CN112920991B

CN112920991B - Exosome secretion inducer, induction medium, and exosome production method and application using exosome secretion inducer

Info

Publication number: CN112920991B
Application number: CN202011636845.1A
Authority: CN
Inventors: 张建民; 王靓; 曲典; 林埕宇
Original assignee: Guodian Beijing Medicine Technology Co ltd
Current assignee: Guodian Beijing Medicine Technology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-06-03
Anticipated expiration: 2040-12-31
Also published as: CN114836375A; CN112920991A; CN114836375B; WO2022143590A1

Abstract

The invention discloses an exosome secretion inducer, an induction medium, and a production method and application of exosomes using the same, and belongs to the technical field of biology. The exosome secretion inducing medium provided by the invention only comprises a basic medium and four additives: l-ascorbic acid or its salt, selenium or its salt, NaHCO₃And Insulin, which has simple components, but can obviously improve the yield of exosomes secreted by stem cells, thereby obviously reducing the production cost of exosomes; the obtained exosome can be produced to more effectively protect neurons from damage and improve the condition of aged cells.

Description

Exosome secretion inducer, induction medium, and exosome production method and application using exosome secretion inducer

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an exosome secretion inducer, an induction medium, a production method of an exosome using the exosome secretion inducer and application of the exosome, and especially relates to application of the exosome in preparation of a medicament for improving the condition of an aged cell and/or protecting neurons.

Background

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

Since exosomes derived from different tissues have specific protein molecules and key molecules for performing functions thereof, exosomes derived from stem cells have important therapeutic effects of stem cells and are safer, can effectively transport bioactive substances such as RNA, proteins and the like with disease therapeutic effects, and have important biological functions of inhibiting apoptosis, inhibiting inflammatory reaction, promoting angiogenesis, inhibiting fibrosis, improving tissue repair potential and the like. In addition, exosomes can penetrate blood brain barriers and can deliver various therapeutic molecules such as small molecular drugs and the like, and the exosomes have very wide clinical application prospects, so that the exosomes are particularly important to be efficiently produced and obtained from stem cells. The existing exosome acquisition mode is mainly cell autocrine, but the yield is very limited.

Disclosure of Invention

In view of one or more of the problems presented in the prior art, one aspect of the present invention provides an exosome secretion-inducing agent comprising the following ingredients in the concentrations used:

in another aspect, the present invention provides an exosome secretion inducing medium comprising a basal medium and the following concentrations of ingredients:

and the exosome secretion-inducing medium does not comprise a combination of Transferrin, FGF2 and TGF β 1/NODAL.

The basic medium was DMEM/F12.

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

1) carrying out adherent culture on stem cells with logarithmic growth phase and good growth state until the coverage rate reaches 60-70%;

2) performing exosome secretion induction culture on the stem cells subjected to adherent culture in the step 1) for 20-30 hours by using the exosome secretion induction culture medium, and collecting supernatant to obtain cell culture solution containing exosomes;

3) separating the cell culture solution containing the exosomes harvested in the step 2) to obtain exosomes;

preferably, repeating the step 2) for 2-3 times, combining cell culture solution containing the exosomes harvested each time, and separating the exosomes from the combined cell culture solution to obtain the exosomes.

In the above method, the stem cell in step 1) is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell and a mesenchymal stem cell.

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

The exosome or cell culture solution containing the exosome produced by the method is also within the protection scope of the invention, the number of the exosome reaches the secretion amount of the exosome in stem cells more than or equal to 1000/cell, the particle size of the exosome is 30-150 nm (mainly 50-80 nm), the proportion of CD9 in the surface marker is more than or equal to 23%, and the proportion of CD63 in the surface marker is more than or equal to 10%.

The application of the exosome in preparing the medicine for improving the condition of the aged cells and/or protecting the neurons also belongs to the content of the invention. Based on this, the present invention also provides a medicament for improving the condition of senescent cells and/or protecting neurons, which comprises the exosomes described above.

