WO2022143590A1 - Exosome secretion inducer and exosome secretion inducing culture medium, exosome production method using same, and application - Google Patents

Abstract

Provided are an exosome secretion inducer and an exosome secretion inducing culture medium, an exosome production method using same, and an application. The exosome secretion inducing culture medium only comprises a basic culture medium and four additives: L-ascorbic acid or a salt thereof, selenium or a salt thereof, NaHCO3, and insulin. The culture medium can improve the yield of exosomes secreted by stem cells; and the exosomes obtained by production can protect neurons from damage and improve senescent cell conditions.

Description

A kind of exosome secretion inducer and induction medium and production method and application of exosome using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Chinese Patent Application No. 202011636845.1 filed on December 31, 2020, the entire contents of which are incorporated herein by reference.

technical field

The invention belongs to the field of biotechnology, and in particular relates to an exosome secretion inducer, an induction medium, and a production method and application of exosomes using the same, in particular to the preparation of exosomes to improve the condition of senescent cells and/or protect nerve cells Metapharmaceutical applications.

Background technique

Exosomes are specifically secreted small membrane vesicles (30-150 nm) containing complex RNAs and proteins, which are involved in intercellular communication and regulate the normal function and damage repair of various tissues and organs.

Since exosomes derived from different tissues have their specific protein molecules and key molecules for their functions, exosomes derived from stem cells have an important therapeutic effect of stem cells and are safer, and can effectively transport and treat diseases. Bioactive substances such as RNA and protein have important biological functions such as inhibiting apoptosis, inhibiting inflammatory response, promoting angiogenesis, inhibiting fibrosis, and improving tissue repair potential. In addition, exosomes can also penetrate the blood-brain barrier and can deliver various therapeutic molecules such as small molecule drugs, which have very broad clinical application prospects. Therefore, it is particularly important to efficiently produce this type of exosomes from stem cells. Existing exosomes are mainly obtained by cell autocrine, but their production is very limited.

SUMMARY OF THE INVENTION

In view of one or more problems existing in the prior art, one aspect of the present invention provides an exosome secretion inducer, which comprises the following components in use concentrations:

Another aspect of the present invention provides an exosome secretion induction medium, which comprises a basal medium and the following concentrations of components:

And the exosome secretion induction medium does not contain the combination of Transferrin, FGF2 and TGFβ1/NODAL.

In some embodiments, the basal medium is DMEM/F12.

Another aspect of the present invention also provides a method for producing exosomes, comprising the following steps:

1) Adherently culture the stem cells in logarithmic growth phase and in good growth state until the coverage rate reaches 60%-70%;

2) Use the above-mentioned exosome secretion induction medium to conduct exosome secretion induction culture for the adherent cultured stem cells in step 1) for 20-30 hours, and harvest the supernatant to harvest the exosome-containing cell culture medium;

3) separating and obtaining exosomes from the cell culture solution containing exosomes harvested in step 2);

Preferably, step 2) is repeated 2-3 times, the cell culture medium containing exosomes harvested each time is combined, and exosomes are separated from the combined cell culture medium.

In some embodiments, the stem cells in step 1) are selected from embryonic stem cells, induced pluripotent stem cells and mesenchymal stem cells.

In some embodiments, the separation method in step 3) includes differential centrifugation, ultrafiltration centrifugation, density gradient centrifugation, precipitation, magnetic bead immunoassay, PS affinity, and chromatography.

In some embodiments, the exosomes or cell culture fluid containing exosomes produced by the method are also within the protection scope of the present invention, and the amount of the exosomes reaches the amount of exosomes secreted in stem cells ≥1000 cells/cell, the particle size of the exosomes is 30-150 nm (mainly 50-80 nm), and among the surface markers, CD9 accounts for ≥23%, and CD63 accounts for ≥10%.

In some embodiments, the application of the exosomes in the preparation of medicines for improving the condition of senescent cells and/or protecting neurons also belongs to the content of the present invention. Based on this, the present invention also provides a medicine for improving the condition of senescent cells and/or protecting neurons, which comprises the above-mentioned exosomes.

