CN114934010B

CN114934010B - Application of erythrocyte membrane in increasing exosome yield

Info

Publication number: CN114934010B
Application number: CN202210789268.2A
Authority: CN
Inventors: 杨国栋; 张荣鑫; 孙汶齐; 韦梦影; 袁丽君; 王辰; 李者龙; 卜特
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2023-06-27
Anticipated expiration: 2042-07-05
Also published as: CN114934010A

Abstract

The invention belongs to the technical field of exosome preparation, and particularly relates to application of erythrocyte membranes in increasing exosome yield. According to the invention, the erythrocyte membrane is added in the reagent for preparing the exosomes by culturing, the endocytosis of the erythrocyte membrane and the membrane component supplementation are utilized by the cells, the generation of the exosomes is promoted, the yield of the exosomes can be remarkably increased, the yield is approximately 7 times that of the exosomes prepared by adopting the conventional reagent without adding the erythrocyte membrane, and in addition, the loading efficiency, the delivery efficiency and the treatment effect of the exosomes are not different from those of the exosomes prepared by adopting the conventional culture medium.

Description

Application of erythrocyte membrane in increasing exosome yield

Technical Field

The invention belongs to the technical field of exosome preparation, and particularly relates to application of erythrocyte membranes in increasing exosome yield.

Background

Exosomes are nanoscale vesicles produced by cells, have good biocompatibility, can carry multiple bioactive substances and transfer information among cells, and therefore play an important role in drug delivery. As a drug delivery vehicle, a key problem faced in clinical transformation is low exosome production and difficulty in mass production to achieve clinical transformation.

Exosomes are endosomes that originate from the formation during endocytosis of cells. The cell membrane is sprouted inwards to form intracellular vesicles, the vesicles are combined with early endosomes in the cells to form a part of the early endosomes, the early endosomes are gradually matured into late endosomes under the action of various proteins and pH change, the late endosomes are continuously matured to form multi-vesicles (MVBs), the MVBs are invaginated in cavities to form intracavity vesicles (ILVs), and the ILVs are finally released when the MVB is fused with a plasma membrane, namely exosomes.

There are many reagents for preparing exosomes available, such as various commercially available kits that are continually improved, and are well established. However, the reagents for extracting and preparing exosomes in the prior art have problems that the exosome yield is low, or the loading efficiency or the delivery efficiency is low although the exosome yield is relatively high.

Disclosure of Invention

The invention aims to provide an application of erythrocyte membranes in increasing the output of exosomes, and the exosomes are prepared by adopting a reagent added with the erythrocyte membranes, so that the output of the exosomes can be obviously increased, and the loading efficiency, the delivery efficiency and the treatment effect of the exosomes are ensured.

The invention provides an application of erythrocyte membranes in increasing exosome yield.

The invention also provides an agent for increasing the yield of exosomes, wherein the agent comprises erythrocyte membranes and auxiliary materials acceptable in the field of agents.

Preferably, the mass volume ratio of the erythrocyte membrane to the auxiliary materials is 20-80 mug: 1mL.

Preferably, the mass volume ratio of the erythrocyte membrane to the auxiliary materials is 80 mug: 1mL.

Preferably, the particle size of the erythrocyte membrane is 0-400 nm.

Preferably, the reagent comprises a culture medium or a kit.

Preferably, the adjunct comprises a cell culture medium.

The invention also provides a method for increasing the yield of exosomes, which cultures exosome donor cells by adopting the reagent according to the technical scheme, and collects exosomes.

Preferably, the exosome donor cells are first cultured in the reagent for 12 hours, the resulting culture fraction is second cultured in exosome-free serum medium, and the exosomes are collected after 48 hours.

Preferably, the exosome donor cells comprise AmL12 cells.

The beneficial effects are that:

the invention provides an application of erythrocyte membrane in increasing exosome yield, which promotes exosome generation by adding erythrocyte membrane in reagent for preparing exosome by culturing and utilizing endocytosis of cells to erythrocyte membrane and membrane component supplementation. The reagent provided by the invention is used for culturing exosome donor cells, so that the yield of exosome can be obviously increased, and is approximately 7 times that of exosome prepared by adopting a conventional reagent without adding erythrocyte membranes.