Based on the technical scheme, the invention provides an exosome secretion inducer for efficient exosome production for the first time, which can only contain L-ascorbic acid or salt thereof, selenium or salt thereof and NaHCO₃And insulin, therefore, the composition is simple, and when the exosome secretion induction medium is combined with a basic medium (such as DMEM/F12) for culturing stem cells (such as ESCs or iPSCs and the like) to form an exosome secretion induction medium for inducing and culturing the stem cells with good adherent production, the exosome secretion in the stem cells can be remarkably promoted, so that the yield of exosomes is remarkably improved. The results of the examples show that the yield (namely the secretion amount) of the exosome for inducing and culturing the iPSC cell with good adherence by using the exosome secretion inducing culture medium provided by the invention is more than 1000 exosomes/cell. The results of the examples also show that the exosome produced by the invention can be used in a cerebral apoplexy neuron model (glucose deprivation test, OGD) and a cerebral apoplexy neuron model (glucose deprivation test)Test) and can effectively protect rat cortical neurons and human cortical neuron axon rupture caused by oxidative damage.

Drawings

FIG. 1 is a bar graph of exosome yields of iPSC cells cultured in GDEV medium, E8 medium and MSC cells cultured in MSC medium, respectively;

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

FIG. 3 is a histogram of the nanoflow detection exosome surface markers CD9 and CD 63;

FIG. 4 is an electron micrograph of exosomes;

FIG. 5 is a photograph of a staining of Hoechst 33342 with human skin fibroblasts, umbilical cord mesenchymal stem cells and exosomes taken into neuronal cells;

FIG. 6 is a histogram of dead cell statistics in a neuron model of stroke;

FIG. 7 is a photograph of simulated Calcein AM/Hoechst staining of human cortical neuron injury;

FIG. 8 is a histogram of statistics of human cortical neuron length;

fig. 9 is a mouse survival curve in a cerebral stroke neuron model;

fig. 10 is a statistical graph of the mouse cerebral infarction area in the neuron model of cerebral apoplexy.

Detailed Description

Aiming at the defects of low yield of cell autocrine exosomes and no exosome secretion inducer and exosome secretion inducing culture medium for exosome production in the prior art, the invention aims to provide an exosome secretion inducer and an exosome secretion inducing culture medium which are used for efficiently producing exosomes from stem cells (ESC or iPSC and the like) and have simple components, and also provides a method for producing exosomes by using the inducing culture medium and application of the exosomes obtained by production.

Guokai Chen et al (reference 1: Guokai Chen et al, chemical defined conditions for Human iPSC derivative and culture, NATURE METHODS, 2011.4.10 days) and Senquan Liu et al (reference 2: Senquan Liu et al, high pure Purified Human Ex)the Stem Cells (ESCs or iPSCs) were routinely cultured using E8 medium (ESCs or iPSCs) which has been shown to improve the efficiency of derivation of Stem Cells ESCs and iPSCs, E8 medium comprising: DMEM/F12, L-Ascorbic Acid (L-Ascorbic Acid), Selenium (Selenium), Transferrin (Transferrin), NaHCO₃Lnulin (Insulin), FGF2, TGF β 1/NODAL, wherein lnulin and FGF2 are important for cell survival and proliferation, L-ascorbyl Acid promotes proliferation of ESCs, Selenium is essential for continuous culture propagation, Transferrin contributes to increased cloning efficiency, TGF β 1/NODAL contributes to long-term culture stability; however, the E8 medium is mainly used for propagation culture of stem cells, and the yield of exosome produced by the cultured stem cells is still low, so that the requirement for efficient exosome production is difficult to meet. Based on the E8 medium disclosed in documents 1 and 2, the inventors have surprisingly found that when only L-Ascorbic Acid, Selenium, Transferrin, NaHCO among the components of the E8 medium are used₃And when the four components of the Insulin are matched and used for a basic culture medium for stem cell culture, the stem cells with good adherent growth can be effectively induced to secrete a large amount of exosomes, so that the yield of the exosomes is obviously improved, and the requirement for efficiently producing the exosomes is met.

The present invention will be described in detail with reference to the following embodiments and drawings.

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, 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 as restrictive.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure only and should not be limiting as to the source of the biological material of the present invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

In the following examples, exosomes were produced by taking induced pluripotent stem cells (ipscs), which were produced by introducing some pluripotent genes into cells such as skin, and were made to have a stem cell function by "initializing" ordinary somatic cells. The ipscs are very similar to Embryonic Stem Cells (ESCs) in terms of cell morphology, growth characteristics, stem cell marker expression, and the like, and are almost identical to ESCs in terms of DNA methylation patterns, gene expression profiles, chromatin states, chimeric animal formation, and the like, and thus the object of the present invention can be achieved by using ESCs as well.