Based on the above technical solutions, the present invention provides for the first time an exosome secretion inducer for high-efficiency exosome production, which can only contain four components of L-ascorbic acid or its salt, selenium or its salt, NaHCO ₃ and insulin, Therefore, the composition is simple, and when it is combined with a basal medium (such as DMEM/F12) for the culture of stem cells (such as ESC or iPSC, etc.) to form an exosome secretion induction medium to induce the culture of well-adherent stem cells, It can significantly promote the secretion of exosomes in stem cells, thereby significantly increasing the production of exosomes. The results of the examples show that, using the exosome secretion induction medium provided by the present invention to induce the production of exosomes (ie, the secretion amount) of well-adhered iPSC cells for culturing well-adhered iPSC cells is as high as 1000/cell or more. The results of the examples also show that the exosomes produced by the present invention can effectively protect rat cortical neurons in the stroke neuron model (glucose and oxygen deprivation test, OGD) and the stroke neuron model (glucose and oxygen deprivation test). At the same time, it can effectively protect the axonal rupture of human cortical neurons caused by oxidative damage.

Description of drawings

Figure 1 is a bar graph showing the exosome yields of iPSC cells cultured in GDEV medium, E8 medium and MSC cells cultured in MSC medium;

Figure 2 is a graph showing the relationship between particle concentration and particle size distribution in exosome solution;

Fig. 3 is a histogram of the exosome surface markers CD9 and CD63 detected by nanoflow cytometry;

Fig. 4 is the electron microscope photograph of exosome;

Figure 5 is a photo of Hoechst33342 staining of exosomes taken up by human skin fibroblasts, umbilical cord mesenchymal stem cells and neuronal cells;

Figure 6 is a histogram of dead cells statistics in a neuron model of stroke;

Figure 7 is a photo of Calcein AM/Hoechst staining simulating human cortical neuron damage;

Fig. 8 is a histogram of the length statistics of human cortical neurons;

Figure 9 is a mouse survival curve in a stroke neuron model;

Figure 10 is a statistical diagram of the area of cerebral infarction in mice in the neuron model of stroke.

Detailed ways

Aiming at the defect of low yield of autocrine exosomes existing in the prior art, the present invention aims to provide an exosome with simple components for the efficient production of exosomes from stem cells (ESC or iPSC, etc.). The body secretion inducer and the exosome secretion induction medium also provide a method for producing exosomes using the induction medium and applications of the obtained exosomes.

Guokai Chen et al. (Reference 1: Guokai Chen et al. Chemically defined conditions for human iPSC derivation and culture, NATURE METHODS, April 10, 2011) routinely culture stem cells (ESC or iPSC) in E8 medium to improve stem cell ESC and iPSC The derivation efficiency of E8 medium contains: DMEM/F12, L-Ascorbic Acid (L-ascorbic acid), Selenium (selenium), Transferrin (transferrin), NaHCO ₃ , Insulin (insulin), FGF2, TGFβ1/NODAL, among which Insulin and FGF2 are important for cell survival and proliferation, L-Ascorbic Acid promotes ESC proliferation, Selenium is essential for sustained culture expansion, Transferrin helps improve cloning efficiency, and TGFβ1/NODAL contributes to long-term culture stability.

The content of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

The ways of obtaining various biological materials described in the examples are only to provide a way to obtain experimentally to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the examples.

In the following examples, induced pluripotent stem cells (iPSCs) are used as an example to produce exosomes, and some pluripotent genes are introduced into cells such as skin. By "initializing" ordinary somatic cells, iPSC cells with stem cell functions are obtained. iPSCs are not only very similar to embryonic stem cells (ESCs) in terms of cell morphology, growth characteristics, stem cell marker expression, etc., but also almost identical to ESCs in terms of DNA methylation patterns, gene expression profiles, chromatin status, and the formation of chimeric animals. It is exactly the same, so the purpose of the present invention can be achieved by replacing the iPSC with the ESC in the implementation.

Example 1: Exosome secretion induction medium and method of use

In order to adapt to high-yield exosome production, this example provides an exosome secretion induction medium for efficient production of exosomes.

