CN117568270A - High-efficiency release agent for steady-state mesenchymal stem cell exosome and application thereof - Google Patents
High-efficiency release agent for steady-state mesenchymal stem cell exosome and application thereof Download PDFInfo
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- CN117568270A CN117568270A CN202311550582.6A CN202311550582A CN117568270A CN 117568270 A CN117568270 A CN 117568270A CN 202311550582 A CN202311550582 A CN 202311550582A CN 117568270 A CN117568270 A CN 117568270A
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a steady-state mesenchymal stem cell exosome high-efficiency release agent and application thereof. The invention provides a steady-state mesenchymal stem cell exosome high-efficiency release agent, which comprises monensin sodium, gentamicin, ESCRT related proteins, alix, HSP90, sphingomyelin, glycosphingolipids, cholesterol, phosphatidylinositol phosphate, ceramide, phosphatidylserine, curcumin, EGCG, resveratrol, sulforaphane, beta-catenin, rabs family proteins and integrins. The steady-state mesenchymal stem cell exosome high-efficiency releasing agent not only effectively promotes the release of the mesenchymal stem cell exosome, but also overcomes the defects of less exosome acquisition, low acquisition rate, insufficient purity and the like in the traditional method for acquiring the exosome by the mesenchymal stem cell in vitro culture, so that the mesenchymal stem cell can stably and efficiently release the exosome.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a steady-state mesenchymal stem cell exosome high-efficiency release agent and application thereof.
Background
Exosomes (Exosomes) are vesicles (Extracellular Vesicles, EVs) secreted by cells to the outside of the cell, having a size of 30-150nm, a bilayer membrane structure and a tea-tray-like morphology, containing abundant contents (including nucleic acids, proteins, lipids, etc.), involved in the transfer of molecules between cells. Is widely present in cell culture supernatants as well as in various body fluids, which makes exosomes attractive in liquid biopsies, useful for longitudinal sampling to follow disease progression. The exosome biogenesis can capture complex extracellular and intracellular molecules, and can be used for comprehensive and multiparameter diagnostic detection. The surface proteins of the exosomes also contribute to their immunocapture and enrichment. In recent years, exosomes have received widespread attention in biological and medical research, for example, as biomarkers of disease for diagnosing and monitoring a variety of diseases, such as tumors, neurodegenerative diseases; the target cell carries beneficial bioactive molecules such as growth factors, microRNA and the like, and the aim of treating diseases is achieved by regulating the biological functions of the target cell. In addition, the exosomes can also be used as a drug delivery carrier, so that the bioavailability and the therapeutic effect of the drug are improved.
Biomedical treatment technology based on exosomes is mature day by day, can transmit information among cells, thereby adjust cell behavior and tissue structure, promote tissue repair regeneration, can directly target focus treatment disease, and compared with the current widely applied liposome drug-loaded medicine, exosome drug-loaded medicine has advantages of good biocompatibility, strong stability, low immune rejection and easy absorption by cells, etc., however extraction conditions of exosomes are harsh and the cost is high, so that a simple, rapid and low-cost method for stimulating exosome yield has important social and economic significance and clinical value.
Exosomes are a recent emerging scientific research hotspot. Although many of the mechanisms of its secretory process are not yet defined, regulating secretion of exosomes in cells is a complex process. At present, methods for increasing the release amount of extracellular bodies include a low-intensity pulse ultrasonic stimulation method, an LED different light irradiation stimulation method and a special culture medium treatment method in addition to changing gene expression. However, these methods have the disadvantages of complicated operation, easy damage to vesicle (exosome) membrane structure, low exosome acquisition rate, more residual impurity proteins, and the like, and have the problems of incapability of preserving the complete bioactivity of stem cells, influence on subsequent differentiation, and the like. Therefore, a method for increasing the content of exosomes released from mesenchymal stem cells is needed to solve the above-mentioned problems.