Secondly, the erythrocyte membrane has wide sources, easy material obtaining, high safety, no numerous organelles, simple preparation work and all functions of the membrane; meanwhile, the erythrocyte membrane has very obvious similarity with the exosome membrane, has high biocompatibility and small influence on receptor cells, and can further solve the problems of exosome loading efficiency and delivery efficiency caused by increasing the yield of exosome. Therefore, the technical scheme provided by the invention is an effective method for solving the problem of low exosome yield, and provides an effective means for solving the bottleneck problem faced by the exosome drug delivery function.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIGS. 1 to 4 are graphs showing NTA detection results of exosomes prepared using the culture media of example 1, example 2, example 3 and control group, respectively;

FIG. 5 shows the results of an exocrine sign analysis, wherein Ctrl@Exo ^Ldlr And RCM@Exo ^Ldlr Representing untreated groups, control exosome load Ldlr and mass produced exosome load Ldlr, respectively;

FIGS. 6 to 8 show the results of three biological replicates of exosomes prepared using the medium of example 1;

FIGS. 9-11 are results of three biological replicates of exosomes prepared using the medium in the control group;

FIG. 12 is a graph showing the results of qPCR detection of the efficiency of exosome loading Ldlr in PBS, ctrl@Exo ^Ldlr And RCM@Exo ^Ldlr Respectively representing a control group, a control exosome load Ldlr group and a mass production exosome load Ldlr group;

FIG. 13 shows the result of red O staining of liver viscera, wherein the scale in the first row is 200 μm, the magnification is 5.0×, the scale in the second row is 50 μm, the magnification is 20.0×, PBS, ctrl@Exo from left to right ^Ldlr And RCM@Exo ^Ldlr Respectively representing a control group, a control exosome load Ldlr group and a mass production exosome load Ldlr group;

FIG. 14 shows the result of red O staining of common vascular artery with PBS, ctrl@Exo ^Ldlr And RCM@Exo ^Ldlr The untreated group, the control exosome-loaded Ldlr-treated group and the mass-produced exosome-loaded Ldlr-treated group are represented, respectively.

FIG. 15 shows the results of toxicity analysis by HE staining with PBS, ctrl@Exo ^Ldlr And RCM@Exo ^Ldlr The control group, the control exosome-loaded Ldlr group and the mass-produced exosome-loaded Ldlr group represent aortic root tissue, heart tissue, liver tissue, spleen tissue, lung tissue and kidney tissue in this order from top to bottom, respectively.

Detailed Description

The invention provides an application of erythrocyte membranes in increasing exosome yield. The invention promotes the generation of exosomes by adding erythrocyte membranes in the reagent and utilizing endocytosis of the erythrocyte membranes by the cells and membrane component supplementation. The reagent provided by the invention is used for culturing exosome donor cells, so that the yield of exosome can be obviously increased, and is approximately 7 times that of exosome prepared by adopting a conventional reagent without adding erythrocyte membranes.

The mass volume ratio of the erythrocyte membrane to the auxiliary materials in the reagent is preferably 20-80 mug: 1mL, more preferably 40 to 80. Mu.g: 1mL, more preferably 80 μg:1mL. The erythrocyte membrane of the present invention is preferably erythrocyte membrane fragment particles, and the particle diameter of the erythrocyte membrane fragment particles is preferably 0 to 400nm, more preferably 40 to 200nm, and even more preferably 150nm. The erythrocyte fragment particles with the particle size are easier to be endocytosed by cells, and promote the generation of exosomes. The reagent of the present invention preferably comprises a culture medium or a kit, more preferably comprises a culture medium. The adjuvants of the invention preferably comprise a cell culture medium. The type of the cell culture medium is not particularly limited, and the preparation of the exosomes is required to be regulated conventionally, and the reagent for increasing the yield of the exosomes is prepared by mixing the commercial conventional DMEM culture medium serving as an auxiliary material with erythrocyte membranes in the following examples of the invention, but cannot be considered as the whole protection scope of the invention.

The invention also provides a preparation method of the agent for increasing the yield of exosomes, which comprises the following steps: mixing the resuspended erythrocyte pellet solution with auxiliary materials to obtain the agent for increasing the exosome yield.

The auxiliary material of the present invention preferably comprises a conventional cell culture medium, more preferably comprises a DMEM medium or a 1640 medium, and even more preferably comprises a DMEM medium. The mixing mode is not particularly limited in the present invention, and a conventional mixing mode in the art may be adopted. The suspension of the present invention for preparing the suspension of erythrocyte pellet solution is preferably PBS buffer, more preferably 1 XPBS buffer. The method for extracting the erythrocyte membrane is not particularly limited, and the method can be used for preparing the erythrocyte membrane by adopting a conventional preparation method in the field.