Example 1: exosome secretion induction culture medium and use method thereof

The embodiment provides an exosome secretion induction culture medium for efficiently producing exosomes, which is suitable for high-yield exosome production.

An exosome secretion inducing medium (designated as GDEV medium) for efficiently producing exosomes from stem cells (ESC or iPSC, etc.) of the present invention is prepared, and its composition comprises: DMEM/F12(Gibco 11330032) as basal medium and an exosome secretion inducer. The exosome secretion inducer is a component with the following use concentration: L-Ascorbic acid 2phosphate magnesium salt (magnesium L-ascorbate diphosphate, Sigma-Aldrich A8960) at a concentration of 64mg/L, sodium selenium (sodium selenium, Sigma-Aldrich S5261) at a concentration of 14. mu.g/L, NaHCO at a concentration of 543mg/L₃(Sigma-Aldrich S5761), Insulin (Insulin, Puxitang I10022) at a concentration of 19.4 mg/L.

As a control, the culture medium E8 was prepared according to the method disclosed in document 1, specifically: DMEM/F12, 64mg/L L-Ascorbic acid 2phosphate magnesium salt (L-Ascorbic acid magnesium diphosphate), 14. mu.g/L sodium selenium, 100. mu.g/L FGF2 (fibroblast growth factor 2), 19.4mg/L insulin, 543mg/L NaHCO₃10.7mg/L transferrin (transferrin), 2. mu.g/L TGF beta 1 (transforming growth factor-. beta.1) or 100. mu.g/L Nodal.

The method comprises the following steps of respectively carrying out induction culture on iPSC cells with the adherence coverage rate of 60-70% by using the prepared GDEV culture medium and E8 culture medium, then collecting exosomes in iPSC cell culture supernatant and detecting the content of the exosomes.

1.1 cell culture

The iPSC cells (product of national classic (Beijing) pharmaceutical technology Co., Ltd.) were subjected to amplification culture (for example, using Essential 8)^TMThe Medium kit (purchased from Thermo Fisher, goods number: A1517001) is subjected to conventional amplification culture) to logarithmic growth phase and has good state, the Medium kit is digested into small cell clusters (3-10 cells) according to the technical manual of the Versene Solution kit (purchased from Thermo Fisher, goods number 15040066), and then the small cell clusters are paved in 10cm cell culture dishes coated by Vitronectin (purchased from Recombinant Human vironectin, goods number 140-09), the coverage rate after adherence reaches 60% -70%, 37 ℃, 95% air and 5% CO₂The culture was performed overnight under an atmosphere. If the iPSC adherence condition is good, the liquid change is GDEV culture medium or E8 culture medium, the liquid change is carried out 24 hours later, cell culture supernatant is collected, the operation of the previous step is repeated, and one batch of cells can be collected for 2-3 times.

Synchronous utilization of MSC

The XF human mesenchymal stem cell serum-free medium (BI, named MSC medium in the examples) of (1) cultures human mesenchymal stem cells (MSC, obtained according to a conventional isolation method) to obtain a cell culture supernatant.

1.2 collecting exosomes in cell culture supernatant

Cell culture supernatants obtained by inducing and culturing the cells by using the GDEV culture medium, the E8 culture medium and the MSC culture medium in the step 1.1 are respectively centrifuged at the room temperature for 15min under the condition of 3000g, and dead cells and cell debris are removed. Collecting supernatant, and filtering with 0.22 μm filter; the filtrate was transferred to an Amicon Ultra-15(100kDa) or Centricon Plus-70(100kDa) ultrafiltration tube for concentration and centrifuged at 3000g at room temperature for 30 min. The concentrate was mixed with DPBS (dunaliella phosphate buffer) in a ratio of about 1: 100 and then concentrated again using the same apparatus. So as to obtain relatively pure exosome solution. The step adopts ultrafiltration to separate exosome in cell culture supernatant. The exosome in the cell culture supernatant may also be isolated by differential centrifugation, density gradient centrifugation, precipitation, magnetic bead immunization, PS affinity, chromatography, or the like.