Disclosure of Invention
In order to solve the problems, the invention provides a steady-state mesenchymal stem cell exosome high-efficiency release agent and application thereof. The steady-state high-efficiency release agent for promoting the exosome of the mesenchymal stem cells ensures that the mesenchymal stem cells stably and efficiently release the exosome while maintaining the original bioactivity of the stem cells in the in-vitro culture process.
The invention provides a steady-state mesenchymal stem cell exosome high-efficiency release agent, which comprises the following components: sodium monensin, gentamicin, ESCRT related proteins, alix, HSP90, sphingomyelin, glycosphingolipids, cholesterol, phosphatidylinositol phosphates, ceramides, phosphatidylserine, curcumin, EGCG, resveratrol, sulforaphane, β -catenin, rabs family proteins, integrins.
Preferably, the concentration of each component in the steady-state mesenchymal stem cell exosome high-efficiency release agent is respectively as follows: 2.0 to 8.0 mu mol/mL of monensin sodium, 1 to 100 mu g/mL of gentamicin, 10 to 150ng/mL of ESCRT related protein, 10 to 60pg/mL of Alix, 5 to 30ng/mL of HSP, 20 to 600 mu g/mL of sphingomyelin, 20 to 300 mu g/mL of glycosphingolipid, 1 to 100mg/mL of cholesterol, 5 to 300 mu g/mL of phosphatidylinositol, 5 to 300mg/mL of ceramide, 10 to 100mg/mL of phosphatidylserine, 5 to 50 mu g/mL of curcumin, 10 to 150mg/mL of EGCG, 10 to 100 mu g/mL of resveratrol, 1 to 10 mu mol/mL of glucoraphanin, 5 to 100IU/mL of beta-catenin, 5 to 100IU/mL of Rabs family protein, and 0.5 to 8.0 mu g/mL of integrin.
Preferably, the concentration of each component in the steady-state mesenchymal stem cell exosome high-efficiency release agent is respectively as follows: 1.0 to 5.0 mu mol/mL of monensin sodium, 10 to 50 mu g/mL of gentamicin, 50 to 100 mu g/mL of ESCRT related protein, 20 to 50 mu mol/mL of Alix, 10 to 15 mu g/mL of HSP902.5, 50 to 400 mu g/mL of sphingomyelin, 10 to 200 mu g/mL of glycosphingolipids, 10 to 50mg/mL of cholesterol, 10 to 200 mu g/mL of phosphatidylinositol, 10 to 400mg/mL of ceramide, 20 to 50mg/mL of phosphatidylserine, 10 to 30 mu g/mL of curcumin, 20 to 125mg/mL of EGCG, 20 to 40 mu g/mL of resveratrol, 1.5 to 3.5 mu mol/mL of glucoraphanin, 10 to 60IU/mL of beta-catenin, 20 to 50IU/mL of Rabs family protein, and 1.0 to 4.0 mu g/mL of integrin.
Preferably, the solvent of the steady-state mesenchymal stem cell exosome high-efficiency releasing agent is a serum-free culture medium.
Preferably, the mesenchymal stem cells comprise: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells and dental pulp mesenchymal stem cells.
The invention also provides application of the steady-state mesenchymal stem cell exosome high-efficiency release agent in exosome release promotion of mesenchymal stem cells.
The invention also provides application of the steady-state mesenchymal stem cell exosome high-efficiency releasing agent in maintaining mesenchymal stem cell proliferation capacity.
The invention also provides application of the steady-state mesenchymal stem cell exosome high-efficiency releasing agent in maintaining the multidirectional differentiation potential of mesenchymal stem cells.
The invention also provides a mesenchymal stem cell serum-free medium containing the steady-state mesenchymal stem cell exosome high-efficiency release agent.
Preferably, the volume ratio of the steady-state mesenchymal stem cell exosome high-efficiency release agent to the mesenchymal stem cell serum-free medium is 1:50.
