WO2022042739A1

WO2022042739A1 - Method for promoting endothelial cells to produce exosomes, and exosome preparation and application thereof

Info

Publication number: WO2022042739A1
Application number: PCT/CN2021/115532
Authority: WO
Inventors: 仉红刚; 张秋菊; 李炳蔚; 修瑞娟
Original assignee: 中国医学科学院微循环研究所
Priority date: 2020-08-31
Filing date: 2021-08-31
Publication date: 2022-03-03
Also published as: CN112029705A; CN112029705B

Abstract

Provided are a method for promoting endothelial cells to produce exosomes, and an exosome preparation and an application thereof. The method promoting endothelial cells to produce exosomes comprises the following operation steps: step 1, taking human umbilical vein endothelial cell strains for adherent culture and passage; step 2, adding panax notoginseng saponins into a culture solution of adherent cells cultured in step 1, and then continuing to perform culturing in an incubator; and step 3, separating and purifying the exosomes from the culture solution obtained in step 2.

Description

Methods for promoting endothelial cell production of exosomes, exosome preparations and applications

technical field

The invention belongs to the field of biomedicine, and in particular relates to a method for promoting endothelial cells to produce exosomes, exosome preparations and applications.

Background technique

At present, biotherapy has become an important research, development and application content in the field of biomedicine. With the rapid development of biological cell technology and industry around the world, my country's national and local authorities have successively issued a number of support policies to support the development of cell therapy. .

Extracellular vesicles are important mediators for protein, mRNA, miRNA, and lipid transport to complete intercellular communication pathways, and are classified into three categories according to their size and occurrence, including exosomes, microvesicles, and apoptotic bodies. Among them, exosomes are packaging vesicles with a diameter of about 40-100 nm, which are secreted by a variety of cells and contain specific proteins, lipids, cytokines or genetic materials. Exosomes derived from different tissues not only have their specific proteins and nucleic acids, but also contain key molecules for their functions.

Exosomes are nanometer-sized membrane-enclosed vesicles that contain biologically active molecules, including proteins, RNAs, and miRNAs. They act as messengers to regulate the function of neighboring cells and distant target cells. Preclinical studies have shown that, under certain conditions, exogenously administered exosomes can re-direct cellular activities to achieve their therapeutic potential. Delivery mediated by exosomes has several advantages: 1. Highly biocompatible if they are derived from appropriate cells [e.g. mesenchymal stem cells (MSCs) or immature dendritic cells (DC)] , it is immune inert at the same time; 2. It can pass through the thick tissue barriers of the human body, such as the blood-brain barrier (BBB). Normally, exosomes can be directly used as potential therapeutic agents.

Exosomes have a very broad therapeutic prospect and market, but the yield is an important issue restricting their application. Internationally, effective technical methods to improve the yield of exosomes are also being sought. So far at home and abroad, only James Lee's research group from the School of Chemistry and Bioengineering at The Ohio State University has invented a cell nanoscale biochip, which can improve the efficiency of exosome production and nucleic acid encapsulation by orders of magnitude. These results were published in Nature Biomedical Engineering on December 16, 2019 (Zhaogang Yang, et al. Large-scale generation of functional mRNA- encapsulating exosomes via cellular nanoporation), but the technology to increase the production of cellular exosomes by biopharmaceutical-induced methods is rarely reported.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a preparation method for improving the yield of endothelial cell exosome active preparations.

In order to achieve the above object, the technical scheme of the present invention is as follows: a method for promoting endothelial cells to produce exosomes, which is characterized in that comprising the following operation steps:

Step 1: Take the human umbilical vein endothelial cell line for adherent culture and passage;

Step 2: Add Panax notoginseng saponins to the culture solution of the adherent cells cultured in Step 1, and then continue to culture in the incubator;

Step 3: Separate and purify exosomes from the culture medium obtained in step 2.

The endothelial cells subcultured in step 1 in the above technical solution are human umbilical vein endothelial cell lines.

In the above technical solution, the culture medium in step 1 is endothelial cell culture medium.

In the above technical solution, the concentration of Panax notoginseng saponins in the culture solution in step 2 is 0.4 mg/mL, and the culture solution is continuously cultured for 24 hours after adding Panax notoginseng saponins.