1.3 exosome yield detection

Respectively taking 20 μ l of the exosome solution collected in the step 1.2 and obtained by culturing in GDEV culture medium, E8 culture medium and MSC culture medium, diluting to 100 μ l with PBS, adding a nano-flow detector (brand: NANOFCM), and operating according to the instrument technical manual to obtain the respective exosome yield.

As a result, as shown in table 1 below and fig. 1, it can be seen that more exosomes (1013.9/cell) can be obtained by induction-culturing ipscs using GDEV medium, which is 5.4 times the number of exosomes (187.5/cell) obtained by induction-culturing iPSC cells using E8 medium disclosed in document 1. Meanwhile, the yield of exosomes obtained by induction culture of ipscs by using a GDEV culture medium and the yield of exosomes obtained by induction culture of MSCs by using an MSC culture medium were compared, and the former was found to be 59 times as high as the latter. GDEV medium was shown to significantly improve the production of exosomes by stem cells (e.g., ipscs) relative to E8 medium and MSC medium. On the other hand, the GDEV culture medium only includes five components in composition, which is simple compared to the E8 culture medium including eight components, but can greatly improve the yield of exosomes, thereby significantly reducing the production cost of exosomes.

Table 1: production of exosomes by culturing cells in different media

	GDEV culture medium	E8 Medium	MSC culture medium
				Exosome yield (cell/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

Taking 20 mu l of exosome solution obtained by induced culture of iPSC (isopropyl-beta-D-alanine) by a GDEV (GDEV) culture medium, diluting the exosome solution to 100 mu l by PBS, adding a nano-flow detector (brand: NANOFCM), operating according to an instrument and technology manual, and obtaining a particle size distribution diagram as shown in figure 2, so that the concentration of particles with the particle size of 50-80 nm (about 60nm) in the exosome solution obtained by production is the highest, the particle size range (30-150 nm) of the exosomes secreted by the iPSC is met, and the exosomes are proved to be obtained. The grain size of exosome obtained by induced culture of iPSC in GDEV culture medium is mainly 50-80 nm by adopting a nano-flow detector, and when the exosome is detected by adopting other methods, the grain size in other ranges of 30-150 nm can be obtained.

1.4.2 Nanoflow detection of exosome surface markers

To 10 μ g of an exosome solution obtained by induction-culturing ipscs from GDEV medium, 1 μ l of an antibody (CD9 antibody or CD63 antibody) was added. Adding antibody, covering the centrifuge tube tightly, mixing with vortex oscillator for 1min, and incubating at 37 deg.C in dark for 30 min; adding 1mL of 1 XPBS into the incubated exosome-antibody complex, uniformly mixing, and extracting exosomes again according to the exosome separation method in the step 1.2 to remove free dyes; and (4) carrying out nano-flow detection on the marked exosome.

The results are shown in fig. 3, which is a histogram of the exosome surface markers CD9 and CD63 detected by nano-flow, and it can be seen that the proportions of CD9 and CD63 positive particles in the labeled exosome surface markers in the total particle number are 25.8% and 11.9%, respectively, which proves that exosomes are indeed obtained by induction culture of ipscs in GDEV culture medium.

1.4.3 Electron microscopy of exosome morphology

(1) The exosomes were immobilized on loaded copper mesh: mixing 50 mu l of exosome solution obtained by induced culture of IPSC (iPSC) by a GDEV culture medium with equivalent 4% PFA (paraformaldehyde) fixing solution to obtain exosome suspension, and adding 5 mu l of exosome suspension to a Formvar-carbon sample-loading copper net; 100 μ l PBS was added to the sealing membrane. Washing the copper mesh (Formvar membrane face down) on the PBS drop with tweezers; placing copper net on 50 μ l 1% glutaraldehyde drop for 5 min; put in 100. mu.l ddH₂In O for 2min (8 washes).

(2) Carrying out negative staining treatment on exosomes and carrying out electron microscopy detection: placing the copper net on 50 μ l uranium oxalate dioxygen liquid drop for 5 min; placing the copper net on 50 μ l methyl cellulose drop for 10min, and operating on ice; placing a copper net on a stainless steel ring at the top end of the sample table, and sucking redundant liquid on filter paper; drying in air for 5min to 10 min; an electron micrograph was taken at 80 kV.