The invention provides a steady-state mesenchymal stem cell exosome high-efficiency release agent, wherein sodium monensin in the steady-state mesenchymal stem cell exosome high-efficiency release agent is used as a sodium ion carrier to influence the balance of sodium ions and calcium in exosome secretion and promote the generation and release of exosome; gentamicin can interfere the uptake of calcium ions by the endoplasmic reticulum of cells, thereby affecting the generation of exosomes; the ESCRT related proteins help to deform the membrane into buds with chelate vesicles, target endocytic membranes, assemble other ESCRT complexes by interaction, promote exosome formation; alix is involved in regulating endocytic membrane transport, cell adhesion formed by interaction with ESCRT (endosomal sorting complex required for transport), endophilin and CIN85 (Cbl 85kDa interacting protein), promoting exosome biogenesis; HSP90 is able to bind to and alter the structure of cellular membranes, interact directly with cellular membranes, stimulate fusion of vesicles-encapsulating small bodies with cytoplasmic membranes, and thereby promote release of exosomes; sphingomyelin, glycosphingolipids and cholesterol are key substances for the formation of exosome phospholipid bilayer, involved in the early endosome development to mature multivesicular body; phosphatidylinositol phosphate, ceramide and phosphatidylserine as essential components of exosomes play a guiding role in vesicle formation and transport, and play a promoting role in exosomes formation and transport; curcumin, EGCG, resveratrol and sulforaphane can inhibit mTOR signal path in the formation process of exosomes, inhibit protein decomposition and autophagy, and promote exosome secretion; beta-catenin participates in signal transduction generated by exosomes, thereby promoting the generation of exosomes; rab family proteins are also present in exosomes and are involved in the biogenesis, release, transport and docking of MVB to the plasma membrane of exosomes; integrins are involved in intercellular interactions, stimulating exosome production. The steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided by the invention not only effectively maintains normal mesenchymal stem cell in-vitro proliferation culture, but also overcomes the defects of low acquisition rate, low purity, large batch-to-batch difference and the like of the traditional mesenchymal stem cell exosome, so that the mesenchymal stem cell can stably secrete the exosome. The components used by the release promoter are a premium grade cell factor and a medical grade recombinant protein or reagent, no exogenous animal source protein is introduced, the components are definite, the subsequent related research work is convenient to develop, and researchers can culture mesenchymal stem cells for exosome culture by using the release promoter.
The results of the examples show that the high-efficiency release agent for promoting the mesenchymal stem cell exosome stably improves the content of the exosome under the condition of ensuring the steady-state development of the mesenchymal stem cell under the synergistic effect of a plurality of components.
Drawings
FIG. 1 is a transmission electron microscope of umbilical cord mesenchymal stem cell exosomes using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided in the present invention in example 1;
FIG. 2 is a flow chart of the P3 generation umbilical cord mesenchymal stem cells using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided by the present invention in example 3 (detection surface antigens are CD34, CD44, CD105, CD73, HLA-DR, CD45, CD19, CD90, CD11 b);
FIG. 3 is a flow chart of the mesenchymal stem cells of the P20 generation using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided by the present invention in example 3 (detection surface antigens are CD34, CD44, CD105, CD73, HLA-DR, CD45, CD19, CD90, CD11 b);
FIG. 4 is a diagram showing the differentiation of umbilical cord mesenchymal stem cells into lipid-forming cells using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided in example 3;
FIG. 5 is a diagram showing the differentiation of umbilical cord mesenchymal stem cells into osteoblasts using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided in example 3;
FIG. 6 is a graph showing the differentiation of umbilical cord mesenchymal stem cells into chondrocytes using the steady-state mesenchymal stem cell exosome high-efficiency releasing agent provided by the present invention in example 3;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were all purchased from conventional reagent manufacturers.
The experimental environment, experimental materials and instrument equipment required to be prompted and described in the invention are as follows:
1. experimental environment: operating in a clean bench in a laboratory in a GMP environment.