The present invention preferably further comprises culturing the exosome donor cells to a density of 80% to 85%, more preferably to 80%, prior to culturing the exosome donor cells with the agent. The culture medium and conditions of the culture are not particularly limited, and conventional culture medium and culture conditions in the art can be adopted, such as DMEM culture medium is adopted in the embodiment of the invention, the culture temperature is 37 ℃, and CO ₂ Is of the body of (2)The integral percentage content is 5 percent. The exosome donor cells cultured to the density of 80-85% are better in state, the number of cells capable of endocytosis is more, and the yield of exosome can be increased in an auxiliary manner. The exosome donor cells of the invention preferably comprise AmL12 cells.

After culturing the exosome donor cells to a density of 80% -85%, the present invention preferably cultures the exosome donor cells cultured to a density of 80% -85% in the agent for increasing exosome yield for 12 hours to obtain a culture component. The ratio of the number volume of the exosome donor cells cultured to 80-85% by density to the reagent is preferably 8×10 ⁵ The following steps: 3mL. The temperature of the first culture according to the invention is preferably 37 ℃; CO ₂ Preferably 5% by volume.

After the culture component is obtained, the culture component is subjected to secondary culture in an exosome-free serum culture medium, and cell supernatants are collected after 48 hours.

After obtaining the cell supernatant, the invention preferably carries out centrifugation and filtration on the cell supernatant to obtain a filtrate, namely an exosome. The rotational speed of the centrifugal machine is preferably 1000-3000 rpm, more preferably 1000rpm or 3000rpm; the time of the centrifugation is preferably 10 to 20 minutes, more preferably 10 minutes or 20 minutes. The pore size of the filter for the filtration of the present invention is preferably 0.22 to 0.45. Mu.m, more preferably 0.22 μm or 0.45. Mu.m.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

The reagents and test methods used in the examples described below, unless otherwise specified, were those conventional in the art.

Example 1

1. A culture medium for increasing exosome production, comprising the following components:

240 μg of PBS-resuspended erythrocyte membrane particles were added to 3mL DMEM medium (Gibco, USA).

2.A method for preparing a culture medium for increasing the yield of exosomes, which comprises the following steps:

2.1 extraction of erythrocyte membranes

1) Anticoagulation with EDTA, centrifugation at 4℃at 3000rpm/min for 10min, collection of erythrocytes and washing with 1 XPBS for 5 times;

2) According to PBS: distilled water = 1:9, preparing a lysate by volume ratio, and standing the red blood cells obtained in the step 1) at 4 ℃ for 2 hours to cause hemolysis of the red blood cells;

3) Centrifuging at 4 ℃ at 12000rpm/min for 15min, discarding supernatant, washing the obtained precipitate with lysate for 3-5 times, and re-suspending with 1 XPBS;

4) Shearing with a handheld high-speed homogenizer at 10000rpm/min for 15min in an ice-water bath, collecting the product, controlling erythrocyte membrane fragments to be within 200nm by particle size measurement, and using one part of the product after PBS (phosphate buffer solution) resuspension for measuring the size, and using one part of the product for quantitatively detecting the concentration of the product, wherein the concentration is 2 mug/mu L.

2.2 preparation of culture Medium for increasing exosome yield

The red blood cell particles resuspended in PBS at a concentration of 2. Mu.g/. Mu.L obtained in 120. Mu.L 2.1 were mixed with 3mL of DMEM medium to obtain a medium for increasing the production of exosomes, the concentration of the red blood cell membrane particles in the medium being 80. Mu.g/mL.

Example 2

to 3mL of DMEM medium (Gibco, USA) was added 120. Mu.g of PBS resuspended erythrocyte membrane particles and 60. Mu.L of 1 XPBS buffer (PBS buffer was added in examples 2 and 3 to ensure that the total volume added remained consistent when the amount was measured).

2.A method for preparing a culture medium for increasing the yield of exosomes was the same as in example 1, and the concentration of erythrocyte membrane particles in the prepared culture medium was 40 μg/mL.

Example 3

to 3mL of DMEM medium (Gibco, USA) was added 60. Mu.g of PBS-resuspended erythrocyte pellet and 90. Mu.L of 1 XPBS buffer.

2.A method for preparing a culture medium for increasing the yield of exosomes was the same as in example 1, and the concentration of erythrocyte membrane particles in the prepared culture medium was 20 μg/mL.

Example 4

A method for increasing exosome production, comprising the steps of:

1) Culturing AmL12 cells in DMEM medium to a density of 80%, at 37deg.C, CO ₂ Is 5% by volume;

2) Culturing the exosome donor cells obtained in step 1) by using the culture mediums of examples 1 to 3, respectively, and culturing the exosome donor cells in a control group by using DMEM; the number to volume ratio of exosome donor cells to medium in examples 1 to 3 and the control group were all 8X 10 ⁵ The following steps: 3mL;

3) At the 12 th hour of culture, the cells are respectively replaced with exosome-free serum culture medium;

4) Collecting cell supernatant after 48h, centrifuging to remove cell debris, and filtering with 0.22 μm filter to obtain filtrate as exosome;

5) The collected exosomes were subjected to NTA detection, wherein the exosomes collected in examples 1 to 3 and the control group were designated as RCM-Exo, RCM-Exo-1, RCM-Exo-2 and Exo, respectively.