The specific steps for the separation and purification of exosomes in step 3 described in the above technical solution are as follows: collect the supernatant of the culture medium after the culture in step 2 is completed, centrifuge at 3000g for 10 min at 4°C, and take the supernatant after centrifugation. Add ECS reagent to the supernatant after centrifugation and mix well. After mixing, let it stand at 2-8°C for 1-3 hours, wherein the amount of ECS reagent added is the amount of the supernatant after centrifugation. The volume ratio is 1:4. After standing, it is centrifuged at 10000g for 60min at 4°C. After centrifugation, the supernatant is discarded, and the precipitate obtained by adding PBS solution is evenly piped and centrifuged. The amount of the PBS solution is ECS reagent. 1/25 of the dosage, then centrifuge at 12000g for 2min at 4°C, take the supernatant, transfer the obtained supernatant into the upper chamber of the EPF column, centrifuge at 3000g for 10min at 4°C, and collect the EPF column after centrifugation The liquid at the bottom of the tube, the collected liquid is the purified exosomes.

Another object of the present invention is to provide an exosome active preparation prepared by the above method.

The third object of the present invention is to apply the exosome active preparation prepared above in the preparation of a drug for improving microvascular function.

Compared with the prior art, the beneficial effect of the technical solution of the present invention is that: by adding Panax notoginseng saponins to the culture solution of human umbilical vein endothelial cells, the human umbilical vein endothelial cells are promoted to secrete a large amount of exosomes, so that the exosomes can be increased. The preparation of exosomes is more convenient, the yield is higher, and the method has good specificity, strong specificity and high accuracy. Therefore, the method provided by the present invention can simply and efficiently provide the biological quantity of exosome-targeted therapeutic drugs, This is of great significance for the research, development and clinical application of endothelial cell exosome-targeted therapeutic drugs.

Description of drawings

Fig. 1 is the standard curve prepared in the embodiment of the present invention.

detailed description

The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

1. Solution preparation

The preparation concentration of the stock solution of TNF-α is 10 μg/mL;

Due to the low solubility of Panax notoginseng saponins, it needs to be slowly dissolved at 37°C to make a 40mg/mL panax notoginseng saponin solution (100mg panax notoginseng saponins powder is added to 2.5mL endothelial cell culture medium). Store at 4°C, and it is best to use it within a short period of time (2-4 weeks). Pay attention to whether there is any precipitation of Panax notoginseng saponins each time you use it.

2. Subculture of Human Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells (purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences) were used for primary culture in endothelial cell culture medium (ECM, produced in Sciencell, San Diego, CA). After subculturing for 2-3 days, remove the supernatant and add PBS to rinse at least twice, then add digestion solution (0.25% trypsin-0.02% EDTA mixed digestion solution) for digestion, and the amount of digestion solution added to cover the adherent After the digestion is complete, the digestion solution is removed, and the endothelial cell medium is added, and at the same time, the human umbilical vein endothelial cells are evenly distributed in the medium by pipetting to obtain the subculture of human umbilical vein endothelial cells.

3. Group experiment

Take three culture dishes and add an equal amount of the above-mentioned subculture of human umbilical vein endothelial cells respectively, and the three culture dishes are respectively the blank group, the control group and the experimental group. The diluent of the solution made the concentration of TNF-α in the control group to be 10ng/mL, and the diluent of the total saponins of Panax notoginseng was added to the experimental group, so that the concentration of total saponins of Panax notoginseng in the experimental group was 0.4mg/mL, and then the The three petri dishes were incubated for 24 h.

4. Collection and purification of exosomes

4.1 Sample Pretreatment

4.1.1 Sampling: Take 20 mL of the supernatant of the cell culture solution from the blank group, the control group and the experimental group as a sample (if it is a frozen sample, take it out of the refrigerator and thaw it in a 25°C water bath. put the sample on ice; if it is a fresh sample, collect the sample and put it on ice);

4.1.2 Centrifuge to remove cell debris: Transfer each sample to a centrifuge tube, centrifuge at 3000g for 10 min at 4°C to remove cell debris from the sample, and transfer the supernatant in each centrifuge tube to a round bottom. in a centrifuge tube;

4.2 Extraction of exosomes

4.2.1 Supernatant pretreatment: add 5 mL of Exosomoe Concentration Solution (ECS reagent) to each round-bottomed centrifuge tube, then close the cap of the centrifuge tube, mix for 1 min with a vortex shaker, and then incubate at 2-8°C Stand for 2h in the environment;