2. Reagent: BC-T4& BC-T4 PhenolRed (-) serum-free medium, phosphate buffer PBS (Pinctada Biotechnology Co., ltd.), r-TE recombinant pancreatin, S-TI pancreatin stop (CSTIS of cell science research Co., ltd.); mesenchymal stem cell culture medium additive MSC-XF (nacreous biotechnology limited);
3. instrument and apparatus: centrifuges (Thermo, U.S.), T75 flasks (TC treatment), CO 2 Incubator (Sanyang, china), ultra clean bench (Zhijing, china), inverted microscope.
Example 1
(1) Preparation of release-promoting agent
The steady-state mesenchymal stem cell exosome high-efficiency release agent consists of the following components in concentration: 3.0 mu mol/mL of monensin sodium, 30 mu g/mL of gentamicin, 60ng/mL of ESCRT related protein, 35pg/mL of Alix, 909 ng/mL of HSP, 200 mu g/mL of sphingomyelin, 100 mu g/mL of glycosphingolipid, 30mg/mL of cholesterol, 100 mu g/mL of phosphatidylinositol, 200mg/mL of ceramide, 35mg/mL of phosphatidylserine, 20 mu g/mL of curcumin, 80mg/mL of EGCG, 30 mu g/mL of resveratrol, 2.5 mu mol/mL of glucoraphanin, 35IU/mL of beta-catenin, 35IU/mL of Rabs family protein and 2.5 mu g/mL of integrin are added into a culture system according to the volume ratio of 1:50 when in use, wherein the solvent is BC-T4 serum preparation medium;
(2) Stem cell resuscitation and culture
1) Preheating a constant-temperature automatic heater to 37 ℃;
2) A 50mL centrifuge tube was prepared and the cell fluid was frozen: BC-T4 medium = 1:9 medium was taken and preheated to 37 ℃;
3) Taking out the frozen stem cells from the liquid nitrogen tank, and rapidly placing the frozen stem cells in a constant-temperature automatic heating instrument until the ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2min;
4) Preparation of complete medium: adding MSC-XF additive into BC-T4 according to the proportion of 50:1 to prepare a complete culture medium;
5) Transferring the dissolved stem cell suspension to a centrifuge tube which is prepared in advance and filled with a stem cell complete culture medium by using a gun head, centrifuging for 6min at the normal temperature at 250 Xg, and discarding the supernatant;
6) Taking stem cells from complete culture medium of stem cells, suspending the stem cells back to obtain cell suspension, taking 50 mu l of cell suspension, uniformly mixing and dyeing with 50 mu l of trypan blue dyeing liquid with the mass percentage concentration of 0.4%, detecting cell activity and counting cells, and obtaining the cell suspension according to the mass percentage concentration of 5000-10000 cells/cm 2 Inoculating the seed at a density of (2);
7) The stem cells are resuspended in 15mL of complete medium at 5000-10000 cells/cm 2 Is added into a T75 culture flask to uniformly spread stem cells on the bottom of the culture flask at 37 ℃ and 5 percent CO 2 Culturing in an incubator;
8) When the cell confluency is about 60-70%, exosome releasing agent is added, and then the mixture is continuously put into the mixture at 37 ℃ and 5% CO 2 Culturing in an incubator for 24-48 hours;
(3) Cell collection and supernatant
And collecting cell culture supernatant for exosome extraction and purification when the cell confluence reaches about 90%, digesting stem cells by using recombinant pancreatin, and performing centrifugal treatment to collect and count cells. .
(4) Exosome extraction and purification
1) Collecting cell supernatant, centrifuging for 10min, removing cells, centrifuging, and collecting supernatant.
2) 2000g, centrifuging for 10min, removing dead cell debris, centrifuging to obtain supernatant, and filtering with a cell sieve.
3) 10000g, centrifuging at 4deg.C for 30min, removing large membrane bubbles, and collecting supernatant;
4) 100000g, centrifuging at 4deg.C for 90min, pouring the supernatant, reversely buckling the centrifuge tube on sterile filter paper, removing residual liquid as much as possible, adding 2ml of ice-bath physiological saline, suspending again, collecting liquid in the same centrifuge tube after 300g and 3min, and supplementing physiological saline to 22ml.