The exosome secretion induction medium (named GDEV medium) formulated for the efficient production of exosomes from stem cells (ESC or iPSC, etc.) of the present invention, the composition of which comprises DMEM/F12 (Gibco11330032) and Exosome secretion inducers. Among them, the exosome secretion inducer is composed of the following four components at the concentration used: L-Ascorbic acid 2 phosphate magnesium salt (L-ascorbic acid diphosphate magnesium salt, Sigma-Aldrich A8960) at a concentration of 64 mg/L, a concentration of Sodium selenium (sodium selenium, Sigma-Aldrich S5261) at a concentration of 14 μg/L, NaHCO ₃ (Sigma-Aldrich S5761) at a concentration of 543 mg/L, and Insulin (insulin, Proceeding I10022) at a concentration of 19.4 mg/L.

As a control, its E8 medium was prepared according to the method disclosed in Document 1, specifically: DMEM/F12, 64 mg/L L-Ascorbic acid 2 phosphate magnesium salt, 14 μg/L sodium selenium, 100 μg /L FGF2 (fibroblast growth factor 2), 19.4 mg/L insulin, 543 mg/L NaHCO ₃ , 10.7 mg/L transferrin (transferrin), 2 μg/L TGFβ1 (transforming growth factor-β1) or 100 μg/ L Nodal.

The above-prepared GDEV medium and E8 medium were used to induce the culture of iPSC cells with an adherent coverage of 60%-70%, respectively, and then collect the exosomes in the supernatant of the iPSC cell culture, and analyze the collected exosomes. The content is detected, and the specific operation includes the following steps.

1.1. Cell culture

Expand iPSC cells (commercial from Guodian (Beijing) Pharmaceutical Technology Co., Ltd.) (for example, use Essential 8 ^TM Medium kit (purchased from thermofisher, product number: A1517001) for routine expansion and culture) to logarithmic growth phase and state Good, digested into small cell clusters (3-10 cells) according to the technical manual of Versene Solution kit (purchased from Thermo Fisher, Cat. No. 15040066), and then plated on Vitronectin (Recombinant Human Vitronectin, purchased from Peprotech Co., Ltd.). , Cat. No. 140-09) coated 10 cm cell culture dish, the coverage rate reached 60%-70% after adherence, and cultured overnight at 37°C in an atmosphere of 95% air and 5% CO ₂ . If the iPSC adheres well, change the medium to GDEV medium or E8 medium. After 24 hours, change the medium to collect the cell culture supernatant. Repeat the previous step, and a batch of cells can be collected 2-3 times.

Synchronous use of MSC

The XF human mesenchymal stem cell serum-free medium (BI, named MSC medium in the examples) was used to culture human mesenchymal stem cells (MSC, obtained according to a conventional separation method) to obtain the cell culture supernatant.

1.2. Collection of exosomes in cell culture supernatant

The cell culture supernatants obtained by inducing cells to be cultured in GDEV medium, E8 medium and MSC medium in the above step 1.1 were taken and centrifuged at 3000 g for 15 min at room temperature to remove dead cells and cell debris. The supernatant was collected and filtered with a 0.22 μm filter; the filtrate was transferred to an Amicon Ultra-15 (100 kDa) or Centricon Plus-70 (100 kDa) ultrafiltration tube, concentrated, and centrifuged at 3000 g at room temperature for 30 min. The concentrate was diluted approximately 1:100 with DPBS (Dulbecco's Phosphate Buffered Saline) and concentrated again using the same apparatus. A relatively pure exosome solution can be obtained. This step uses ultrafiltration to separate exosomes from the cell culture supernatant. Differential centrifugation, density gradient centrifugation, precipitation, magnetic bead immunoassay, PS affinity, chromatography, etc. can also be used to separate exosomes in cell culture supernatants.

1.3. Exosome yield detection

Take 20 μl of the exosome solutions collected in the above step 1.2 and cultured in GDEV medium, E8 medium and MSC medium respectively, dilute to 100 μl with PBS, add nano-flow detector (brand: NANOFCM), according to the instrument technology Manual operation to obtain the respective exosome yields.