The results are shown in fig. 4, which is an electron micrograph of an exosome obtained by induced culture of ipscs in GDEV medium, and it can be seen that the produced exosome has an exosome typical structure (usually, a saucer type or a hemisphere with one side depressed).

Example 2: exosome secretion inducer

The present example provides an exosome secretion inducer for efficient production of exosomes, suitable for high-yield exosome production.

The exosome secretion inducers for efficiently producing exosomes according to the present invention (named GD1, GD2, and GD3, respectively, in which the concentrations of the respective components are in the basal medium DMEM/F12) were prepared according to the following table 2, and ipscs were induced-cultured with each of the exosome secretion inducers and the basal medium DMEM/F12 according to the procedures of steps 1.1 to 1.3 in the above example 1, followed by collecting exosomes in the cell culture supernatant and detecting the exosome content, respectively, and the results are shown in the following table 3.

Table 2: formula of exosome secretion inducer

Table 3: induction of exosome yield in cultured ipscs using GD1, GD2 and GD3 inducers and basal medium DMEM/F12

Induction agent	Basic culture medium	Exosome yield (cell/cell)
			GD1	DMEM/F12	1171
GD2	DMEM/F12	1092
			GD3	DMEM/F12	1208

As can be seen from the results in tables 2 and 3 above, the exosome secretion inducing agent for efficiently producing exosomes provided by the present invention (comprising L-magnesium ascorbate diphosphate used at a concentration of 15-100mg/L, sodium selenium used at a concentration of 2-100 μ g/L, NaHCO used at a concentration of 200-1000 mg/L)₃And insulin with the concentration of 5-30 mg/L) is used in combination with a basal medium DMEM/F12 to induce and culture the iPSC cells with good adherent growth, the secretion of exosomes by the iPSC cells can be remarkably promoted, the yield (namely the secretion) of the exosomes is over 1000/cell, and the purpose of efficiently producing the exosomes is realized. And the invention providesThe provided exosome secretion inducer can only comprise four components, so the components are simple and easy to prepare.

The invention provides an exosome secretion inducer for efficiently producing exosomes for the first time, and the exosome secretion inducer comprises the components of L-ascorbic acid or other salts thereof and selenium or other salts thereof besides the L-ascorbic acid diphosphate magnesium salt and sodium selenium. Wherein L-ascorbic acid (vitamin C) is an essential vitamin for keeping cells in vivo and in vitro healthy growth and maintenance, and is a water-soluble antioxidant which can prevent the peroxidation of esterified and non-esterified unsaturated fatty acids, but in the exosome secretion inducer provided by the invention, when the concentration of the L-ascorbic acid is lower than 15mg/L or higher than 100mg/L, the L-ascorbic acid affects the growth state of stem cells and can possibly cause the secretion amount of exosomes to be reduced; selenium is an essential trace element for normal cell growth and development in vivo and in vitro, can be incorporated into enzymes and protects cells by reducing peroxides, organic hydroperoxides and peroxynitrites into harmless substances, selenium-containing enzymes with various antioxidant functions and different substrate specificities are located in cells, on cell surfaces and outside cells, and the enzymes jointly form a complete oxidative damage defense system, but in the exosome secretion inducer provided by the present invention, when the concentration of the selenium-containing enzymes is lower than 2 mug/L or higher than 100 mug/L, the growth state of stem cells is affected, and the exosome secretion amount is possibly reduced; when the cells are cultured, the cells can grow in an open system (the air on the upper layer of the culture medium and the air in the incubator can be freely exchanged), and the cells need to contain NaHCO with certain concentration₃The pH of the solution is maintained at a concentration such that NaHCO is present in the system₃Too high or too low a concentration may affect the growth state of the cells and thus the secretion of extracellular exosomes; insulin stimulates the absorption of uridine and glucose by cells to synthesize RNA, proteins and lipids, and also binds to insulin receptors on cell membranes to regulate various metabolic pathways in cells, increases the synthesis of fatty acids and glucose, is considered to be a key factor for regulating most of cell growth and differentiation, and plays an important role in cell growth, but in the exosome secretion inducer provided by the present invention, when the concentration thereof is lower than 5mg/L or higher than 30mg/L, cells are inducedMorphological abnormalities and growth rate disorders may occur, which may lead to a decrease in the secretion of exosomes. Therefore, in order to stably improve the yield of the exosome, the use concentration of the L-ascorbic acid or the salt thereof in the exosome secretion inducer provided by the invention is limited within the range of 15-100mg/L, and can be selected from 15-50mg/L, 15-64mg/L, 50-100mg/L, 64-100mg/L and the like; the concentration of selenium or its salt is limited to 2-100 μ g/L, and can be selected from 2-14 μ g/L, 2-20 μ g/L, 14-100 μ g/L, 20-100 μ g/L, etc.; NaHCO 2₃The use concentration is limited within the range of 200-1000mg/L, and can be selected from 200-500mg/L, 200-543mg/L, 500-1000mg/L, 543-1000mg/L, etc.; the concentration of insulin is limited to 5-30mg/L, and may be selected from 5-19.4mg/L, 5-20mg/L, 19.4-30mg/L, 20-30mg/L, etc.