5) Centrifuging again with 100000g at 4deg.C for 90min to remove impurity protein, adding small amount of ice-bath physiological saline, and collecting to obtain exosome.
Comparative example 1
Comparative example 1 differs from example 1 only in that no exosome releasing agent was added when the cell confluency was about 60% to 70%.
The cell morphology and exosome content of example 1 and comparative example 1 are shown in Table 1.
TABLE 1 comparison of cell morphology and exosome content for example 1 and comparative example 1
As is clear from Table 1, there was no significant difference in the number and morphology of cells in example 1 compared with comparative example 1, and the cell morphology was good; the aposome is in a tea-tray shape under the electron microscope, the grain diameter is between 30 and 150nm, and the content is obviously improved, so that the steady-state mesenchymal stem cell aposome high-efficiency release agent provided by the invention can improve the content of the aposome in the stem cell culture process.
Example 2
(1) Preparation of release-promoting agent
The steady-state mesenchymal stem cell exosome high-efficiency release agent consists of the following components in concentration: 3.0 mu mol/mL of monensin sodium, 30 mu g/mL of gentamicin, 60ng/mL of ESCRT related protein, 35pg/mL of Alix, 909 ng/mL of HSP, 200 mu g/mL of sphingomyelin, 100 mu g/mL of glycosphingolipid, 30mg/mL of cholesterol, 100 mu g/mL of phosphatidylinositol, 200mg/mL of ceramide, 35mg/mL of phosphatidylserine, 20 mu g/mL of curcumin, 80mg/mL of EGCG, 30 mu g/mL of resveratrol, 2.5 mu mol/mL of glucoraphanin, 35IU/mL of beta-catenin, 35IU/mL of Rabs family protein and 2.5 mu g/mL of integrin are added into a culture system according to the volume ratio of 1:50 when in use, wherein the solvent is BC-T4 serum preparation medium;
(2) Stem cell resuscitation and culture
1) Preheating a constant-temperature automatic heater to 37 ℃;
2) A 50mL centrifuge tube was prepared and the cell fluid was frozen: BC-T4 medium = 1:9 medium was taken and preheated to 37 ℃;
3) Taking out the frozen stem cells from the liquid nitrogen tank, and rapidly placing the frozen stem cells in a constant-temperature automatic heating instrument until the ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2min;
4) Preparation of complete medium: adding MSC-XF additive into BC-T4 according to the proportion of 50:1 to prepare a complete culture medium;
5) Transferring the dissolved stem cell suspension to a centrifuge tube which is prepared in advance and filled with a stem cell complete culture medium by using a gun head, centrifuging for 6min at the normal temperature at 250 Xg, and discarding the supernatant;
6) Taking stem cells from complete culture medium of stem cells, suspending the stem cells back to obtain cell suspension, taking 50 mu l of cell suspension, uniformly mixing and dyeing with 50 mu l of trypan blue dyeing liquid with the mass percentage concentration of 0.4%, detecting cell activity and counting cells, and obtaining the cell suspension according to the mass percentage concentration of 5000-10000 cells/cm 2 Inoculating the seed at a density of (2);
6) The stem cells are resuspended in 15mL of complete medium at 5000-10000 cells/cm 2 Is added into a T75 culture flask to uniformly spread stem cells on the bottom of the culture flask at 37 ℃ and 5 percent CO 2 Culturing in an incubator;
7) When the cell confluency is about 60-70%, exosome releasing agent is added, and then the mixture is continuously put into the mixture at 37 ℃ and 5% CO 2 Culturing in an incubator for 24-48 hours;
(3) Cell collection and supernatant
And collecting cell culture supernatant for exosome extraction and purification when the cell confluence reaches about 90%, digesting stem cells by using recombinant pancreatin, and performing centrifugal treatment to collect and count cells.
(4) Exosome extraction and purification
1) Collecting cell supernatant, centrifuging for 10min, removing cells, centrifuging, and collecting supernatant.