The results are shown in Table 1 and Figure 1 below. It can be seen that more exosomes (1013.9/cell) can be obtained by inducing and culturing iPSCs in GDEV medium. 5.4 times the number of bodies (187.5/cell). At the same time, the yield of exosomes obtained by inducing and culturing iPSCs in GDEV medium was compared with that obtained by inducing and culturing MSCs in MSC medium. It can be seen that the former is 59 times higher than the latter. It is shown that GDEV medium can significantly increase the yield of exosomes produced by stem cells (such as iPSCs) relative to E8 medium and MSC medium. On the other hand, in terms of composition, the GDEV medium only includes five components, which is simpler than the E8 medium including eight components, but can greatly increase the production of exosomes, thereby significantly reducing exosomes. production cost.

Table 1: Yield of exosome-producing cells cultured in different media

	GDEV medium	E8 medium	MSC medium
Exosome yield (units/cell)	1013.9	187.5	17.1

1.4. Identification of exosomes: particle size analysis, nano-flow detection and electron microscopy analysis

1.4.1. Exosome particle size analysis

Take 20 μl of the exosome solution obtained by inducing and culturing iPSCs in GDEV medium, dilute it to 100 μl with PBS, add a nano-flow detector (brand: NANOFCM), and operate according to the instrument technical manual, as shown in Figure 2, to obtain the particle size distribution It can be seen from the figure that the concentration of particles with a particle size of 50-80 nm (about 60 nm) in the produced exosome solution is the highest, which is in line with the particle size range of exosomes secreted by iPSC (30-150 nm), which proves that exosomes were obtained. In the present invention, the nanometer flow detector is used to detect the particle size of exosomes obtained by inducing and culturing iPSCs in GDEV medium, which is mainly 50-80 nm. size.

1.4.2. Nanofluidic detection of exosome surface markers

Add 1 μl of antibody (CD9 antibody or CD63 antibody) to 10 μg of the exosome solution obtained by inducing iPSCs in GDEV medium. After adding the antibody, cover the centrifuge tube tightly, mix with a vortex shaker for 1 min, and then incubate at 37°C for 30 min in the dark; add 1 mL of 1×PBS to the incubated exosome-antibody complex and mix well, according to the above step 1.2 The exosome isolation method in the method of exosomes was extracted again to remove free dyes; the labeled exosomes were subjected to nanofluidic detection.

The results are shown in Figure 3, which is the histogram of the exosome surface markers CD9 and CD63 detected by nanofluidics. It can be seen that the proportion of CD9 and CD63 positive particles in the total number of particles in the labeled exosome surface markers is 25.8, respectively. % and 11.9%, which proved that exosomes were indeed obtained from iPSCs induced by GDEV medium.

1.4.3. Electron microscope detection of exosome morphology

(1) Immobilize exosomes on the copper-loaded grid: Mix 50 μl of the exosome solution obtained by inducing iPSCs in GDEV medium with an equal amount of 4% PFA fixative solution (paraformaldehyde solution) to obtain an exosome suspension , add 5 μl of the exosome suspension to the Formvar-carbon-loaded copper grid; add 100 μl of PBS to the parafilm. The copper mesh (Formvar membrane side down) was washed on a drop of PBS with tweezers; the copper mesh was placed on a drop of 50 μl 1% glutaraldehyde for 5 min; placed in 100 μl ddH ₂ O for 2 min (8 washes).

(2) Negative staining of exosomes and electron microscope detection: place the copper mesh on 50 μl uranyl oxalate droplets for 5 minutes; place the copper mesh on 50 μl methylcellulose droplets for 10 minutes, and operate on ice; On the stainless steel ring at the top of the sample stage, absorb the excess liquid on the filter paper; dry in air for 5min to 10min; take electron microscope pictures at 80kV.

The results are shown in Figure 4, which is an electron microscope photo of exosomes obtained by inducing and culturing iPSCs in GDEV medium.

Example 2: Exosome secretion inducer

This example provides an exosome secretion inducer for efficient production of exosomes in order to adapt to high-yield exosome production.

The exosome secretion inducers for efficient production of exosomes provided by the present invention (named GD1, GD2 and GD3 respectively, wherein the concentration of each component is the concentration in the basal medium DMEM/F12) are prepared according to the following table 2. , each exosome secretion inducer and basal medium DMEM/F12 were prepared into GDEV medium, and iPSCs were induced and cultured according to the operations of steps 1.1 to 1.3 in the above Example 1, and then the exosomes in the cell culture supernatant were collected, The contents of exosomes were detected separately, and the results are shown in Table 3 below.

