, P is the preservation rate of exosomes after treatment, N t-cal is the calibrated N t Value, N t is the number of particles in the exosome sample after treatment, N is the number of particles in the exosome sample before treatment, and N r N is the number of particles in the exosome rupture sample before treatment. tr is the number of particles in the exosome ruptured sample after treatment;

[0085] The treatment effect was evaluated based on the preservation rate of exosomes after treatment. The higher the preservation rate of exosomes after treatment, the lower the loss rate of exosomes after treatment, and the better the protective effect of the treatment on exosomes.

[0086] Example 2

[0087] 1. Preparation of exosome concentrate: 10L of human neural stem cell culture supernatant (i.e. neural stem cell conditioned medium, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.) was collected and 3D The vivaEXO exosome harvesting system (manufacturer: Huakan Bio) was used to concentrate and purify the exosome concentrate, and the product was stored at -80°C.

[0088] 2. Preparation of lyophilized exosome product NSCE240612F1DG:

[0089] Prepare exosome concentrate according to the method in step 1, add lyophilization protectant (PBS solution containing final concentration of 3% trehalose and 12% mannitol), mix well and dispense into vials, 2 ml / bottle, take 1 bottle marked as NSCE240612F1 for sampling and inspection (the rest are stored at -80°C); the remaining bottles are freeze-dried using a vacuum freeze dryer (Boyikang Pilot5-8ES), and after freeze-drying, they are capped and taken out, marked as NSCE240612F1DG for sampling and inspection, and the products are stored at 2-8°C.

[0090] 3. Exosome membrane rupture

[0091] 1) Reagents used: High-efficiency RIPA tissue / cell lysis buffer, with a tube of PMSF (0.3mL / 1.5mL), brand: Solarbio, item number: R0010.

[0092] 2) Reagent preparation: According to the usage amount, add 10 μL PMSF to every 1 mL RIPA to make the final concentration of PMSF 1 mM, mix well and set aside.

[0093] 3) Membrane rupture and inspection:

[0094] Take NSCE240612F1 prepared in step 2, 100 μL / tube, take 2 tubes, and perform the following treatment:

[0095] Take one tube and add PBS buffer at a volume ratio of 1:1 (NSCE240612F1 sample), take another tube and add 100 μL of the prepared high-efficiency RIPA tissue / cell lysis buffer at a volume ratio of 1:1, mix well, and lyse on ice for 25 minutes (NSCE240612F1 membrane rupture sample), and send it to Heyuan Biotechnology (Shanghai) Co., Ltd. for NTA nanoparticle counting.

[0096] Take the exosome lyophilized powder NSCE240612F1DG prepared in step 2, reconstitute it with 2 mL of sterile water for injection, mix thoroughly, and obtain an exosome lyophilized powder solution. Take 2 vials at 100 μL / vial and perform the following treatments:

[0097] Take one tube and add PBS buffer at a volume ratio of 1:1 (NSCE240612F1DG sample), and take another tube and add 100 μL of the prepared high-efficiency RIPA tissue / cell lysis buffer at a volume ratio of 1:1, mix well, and lyse on ice for 25 minutes (NSCE240612F1DG membrane rupture sample), and send it to Heyuan Biotechnology (Shanghai) Co., Ltd. for NTA nanoparticle counting.

[0098] The results are shown in Table 1 and Figure 1 to Figure 4 .

[0099] Table 1 NTA nanoparticle counting results of different samples

[0100] batch number Particle size, percentage concentration NSCE240612F1 30-200nm, 94.5% <![CDATA[4.36×10 8 particles / mL]]> NSCE240612F1 membrane rupture 30-200nm, 98.7% <![CDATA[7.73×10 9 particles / mL]]> NSCE240612F1DG 30-200nm, 90.1% <![CDATA[8.25×10 8 particles / mL]]> NSCE240612F1DG membrane rupture 30-200nm, 91.5% <![CDATA[6.11×10 9 particles / mL]]>

[0101] According to the method of Example 1, after the exosomes are freeze-dried, the calibrated N t The value is N t-cal :

[0102] N t-cal =N t ×(N tr / N r )=6.52×10 8 ;

[0103] Exosome preservation rate P = (N t-cal -N r ) / (NN r )×100%=97%;

[0104] Exosome loss rate 100% - P = 3%.