Example 5

The exosomes RCM-Exo, RCM-Exo-1, RCM-Exo-2 and Exo collected in example 4 were each subjected to NTA detection using a nanoparticle tracking analyzer (ZetaView, particle Metrix) as follows:

1. opening an instrument power supply and computer software to confirm normal connection;

2. injecting 1mL of purified water into the sample pipeline to clean the pipeline;

3. adding 1mL of standard substance instrument for calibration;

4. the standard was washed off using 1ml pbs;

5. taking out the collected supernatant, respectively diluting each group of samples by 100 times, sucking 1mL, and adding the samples into a sample pipeline;

6. focusing a sample;

7. clicking on "Checkparticle Drift at V" determines that the sample is performing brownian motion;

8. clicking 'Measurement', selecting EV parameter in SOP, and measuring.

Wherein the results of RCM-Exo, RCM-Exo-1, RCM-Exo-2 and Exo are shown in FIGS. 1 to 4, respectively:

from the peak graphs at the lower left hand corner in FIGS. 1 to 4, it can be seen that the particle size on the abscissa and the number of exosomes on the ordinate are shown, and that RCM-Exo, RCM-Exo-1, RCM-Exo-2 and Exo peaks at 140nm, 150nm, 140nm and 140nm in this order, namely, the number of exosomes at 140nm, 150nm, 140nm and 140nm in this order of RCM-Exo, RCM-Exo-1, RCM-Exo-2 and Exo is the largest, and particularly the exosome produced in example 1 is higher in yield (obtained by peak area recognition, the larger the peak area, the higher the exosome yield is). The exosomes prepared by using the culture medium in examples 1 to 3 and exosomes prepared by using the conventional culture medium in the control group are all in the known particle size range, and the physical characteristics of the exosomes are not affected by the exosomes prepared by using the culture medium provided by the invention.

Example 6

Verification of exosome RCM-Exo loading efficiency, delivery efficiency and therapeutic Effect

Familial Hypercholesterolemia (FH) is a genetic disease of single gene (Ldlr gene), and gene therapy is an effective means of treating the disease. This example demonstrates the loading efficiency, delivery efficiency and therapeutic effect of exosomes prepared using the culture medium of the present invention, taking this disease as an example.

1) AML12 cells over-expressing Ldlr (AML 12 cells were purchased from the GmbH of the Living technology Co., ltd., gmbH, procell) were used as exosome donor cells, and PBS, the medium in example 1 and the medium in the control group were added to AML12 cells over-expressing Ldlr at a density of 80%, respectively, and the cells were divided into: PBS, RCM@Exo ^ldlr And Ctrl@Exo ^ldlr Each set was provided with 3 biological replicates;

the preparation method of the AML12 cell over-expressing Ldlr comprises the following steps:

1. constructing an Ldlr plasmid in vitro,

aml12 cells were plated and when the cell density reached 80%, transfected (i.e. overexpressed) as follows:

mu.g of Ldlr plasmid was diluted in 200. Mu.L of PBS buffer to give an Ldlr solution; adding 7.2 mu LHigene transfection reagent (C1506, prilet Gene technologies Co., ltd.) into Ldlr solution, immediately shaking and mixing, and centrifuging at low speed to get liquid at the bottom of the tube; standing at room temperature for 15min; the transfection mixture was added directly to the cell culture medium and mixed well with shaking the petri dish.

2) Exosomes were collected by ultracentrifugation, observed: ctrl@Exo ^ldlr And RCM@Exo ^ldlr Morphology, size and yield changes of exosomes in the group; the analysis results of the exosome are shown in FIG. 5, and the exosome form and size are not changed, wherein Ctrl@Exo is shown in FIG. 5 ^Ldlr And RCM@Exo ^Ldlr The control exosome-loaded Ldlr group and the mass-produced exosome-loaded Ldlr group are represented, respectively.