Table 2: Formulations of exosome secretion inducers

Table 3: Use of GD1, GD2 and GD3 inducers and basal medium DMEM/F12

Induction of exosome production in cultured iPSCs

inducer	basal medium	Exosome yield (units/cell)
GD1	DMEM/F12	1171
GD2	DMEM/F12	1092
GD3	DMEM/F12	1208

As can be seen from the results of Table 2 and Table 3 above, the exosome secretion inducer for efficient production of exosomes provided by the present invention (including L-ascorbic acid diphosphate magnesium salt with a concentration of 15-100mg/L, a concentration of 2-100 μg/L sodium selenium, 200-1000 mg/L NaHCO ₃ and 5-30 mg/L insulin) combined with basal medium DMEM/F12 to induce well-adherent iPSC cells During culture, it can significantly promote the secretion of exosomes from iPSC cells, and the output of exosomes (that is, the secretion amount) is above 1,000 per cell, so as to achieve the purpose of producing exosomes efficiently. And the exosome secretion inducer provided by the present invention can only contain four components, so the components are simple and easy to prepare.

The present invention provides an exosome secretion inducer for the efficient production of exosomes for the first time, and the components contained in the inducer can be, in addition to the above-mentioned L-ascorbic acid diphosphate magnesium salt and sodium selenium, L- Ascorbic acid or other salts thereof and selenium or other salts. Among them, L-ascorbic acid (vitamin C) is an essential vitamin for maintaining healthy growth and maintenance of cells in vivo and in vitro, and is also a water-soluble antioxidant, which can prevent the peroxidation of esterified and non-esterified unsaturated fatty acids, but in the present invention In the provided exosome secretion inducer, when its concentration is lower than 15mg/L or higher than 100mg/L, it will affect the growth state of stem cells, which may lead to the reduction of exosome secretion; selenium is a normal cell in vivo and in vitro. Essential trace element for growth and development, it can be incorporated into enzymes and protect cells by reducing peroxides, organic hydroperoxides, and peroxynitrites to harmless substances, with various antioxidant functions and different substrates Substance-specific selenium-containing enzymes are located in cells, on cell surfaces and outside cells, and these enzymes together constitute a complete oxidative damage defense system, but in the exosome secretion inducer provided by the present invention, when the concentration is lower than 2 μg/ L or higher than 100μg/L will affect the growth state of stem cells, which may lead to a decrease in the secretion of exosomes; during cell culture, cells can grow in an open system (the air above the medium and the air in the incubator can be freely exchanged), A solution containing a certain concentration of NaHCO ₃ is required to maintain pH, so the concentration of NaHCO ₃ in the system is too high or too low, which may affect the growth state of cells, thereby affecting the secretion of cell exosomes; insulin can stimulate cells to uridine and glucose It can also bind to insulin receptors on the cell membrane to regulate various metabolic pathways in cells, increase the synthesis of fatty acids and glucose, and is considered to be used to regulate most cell growth and differentiation. It plays an important role in cell growth, but in the exosome secretion inducer provided by the present invention, when its concentration is lower than 5mg/L or higher than 30mg/L, cells may have abnormal morphology and growth rate The disordered phenomenon may lead to a decrease in the secretion of exosomes. Therefore, in order to stably improve the production of exosomes, the concentration of L-ascorbic acid or its salt in the exosome secretion inducer provided by the present invention is limited within the range of 15-100mg/L, and can be selected as 15-50mg/L, 15-64mg/L, 50-64mg/L, 50-100mg/L, 64-100mg/L, etc.; the use concentration of selenium or its salt is limited to 2-100μg/L, optional 2-14μg/L , 2-20μg/L, 14-20μg/L, 14-100μg/L, 20-100μg/L, etc.; the concentration of NaHCO ₃ is limited to 200-1000mg/L 200-543mg/L, 500-543mg/L, 500-1000mg/L, 543-1000mg/L, etc; -20mg/L, 19.4-20mg/L, 19.4-30mg/L, 20-30mg/L, etc.