2) 2000g, centrifuging for 10min, removing dead cell debris, centrifuging to obtain supernatant, and filtering with a cell sieve.
3) 10000g, centrifuging at 4deg.C for 30min, removing large membrane bubbles, and collecting supernatant;
4) 100000g, centrifuging at 4deg.C for 90min, pouring the supernatant, reversely buckling the centrifuge tube on sterile filter paper, removing residual liquid as much as possible, adding 2ml of ice-bath physiological saline, suspending again, collecting liquid in the same centrifuge tube after 300g and 3min, and supplementing physiological saline to 22ml.
5) Centrifuging again with 100000g at 4deg.C for 90min to remove impurity protein, adding small amount of ice-bath physiological saline, and collecting to obtain exosome.
Comparative example 2:
the difference from example 2 is that the components of the release-promoting agent are different, and the monensin sodium and gentamicin in the release-promoting agent are removed, and the rest is the same as example 2;
comparative example 3:
the difference from example 2 is that the components of the release-promoting agent are different, and ESCRT-related protein, alix and HSP90 in the release-promoting agent are removed, and the rest is the same as example 2;
comparative example 4:
the difference from example 2 is that the components of the release-promoting agent are different, and sphingomyelin, glycosphingolipids, cholesterol, phosphatidylinositol, ceramide and phosphatidylserine in the release-promoting agent are removed, and the rest is the same as example 2;
comparative example 5:
the difference from example 2 is that the components of the release-promoting agent are different, curcumin, EGCG, resveratrol and sulforaphane in the release-promoting agent are removed, and the rest is the same as example 2;
comparative example 6:
the difference from example 2 is that the components of the release-promoting agent are different, and the beta-catenin, rabs family protein and integrin in the release-promoting agent are removed, and the rest is the same as in example 2.
The exosomes of example 2 and comparative examples 2 to 6 were collected, tested and counted, and the statistical results are shown in table 2.
TABLE 2 statistical results of exosome content and cell count in example 2 and comparative examples 2 to 6
As can be seen from Table 2, the exosome content is the highest in example 2, and the exosome content is reduced after the components of the release-promoting agent are reduced in comparative examples 2-6, because the monensin sodium and gentamicin in the release-promoting agent can interfere with the balance of sodium ions and calcium ions in cells to influence the uptake of ions by the cell bodies or the endoplasmic reticulum, thereby promoting the generation and release of exosomes. ESCRT related proteins, alix and HSP90 can affect classical ESCRT pathways, alter the properties of the vesicle plasma membrane in the early stages of exosome formation, and affect exosome production. Sphingomyelin, glycosphingolipids, cholesterol, phosphatidylinositol, ceramide and phosphatidylserine are key substances for the formation of exosome phospholipid bilayer, which promote exosome formation and transport. Curcumin, EGCG, resveratrol and sulforaphane can inhibit mTOR signal path in the formation process of exosome, and inhibit protein decomposition and autophagy. The main role of β -catenin, rabs family proteins and integrins is to participate in the signaling of exosomes production, playing an important role in the biogenesis, release and transport of exosomes. As can be seen from comparative examples 2 to 6, after any component is omitted, the effect of secreting exosomes by the mesenchymal stem cells is inferior to that of example 2 of the present invention, the cell number is not significantly different, and further, the synergistic effect of the components is further demonstrated, and the exosome release performance of the mesenchymal stem cells is improved.