RCM@Exo ^ldlr And Ctrl@Exo ^ldlr The results of the group throughput are shown in figures 6-8 and 9-11, respectively: as can be seen from the data in part original concentration of FIGS. 6-8 and 9-11, RCM@Exo ^ldlr The content of exosomes obtained from 3 biological replicates in the group was 9.3X10, respectively ⁹ Particles/mL、1.6×10 ¹⁰ Particles/mL and 9.2X10 ⁹ Particles/mL, average 1.15X10 ¹⁰ Particles/mL；Ctrl@Exo ^ldlr The content of exosomes obtained from 3 biological replicates in the group was 2.6x10, respectively ⁹ Particles/mL、1.7×10 ⁹ Particles/mL and 9.5X10 ⁸ Particles/mL, average 1.75X10 ⁹ Particles/mL，RCM-Exo ^Ldlr Group comparison Exo ^Ldlr The exosome yield in the group was improved by 6.57 times.

3) qPCR detects the efficiency of exosome loading Ldlr.

The efficiency of exosome loading Ldlr was tested using a conventional qPCR test procedure with GAPDH as the reference gene, wherein the upstream primer for exosome loading Ldlr was 5'-TGACTCAGACGAACAAGGCTG-3' (SEQ ID No. 1) and the downstream primer was 5'-ATCTAGGCAATCTCGGTCTCC-3' (SEQ ID No. 2), and the results are shown in fig. 12. From the above results, it can be seen that the efficiency of loading Ldlr on exosomes prepared by using the culture medium provided by the invention is not different from the loading efficiency of exosomes prepared by using a conventional culture medium, and it is proved that exosomes prepared by using the culture medium provided by the invention do not affect the loading rate of exosomes per se.

4) Ldlr for high fat breeding ^-/- Mice were treated as follows:

PBS, collected RCM@Exo ^ldlr And Ctrl@Exo ^ldlr After quantitative determination of group exosome BCA, ldlr per gram was used ^-/- Mice (Jiangsu Jiugao Biotech Co., ltd.; no. T001464, high fat feeding using the conventional high fat feeding method in the art) were given a dose of 10. Mu.g of exosomes by alternate intraperitoneal administration on the left and right weekly, and the weekly injection time (in this treatment, once weekly administration treatment) was determined, followed by 8 weeks of continuous injection.

The treatment effect of the mice was verified by the oil red O of the liver and the general oil red staining index of the common vascular artery, wherein the results are shown in fig. 13 to 14, wherein the scale in the first row in fig. 13 is 200 μm, the magnification is 5.0×, the scale in the second row of pictures is 50 μm, and the magnification is 20.0×; from left to right, PBS, ctrl@Exo ^Ldlr And RCM@Exo ^Ldlr The control group, the control exosome-loaded Ldlr group and the mass-produced exosome-loaded Ldlr group are represented, respectively.

Toxicity analysis by HE staining was performed as shown in FIG. 15, and as can be seen from FIG. 15, PBS and RCM@Exo were used ^ldlr And Ctrl@Exo ^ldlr Ldlr for high fat breeding in group pair ^-/- After mice are treated, no obvious difference exists from the observation of cell morphology and cell size in each tissue, which indicates that exosomes have no toxic or side effects.

According to FIGS. 13 to 15, it was found that the exosomes prepared using the culture medium provided by the present invention have the same therapeutic effect on mice with familial hypercholesterolemia as those obtained under ordinary conditions, and are nontoxic.

From the above embodiments it can be derived that: the reagent provided by the invention is used for culturing exosome donor cells, so that the yield of exosome can be obviously increased, which is approximately 7 times that of exosome prepared by adopting a conventional reagent without adding erythrocyte membrane, and meanwhile, the loading efficiency, the delivery efficiency and the treatment effect on diseases are not affected.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. Use of fragments of erythrocyte membranes for increasing the production of exosomes, characterized in that said fragments of erythrocyte membranes are co-cultured with exosome donor cells.

2.A method for increasing the production of exosomes, comprising culturing donor cells of the exosomes with an agent comprising fragments of erythrocyte membranes and an adjuvant, and collecting the exosomes.

3. The method of claim 2, wherein the exosome donor cells are first cultured in the reagent for 12 hours, the resulting culture fraction is second cultured in exosome-free serum medium, and the exosomes are collected after 48 hours.

4. The method of claim 2 or 3, wherein the exosome donor cells comprise AmL12 cells.

5. The method according to claim 2, wherein the mass-to-volume ratio of the red cell membrane fragments to the auxiliary material is 20-80 μg:1mL.

6. The method of claim 5, wherein the mass to volume ratio of the red blood cell membrane fragments to the auxiliary material is 80 μg:1mL.

7. The method of claim 2, wherein the particle size of the red cell membrane fragments is 0-400 nm.

8. The method of claim 2, wherein the reagent comprises a culture medium or a kit.

9. The method of any one of claims 2, 5-8, wherein the adjunct comprises a cell culture medium.