Example 3: application of exosome

This example uses exosome solution obtained by induction-culturing ipscs in GDEV medium in the above example 1 to verify its effect in a stroke neuron model (glycoxygen deprivation test, OGD), improvement of senescent cell condition, and a stroke neuron model (glycoxygen deprivation test).

3.1 uptake of exosomes

1) The Exosome solution was labeled with PKH26 according to the technical manual for the PKH26 Red Fluorescent Cell Linker Mini Kit (available from Sigma, cat # Mini26) Kit, and excess dye was removed using the technical manual for the Exosome Spin Columns (MW 3000) (available from Invitrogen, cat # 4484449) Kit to obtain a labeled Exosome solution.

2) According to exosomes: the cells were 10000: 1, adding the marked exosome solution into human skin fibroblasts, umbilical cord mesenchymal stem cells and neuronal cells (a commodity of national classic (Beijing) medicine and technology Co., Ltd., a human skin fibroblast culture medium is DMEM + 15% FBS + NEAA, an umbilical cord mesenchymal stem cell culture medium is DMEM + 10% FBS, a neuronal cell culture medium is B27+ Neurobasal + GlutaMAX, the culture conditions are 37 ℃ and 5% CO₂) Culture medium, at 37 deg.C, 95% air and 5% CO₂Incubate for 24 hours, wash the cells once with PBS, addHoechst 33342 (purchased from Sigma-Aldrich) stained nuclei to a final concentration of 5. mu.g/ml and photographed under a fluorescent microscope.

Results as shown in fig. 5, showing the cell uptake of exosomes obtained from GDEV culture medium induction culture iPSC in example 1, wherein a is umbilical cord mesenchymal stem cells added with exosomes; b is human skin fibroblast added with exosome; the C amplitude is a human neuron cell added with an exosome; h is a Hoechst 33342 signal, indicating the nucleus; p is the PKH26 signal, indicating an exosome. Obviously, exosomes can be taken into cells by human skin fibroblasts, umbilical cord mesenchymal stem cells and neuronal cells.

3.2 exosomes effectively protect rat cortical neurons in cerebral apoplexy neuron model (sugar oxygen deprivation test, OGD)

(1) After 14 days in vitro culture with primary rat neurons, neuronal cultures were changed to sugar-free anaerobic cultures (95% N in advance)₂/5％CO₂Equilibrium), neurons were placed in the OGD chamber for 90 minutes (10 minutes with 95% N2/5% CO2 air);

(2) after the treatment, the control group (designated as OGD group) was changed to normal neuron culture solution, experimental group 1 (designated as iPSC EV group) was changed to normal neuron culture solution and added with 1 μ g/ml or 0.2 μ g/ml iPSC exosomes (i.e., GDEV exosomes obtained by induction culture of ipscs from GDEV culture medium in example 1), experimental group 2 (designated as MSC group) was changed to normal neuron culture solution and added with 1 μ g/ml or 0.2 μ g/ml MSC exosomes (i.e., MSC exosomes obtained by culture of MSCs from MSC culture medium in example 1), experimental group 3 (designated as E8 group) was changed to normal neuron culture solution and added with 1 μ g/ml or 0.2 μ g/ml E8 exosomes (i.e., e. E8 exosomes obtained by induction culture of ipscs from E8 culture medium in example 1), and then 95% air/5% CO was added.₂After 24 hours of incubation in an incubator, the cells were stained with Hoechst 33342/PI and analyzed for neuronal activity by high content photography.