Example 3: Application of exosomes

In this example, the exosome solution obtained by inducing and culturing iPSCs in the GDEV medium in the above Example 1 was used to verify that it was used in a stroke neuron model (glucose and oxygen deprivation test, OGD), improving the condition of senescent cells, and a stroke neuron model (glucose and oxygen deprivation test). oxygen deprivation test).

3.1. Uptake of exosomes

1) Label the exosome solution with PKH26 according to the technical manual of the PKH26 Red Fluorescent Cell Linker Mini Kit (purchased from Sigma, Cat. No. MINI26), and use Exosome Spin Columns (MW 3000) (purchased from Invitrogen, Cat. No. 4484449) reagent Remove excess dye from the cassette's technical manual to obtain a labeled exosome solution.

2) According to the ratio of exosome:cell to 10000:1, the labeled exosome solution was added to human skin fibroblasts, umbilical cord mesenchymal stem cells and neuronal cells (Guodian (Beijing) Pharmaceutical Technology Co., Ltd. commodity , human skin fibroblast medium is DMEM+15%FBS+NEAA, umbilical cord mesenchymal stem cell medium is DMEM+10%FBS, neuron cell medium is B27+Neurobasal+GlutaMAX, culture conditions are 37 ℃, 5% CO ₂ ) culture medium, placed at 37°C, 95% air and 5% CO ₂ incubator for 24 hours, washed the cells with PBS, and added Hoechst 33342 (purchased from Sigma-Aldrich) to a final concentration of 5 μg/ml Nuclei were stained and photographed under a fluorescence microscope.

The results are shown in Figure 5, which shows the cellular uptake of exosomes obtained by inducing and culturing iPSCs with GDEV medium in Example 1, where Panel A is the umbilical cord mesenchymal stem cells added with exosomes; Panel B is Human skin fibroblasts with added exosomes; Panel C, human neuronal cells with added exosomes; H, Hoechst 33342 signal, indicating the nucleus; P, PKH26 signal, indicating exosomes. It is evident that exosomes can be taken up into cells by human skin fibroblasts, umbilical cord mesenchymal stem cells and neuronal cells.

3.2. Exosomes effectively protect rat cortical neurons in a stroke neuron model (oxygen deprivation test, OGD)

(1) After culturing primary rat neurons in vitro for 14 days, the neuron culture medium was changed to a sugar-free anaerobic medium (balanced with 95% N ₂ /5% CO ₂ in advance), and the neurons were placed in the OGD chamber 90 minutes (10 minutes inflated with 95% N2/5% CO2);

(2) After the treatment, the control group (named OGD group) was replaced with normal neuron culture medium, and the experimental group 1 (named iPSC EV group) was replaced with normal neuron culture medium and 1 μg/ml or 0.2 μg/ml iPSC was added Exosomes (ie, GDEV exosomes obtained by inducing and culturing iPSCs in GDEV medium in Example 1), experimental group 2 (named MSC group) was replaced with normal neuron culture medium and added 1 μg/ml or 0.2 μg/ml MSCs Exosomes (ie, MSC exosomes produced by culturing MSCs in MSC medium in Example 1), experimental group 3 (named E8 group) was replaced with normal neuron culture medium and added 1 μg/ml or 0.2 μg/ml E8 exosomes (ie E8 exosomes produced by inducing and culturing iPSCs in E8 medium in Example 1) were then placed in a 95% air/5% CO ₂ incubator for 24 hours and then stained with Hoechst 33342/PI. , using high-content photography for neuronal activity analysis.

The results are shown in Figure 6, which is a statistical histogram of the proportion of dead cells, in which NO OGD means that the OGD model was not performed, and DPQ means that the inhibitor of DPQ (PARP-1 (polyADP-ribose polymerase-1) was added) ) treated cells, DPQ has been shown to reduce apoptosis under the influence of ischemia. It can be seen that the proportion of dead cells in the control group (OGD group), experimental group 2 (ie, MSC group) and experimental group 3 (ie, E8 group) was significantly reduced. The proportion of dead cells in EV group) was significantly lower than that in experimental group 3 (ie, E8 group) and experimental group 2 (ie, MSC group), indicating that exosomes obtained by inducing iPSC cultured with GDEV medium could more effectively protect large cells in the OGD model. Mouse cortical neurons.