4.2.2 Precipitation of exosomes: Centrifuge the above-treated round-bottom centrifuge tubes at 10,000g for 60 min at 4°C, discard the supernatant, and keep the precipitate, which is rich in exosome particles (Note: as much as possible to absorb net supernatant);

4.2.3 Resuspending exosomes: Take 200μL of PBS and evenly pipet the precipitate obtained by the above centrifugation. After it is evenly suspended in PBS, transfer the resuspension to a new 1.5mL centrifuge tube;

4.2.4 Harvest exosome particles: centrifuge the 1.5mL centrifuge tube containing the resuspension at 12000g for 2 min at 4°C, and retain the supernatant, which is rich in exosome particles;

4.2.5 Purification of exosomes: Transfer the above-mentioned supernatant rich in exosome particles into the upper chamber of Exosomoe Purafication Filter (EPF column), centrifuge at 3000g for 10min at 4°C, and collect the liquid at the bottom of the EPF column after centrifugation. , this liquid is the purified exosome particles.

5. Exosome quantification

First, the BCA protein quantification kit (produced by ^Kangwei Century Biotechnology Co., Ltd., operated according to the product instructions) was used to make a standard curve.

Exosome quantification is the protein content in the exosome lysate to reflect the amount of exosomes, using protein lysis buffer (containing protease inhibitors, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) RIPA cleavage model P0013C The exosomes to be tested were lysed on ice, the protein concentration in the lysing solution was detected by BCA method, and the concentration in the original stored exosome solution was calculated. The calculation results are shown in Table 1.

Table 1 shows the quantitative results of exosome concentration in blank group, control group and experimental group

From Figure 1 and the table are the protein quantification standard curve and exosome quantification results, respectively. The concentrations of exosomes that can be extracted from the culture supernatant of human umbilical vein endothelial cells with different treatments (blank group, control group, and experimental group) are 327.83 μg/mL, 564.54 μg/mL and 2515.03 μg/mL, respectively (this is the concentration after concentration, not the concentration in the original supernatant). It can be seen that the induction of human umbilical vein endothelial cells by Panax notoginseng saponins can significantly increase the production of exosomes (up to 7.69 times that of the control).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims

A method for promoting endothelial cell production of exosomes, comprising the following steps:

Step 1: Take the human umbilical vein endothelial cell line for adherent culture and passage;

Step 2: Add Panax notoginseng saponins to the culture solution of the adherent cells cultured in Step 1, and then continue to culture in the incubator;

Step 3: Separate and purify exosomes from the culture medium obtained in step 2.
The method for promoting endothelial cells to produce exosomes according to claim 1, wherein the endothelial cells subcultured in the step 1 are human umbilical vein endothelial cell lines.
The method for promoting endothelial cell production of exosomes according to claim 1, wherein the culture medium in the step 1 is an endothelial cell culture medium.
The method for promoting endothelial cell production of exosomes according to claim 1, wherein in the step 2, the concentration of Panax notoginseng saponins in the culture solution is 0.4 mg/mL, and the culture solution is added with Panax notoginseng at a concentration of 0.4 mg/mL. After the total saponins were cultured for 24h.
The method for promoting endothelial cell production of exosomes according to claim 1, wherein the specific steps of separating and purifying exosomes in the step 3 are as follows: collecting the supernatant of the culture solution after the culture is completed in the step 2 Centrifuge at 3000g for 10min at 4°C, take the supernatant after centrifugation, add ECS reagent to the supernatant after centrifugation and mix well, after mixing, let stand at 2-8°C for 1-3h , wherein the added amount of the ECS reagent is that the volume ratio of the supernatant after centrifugation is 1:4. After standing, it is centrifuged at 10000g for 60min at 4°C, the supernatant is discarded after centrifugation, and PBS is added. The solution was evenly pipetted and centrifuged the precipitate obtained, the amount of the PBS solution was 1/25 of the amount of the ECS reagent, then centrifuged at 12,000g for 2min at 4°C, the supernatant was taken, and the supernatant was transferred to the upper chamber of the EPF column , centrifuge at 3000g for 10 min at 4°C, and collect the liquid at the bottom of the EPF column after centrifugation. The collected liquid is the purified exosomes.
A kind of exosome active preparation prepared by the method according to any one of claims 1-5.
An application of the exosome active preparation as prepared in claim 6 in the preparation of a drug for improving microvascular function.