The results are shown in FIG. 6, which is a histogram of the statistical proportion of dead cells, where NO OGD indicates that NO OGD model was performed and DPQ indicates that cells were treated with the addition of DPQ (an inhibitor of PARP-1 (poly (ribose-diphosphate) polymerase-1)), which has been shown to reduce apoptosis under the effects of ischemia. It can be seen that the ratio of dead cells is significantly reduced compared to the control group (OGD group), the experimental group 2 (i.e., MSC group) and the experimental group 3 (i.e., E8 group), and the ratio of dead cells in the experimental group 1 (i.e., iPSC EV group) is significantly reduced compared to the experimental group 3 (i.e., E8 group) and the experimental group 2 (i.e., MSC group) under the same exosome addition concentration, which indicates that exosomes obtained by induction culture of ipscs in GDEV medium can more effectively protect rat cortical neurons in the OGD model.

3.3 exosomes can effectively protect neuronal axon rupture caused by oxidative damage

The oxidative damage of nerve cells related to aging is simulated by damaging human cortical neurons by hydrogen peroxide. The method specifically comprises the following steps:

(1) recovering iPSC differentiated cortical neuron of normal human, spreading in 96-well plate, culturing for 7 days, preparing Neurobasal Medium (B27+ Neurobasal + GlutaMAX) and adding H in experimental group 1 except untreated control group₂O₂To a final concentration of 20. mu.M (designated H)₂O₂Group), experimental group 2 was prepared with Neurobasal Medium and added with H₂O₂To a final concentration of 20. mu.M, the iPSC exosomes obtained from the induced culture of iPSC in GDEV medium in example 1 were added to a final concentration of 5. mu.g/ml (designated as H)₂O₂+ EV group), completely replacing the liquid, and establishing a hydrogen peroxide damage model;

(2) after adding hydrogen peroxide for treatment for 16h, using Calcein AM/Hoechst to stain neurons, taking high content pictures, and counting data such as neuron protrusion length.

The results are shown in FIG. 7 and FIG. 8, in which FIG. 7 is a photograph of Calcein AM/Hoechst stained neurons, and FIG. 8 is a histogram of the length of each group of neurons. Can see H₂O₂After the iPSC exosomes are added into the + EV group, the length of the axon of the neuron is obviously longer than that of the group without the exosomes (namely H)₂O₂Group), and thus exosomes, may effectively protect against neuronal axonal rupture caused by oxidative damage.

3.4 exosomes effectively protect rat cortical neurons in cerebral apoplexy neuron model (glucose oxygen deprivation test)

The Middle Cerebral Artery Occlusion (MCAO) modeling scheme is as follows: 18 male C57 mice (8-12 weeks, from Witongli animal technology, Inc.) were molded with MCAO. Meanwhile, 6 male C57 mice (8-12 weeks) were sham-operated as sham-operated controls. Mice were anesthetized with 1.5-2% isoflurane using an anesthetic and were maintained at 37 ℃ throughout the procedure using a heating pad. The wire plug is inserted into the right internal carotid artery through a small incision of the right external carotid artery, and is slowly pushed through the right internal carotid artery until reaching the base of the middle cerebral artery, so that the blood flow entering the cerebral region of the middle cerebral artery is blocked. After blocking for 60min, the wire plug was removed for reperfusion. Blood flow to the brain was monitored throughout the modeling process using a laser doppler blood flow meter.

After the model building is successful, the 6 mice in the GDEV treatment group are injected with 200 mu l of iPSC exosome (250 mu g/ml, namely exosome obtained by induced culture of iPSC in GDEV culture medium in example 1) in tail vein 2h, 24h, 3 days and 5 days after the model building respectively, namely 50 mu g/mouse per time; MSC EV treatment group 6 mice were injected with equal volume of mesenchymal stem cell exosomes (250 μ g/ml, exosomes obtained from MSC culture medium in example 1) at tail vein 2h, 24h, 3 days and 5 days after molding, respectively, 200 μ l, i.e. 50 μ g/mouse; after cerebral apoplexy, 6 mice in an untreated group are injected with equal volume of 200 mu l of DPBS in tail vein after 2h, 24h, 3 days and 5 days of molding respectively; sham group (i.e. no stroke control) did not have any treatment. After the mice are treated according to the corresponding groups, the mice are raised for 8 days, the death condition of the mice is recorded, the mice are sacrificed on the 8 th day, and a survival curve is made.