[0105] In summary, the method provided by the present invention can more effectively handle exosome evaluation, thereby performing quality detection and quality control.

[0106] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for evaluating exosomes by counting exosome nanoparticles, characterized in that: The following steps are involved: The exosome sample to be evaluated is perforated, and the number of particles in the exosome sample before and after perforation is measured, which are recorded as N t and N tr ; The particle size of the particles is 30-200nm or 30-150nm; The membrane-broken particle ratio of the exosome sample to be evaluated was calculated according to formula I: R t =N tr / N t Formula I; Among them, R t is the ratio of membrane-broken particles, N tr N is the number of particles in the exosome sample to be evaluated after membrane rupture. t is the number of particles in the exosome sample to be evaluated before membrane rupture; The exosome samples to be evaluated include any one of 1) to 3): 1) exosome samples from different batches using the same preparation method, 2) exosome samples from the same batch of exosome products treated by different methods, 3) treated exosome samples that need to be quantitatively evaluated; When the exosome sample to be evaluated is 1), the larger the membrane-broken particle ratio is, the higher the purity of the exosomes is or the better the integrity is; When the exosome sample to be evaluated is 2), the larger the membrane-breaking particle ratio is, the lower the loss rate of exosomes after treatment is, and the better the treatment effect is; When the exosome sample to be evaluated is 3), the following steps are also included: The exosome sample before treatment is subjected to membrane rupture, and the number of particles in the exosome sample before treatment and after membrane rupture is determined; the particle size of the particles is 30-200 nm or 30-150 nm; the exosome sample before treatment is an exosome sample from the same batch as the exosome sample to be evaluated before treatment; The preservation rate of the treated exosome samples was calculated according to Formula II and Formula III: P=(N t-cal -N r ) / (NN r )×100% Formula II; N t-cal = N t × (N tr / N r ) Equation Ⅲ; Among them, P is the preservation rate of the processed exosome sample, N t-cal is the calibrated N t Value, N t is the number of particles in the exosome sample to be evaluated before membrane rupture, N is the number of particles in the exosome sample before treatment before membrane rupture, and N r N is the number of particles after the membrane of the exosome sample was ruptured before treatment. tr is the number of particles after membrane rupture of the exosome sample to be evaluated; The protective effect of the treatment on exosomes was quantitatively evaluated based on the preservation rate of the treated exosome samples. The greater the preservation rate of the treated exosomes, the lower the loss rate of the treated exosomes, and the better the protective effect of the treatment on exosomes.

2. The method according to claim 1, characterized in that The treatment includes one or more of storage treatment under different conditions, freeze-drying treatment and purification treatment.

3. The method according to claim 1, characterized in that The determination method includes an exosome nanoparticle counting method.

4. The method according to claim 1 or 3, characterized in that: The method for counting exosome nanoparticles is NTA nanoparticle counting.

5. The method according to claim 1, characterized in that The reagents used for membrane disruption include: tissue and / or cell lysis solution.

6. The method according to claim 5, characterized in that The tissue and / or cell lysate is RIPA lysate.

7. The method according to claim 1 or 5, characterized in that: The membrane rupture time is ≥25min.

8. The method according to claim 1, characterized in that When the treatment methods are ≥2, the preservation rates of exosomes after different treatments are calculated using Formula II and Formula III, respectively.

9. The method according to claim 1, characterized in that: The loss rate = 100% - P.

10. Use of the method according to any one of claims 1 to 9 in exosome quality detection and / or quality control.

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