3.3. Exosomes can effectively protect neurons from axonal rupture caused by oxidative damage

Injury of human cortical neurons by hydrogen peroxide mimics aging-related neuronal oxidative damage. Specifically include the following steps:

(1) Resuscitate normal human iPSC-differentiated cortical neurons, spread them in a 96-well plate, and cultivate them for 7 days. Except for the control group without treatment, the experimental group 1 was prepared with Neurobasal Medium (B27+Neurobasal+GlutaMAX) and added H ₂ O ₂ To the final concentration of 20 μM (named the H ₂ O ₂ group), the experimental group 2 prepared Neurobasal Medium and added H ₂ O ₂ to the final concentration of 20 μM, and at the same time added the iPSC exocytosis obtained by inducing and culturing iPSCs in the GDEV medium in Example 1. body to a final concentration of 5 μg/ml (named as H ₂ O ₂ +EV group), the medium was completely changed, and the injury model of hydrogen peroxide was established;

(2) After adding hydrogen peroxide for 16 hours, the neurons were stained with Calcein AM/Hoechst, high-content photographs were taken, and data such as the length of neuron neurites were counted.

The results are shown in Figures 7 and 8, wherein Figure 7 is a photo of Calcein AM/Hoechst stained neurons, and Figure 8 is a statistical histogram of neuron lengths in each group. It can be seen that after the addition of iPSC exosomes in the H ₂ O ₂ +EV group, the length of neuron axons was significantly longer than that in the non-exosome group (ie, the H ₂ O ₂ group), so exosomes can effectively protect against oxidative damage caused by neuron axon rupture.

3.4. Exosomes effectively protect rat cortical neurons in a stroke neuron model (glucose-oxygen deprivation test)

The middle cerebral artery occlusion (MCAO) modeling protocol was as follows: 18 male C57 mice (8-12 weeks old, purchased from Weitong Lihua Animal Technology Co., Ltd.) were used for MCAO modeling. Meanwhile, 6 male C57 mice (8-12 weeks) were shamed as a sham-operated group control. Mice were anesthetized with 1.5-2% isoflurane using an anesthetic agent and a heating pad was used to keep mice at 37°C throughout the procedure. Insert the suture into the right internal carotid artery through a small incision in the right external carotid artery, and slowly push it through the right internal carotid artery until it reaches the base of the middle cerebral artery, blocking the blood flow into the brain region of the middle cerebral artery. After blocking for 60 min, the suture was removed for reperfusion. Blood flow to the brain was monitored with a laser Doppler flowmeter throughout the modeling process.

After successful modeling, 6 mice in the GDEV treatment group were injected with iPSC exosomes (250 μg/ml, i.e., iPSCs were induced to culture by GDEV medium in Example 1, through the tail vein at 2 h, 24 h, 3 days and 5 days after modeling). obtained exosomes) 200 μl, that is, 50 μg/mice/time; 6 mice in the MSC EV treatment group were injected with equal volumes of mesenchymal stem cell exosomes ( 250 μg/ml, i.e. exosomes obtained by culturing MSCs in MSC medium in Example 1) 200 μl, i.e. 50 μg/mouse/time; 6 mice in the untreated group after stroke were treated at 2h, 24h, 3h after modeling, respectively. The same volume of DPBS 200μl was injected into the tail vein on days and 5; the Sham group (ie, no stroke control) did not receive any treatment. After the mice were treated according to the above corresponding groups, they were raised for 8 days and the death of the mice was recorded. They were killed on the 8th day and the survival curve was made.