The results are shown in fig. 9 and fig. 10, wherein fig. 9 is the results of survival curves of mice in each group, and it can be seen that the survival rate on day 8 of the GDEV-treated group is 83.3%, which is significantly higher than that of the MSC EV-treated group (30%) and the untreated group after stroke (50%). In fig. 10, panel a shows the area of cerebral infarction caused by stroke, which is displayed by TTC staining after the brain is taken from each group of mice after they are sacrificed, wherein the white area is the ischemic infarction area; the B is a histogram for statistics of cerebral infarction areas of mice in each group, and it can be seen that the cerebral infarction areas of the mice in the GDEV treatment group are obviously smaller than those of untreated groups and MSC EV treatment groups after cerebral apoplexy, which indicates that compared with exosomes obtained by culturing MSC in MSC culture medium in example 1, exosomes obtained by induced culture of iPSC in GDEV culture medium can protect rat cortical neurons more effectively in a cerebral apoplexy neuron model (glucose deprivation test).

To sum up, the results of the embodiments 1 to 3 show that the exosomes with higher yield can be obtained by continuously culturing the stem cells with the culture medium (GDEV culture medium) provided by the present invention after the stem cells with logarithmic growth phase and good growth state are cultured in an adherent manner until the coverage rate reaches 60% to 70%, and the yield reaches more than 1000 exosomes/cell; the GDEV culture medium provided by the invention only comprises L-ascorbic acid and salts thereof, selenium and salts thereof and NaHCO on the basis of DMEM/F12 serving as a basic culture medium₃And insulin, so the GDEV culture medium has simple components and easy preparation, and can obviously reduce the production cost of exosomes. On the other hand, compared with the exosome obtained by culturing stem cells by using the existing E8 culture medium and MSC culture medium, the exosome obtained by the method provided by the invention has better effects on protecting neurons from damage and improving the condition of the aged cells, so that the exosome provided by the invention is more suitable for preparing a medicine for improving the condition of the aged cells and/or protecting the neurons.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of producing exosomes comprising the steps of:

1) carrying out adherent culture on stem cells in a logarithmic growth phase and in a good growth state until the coverage rate reaches 60-70%, wherein the stem cells are selected from induced pluripotent stem cells and mesenchymal stem cells;

2) performing exosome secretion induction culture on the stem cells subjected to adherent culture in the step 1) for 20-30 hours by using a basic culture medium containing an exosome secretion inducer, and collecting the supernatant to obtain a cell culture solution containing exosomes;

3) separating the exosome from the cell culture solution containing the exosome harvested in the step 2) to obtain exosomes;

wherein the exosome secretion-inducing agent is a component at the following use concentrations:

l-ascorbic acid or a salt thereof 15-100 mg/L;

selenium or salt thereof is 2-100 mug/L;

NaHCO₃ 200-1000 mg/L；

5-30mg/L of insulin.

2. The method of claim 1, further comprising: and (3) repeating the step 2) for 2-3 times, combining the cell culture solution containing the exosomes harvested each time, and separating the exosomes from the combined cell culture solution to obtain the exosomes.

3. The method according to claim 1 or 2, wherein the basal medium in step 2) is DMEM/F12.

4. The method according to claim 1 or 2, wherein the separation in step 3) comprises differential centrifugation, ultrafiltration centrifugation, density gradient centrifugation, sedimentation, magnetic bead immunization, PS affinity, chromatography.

5. The method according to claim 1 or 2, wherein the number of the exosomes obtained in the step 3) is more than or equal to 1000 exosomes per cell, the particle size of the exosomes is 30-150 nm, the ratio of CD9 in the surface marker is more than or equal to 23%, and the ratio of CD63 in the surface marker is more than or equal to 10%.