The results are shown in Figure 9 and Figure 10, of which Figure 9 shows the results of the survival curves of mice in each group. It can be seen that the survival rate of the GDEV treatment group on the 8th day was 83.3%, which was significantly higher than that of the MSC EV treatment group (30%) and the post-stroke group. Untreated group (50%). Panel A in Figure 10 shows the area of cerebral infarction caused by cerebral apoplexy displayed by TTC staining after the mice were sacrificed in each group, and the white area is the ischemic infarction area; Panel B is the statistics of cerebral infarction area of each group of mice Histogram, it can be seen that the cerebral infarction area of the mice in the GDEV treatment group is significantly smaller than the untreated group and the MSC EV treatment group after stroke, indicating that compared with the exosomes obtained by culturing MSCs in the MSC medium in Example 1, the GDEV medium The exosomes obtained by inducing and culturing iPSC were more effective in protecting rat cortical neurons in a neuron model of stroke (glucose and oxygen deprivation test).

To sum up the results of Examples 1-3, it can be seen that the medium (GDEV medium) provided by the present invention can continue to cultivate stem cells in the logarithmic growth phase and the stem cells in good growth state are adhered and cultured until the coverage reaches 60%-70%. Obtain higher yield exosomes, reaching more than 1000 exosomes/cell; and the GDEV medium provided by the present invention only includes L-ascorbic acid and its salts, selenium and its salts on the basis of DMEM/F12 as a basal medium. The four components of salt, NaHCO ₃ and insulin are used as the main components, so the GDEV medium has simple components and easy preparation, which can significantly reduce the production cost of exosomes. On the other hand, compared with the exosomes obtained by culturing stem cells in the existing E8 medium and MSC medium, the exosomes obtained by the method provided by the present invention are more effective in protecting neurons from damage and improving the condition of senescent cells. Therefore, the exosomes provided by the present invention are more suitable for preparing drugs for improving the condition of senescent cells and/or protecting neurons.

The embodiments described herein are used as examples for illustration, and various modifications or changes made by a skilled person based on the embodiments should also be included within the essential scope of the patent application.

Industrial applicability

The present invention provides an exosome secretion inducer and an induction medium which can significantly increase the production of exosomes secreted by stem cells. The exosome secretion inducer and the induction medium are simple in composition and can significantly reduce the production of exosomes. cost, suitable for industrial applications.

Claims (11)

1. An exosome secretion inducer, wherein the exosome secretion inducer comprises the following components in use concentrations:

   
2. An exosome secretion induction medium, wherein the exosome secretion induction medium comprises a basal medium and the following concentrations of components:

   

   And the exosome secretion induction medium does not contain the combination of Transferrin, FGF2 and TGFβ1/NODAL.
3. The exosome secretion induction medium according to claim 2, wherein the basal medium is DMEM/F12.
4. A method for producing exosomes, comprising the steps of:

   1) Adherently culture the stem cells in logarithmic growth phase and in good growth state until the coverage rate reaches 60%-70%;

   2) Use the exosome secretion induction medium described in claim 2 or 3 to carry out the exosome secretion induction culture of the adherent cultured stem cells in step 1) for 20-30 hours, and take the supernatant to harvest the cells containing exosomes culture medium;

   3) Separating and obtaining exosomes from the cell culture medium containing exosomes harvested in step 2).
5. The method according to claim 4, wherein step 2) is repeated 2-3 times, the cell culture medium containing exosomes harvested each time is combined, and exosomes are separated from the combined cell culture medium.
6. The method according to claim 4 or 5, wherein the stem cells in step 1) are selected from embryonic stem cells, induced pluripotent stem cells and mesenchymal stem cells.
7. The method according to any one of claims 4-6, wherein the method for separation described in step 3) comprises differential centrifugation, ultrafiltration centrifugation, density gradient centrifugation, precipitation, magnetic bead immunoassay, PS Affinity, chromatography.
8. The exosome or exosome-containing cell culture fluid produced by the method of any one of claims 4-7.
9. The exosome or the cell culture solution containing exosomes according to claim 8, wherein the number of the exosomes reaches the secretion amount of exosomes in stem cells ≥ 1000/cell, and the granules of the exosomes The diameter is 30-150nm (mainly 50-80nm), the proportion of CD9 in surface markers is ≥23%, and the proportion of CD63 is ≥10%.
10. The application of the exosome according to claim 8 or 9 in the preparation of a medicine for improving the condition of senescent cells and/or protecting neurons.
11. A medicine for improving the condition of senescent cells and/or protecting neurons, comprising the exosome according to claim 8 or 9.

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