Example 3
(1) Preparation of release-promoting agent
The steady-state mesenchymal stem cell exosome high-efficiency release agent consists of the following components in concentration: 3.0 mu mol/mL of monensin sodium, 30 mu g/mL of gentamicin, 60ng/mL of ESCRT related protein, 35pg/mL of Alix, 909 ng/mL of HSP, 200 mu g/mL of sphingomyelin, 100 mu g/mL of glycosphingolipid, 30mg/mL of cholesterol, 100 mu g/mL of phosphatidylinositol, 200mg/mL of ceramide, 35mg/mL of phosphatidylserine, 20 mu g/mL of curcumin, 80mg/mL of EGCG, 30 mu g/mL of resveratrol, 2.5 mu mol/mL of glucoraphanin, 35IU/mL of beta-catenin, 35IU/mL of Rabs family protein and 2.5 mu g/mL of integrin are added into a culture system according to the volume ratio of 1:50 when in use, wherein the solvent is BC-T4 serum preparation medium;
(2) Stem cell resuscitation and culture
1) Preheating a constant-temperature automatic heater to 37 ℃;
2) A 50mL centrifuge tube was prepared and the cell fluid was frozen: BC-T4 medium = 1:9 medium was taken and preheated to 37 ℃;
3) Taking out the frozen stem cells from the liquid nitrogen tank, and rapidly placing the frozen stem cells in a constant-temperature automatic heating instrument until the ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2min;
4) Preparation of complete medium: adding MSC-XF additive into BC-T4 according to the proportion of 50:1 to prepare a complete culture medium;
5) Transferring the dissolved stem cell suspension to a centrifuge tube which is prepared in advance and filled with a stem cell complete culture medium by using a gun head, centrifuging for 6min at the normal temperature at 250 Xg, and discarding the supernatant;
6) Taking stem cells from complete culture medium of stem cells, suspending the stem cells back to obtain cell suspension, taking 50 mu l of cell suspension, uniformly mixing and dyeing with 50 mu l of trypan blue dyeing liquid with the mass percentage concentration of 0.4%, detecting cell activity and counting cells, and obtaining the cell suspension according to the mass percentage concentration of 5000-10000 cells/cm 2 Inoculating the seed at a density of (2);
6) The stem cells are resuspended in 15mL of complete medium at 5000-10000 cells/cm 2 Is added into a T75 culture flask to uniformly spread stem cells on the bottom of the culture flask at 37 ℃ and 5 percent CO 2 Culturing in an incubator;
7) When the cell confluency is about 60-70%, exosome releasing agent is added, and then the mixture is continuously put into the mixture at 37 ℃ and 5% CO 2 Culturing in an incubator for 24-48 hours;
(3) Cell collection and supernatant
And collecting cell culture supernatant for exosome extraction and purification when the cell confluence reaches about 90%, and carrying out cell collection by digesting stem cells with recombinant pancreatin and carrying out centrifugal treatment.
(4) Exosome extraction and purification
1) Collecting cell supernatant, centrifuging for 10min, removing cells, centrifuging, and collecting supernatant.
2) 2000g, centrifuging for 10min, removing dead cell debris, centrifuging to obtain supernatant, and filtering with a cell sieve.
3) 10000g, centrifuging at 4deg.C for 30min, removing large membrane bubbles, and collecting supernatant;
4) 100000g, centrifuging at 4deg.C for 90min, pouring the supernatant, reversely buckling the centrifuge tube on sterile filter paper, removing residual liquid as much as possible, adding 2ml of ice-bath physiological saline, suspending again, collecting liquid in the same centrifuge tube after 300g and 3min, and supplementing physiological saline to 22ml.
5) Centrifuging again with 100000g at 4deg.C for 90min to remove impurity protein, adding small amount of ice-bath physiological saline, and collecting to obtain exosome.
(3) Exosome detection
Cells from which the supernatant was collected were digested with pancreatin and passaged, and the above procedure was repeated for 20 passages; counting the cells of the 3 rd generation and the 20 th generation stem cells, detecting NTA of exosomes, and comparing the differences of the cells;
(4) Cell phenotype detection
Digestion with pancreatin into single cells, washing the cells with DPBS, and detecting the surface antigens CD34, CD45, CD19, CD73, CD90, CD105, CD44, HLA-DR and CD11b by flow cytometry after counting; and taking the stem cell exosomes of the 3 rd generation and the 20 th generation for detection and comparison.
(5) Stem cell differentiation ability assessment
1) Adipogenic induced differentiation of human umbilical cord mesenchymal stem cells
Selecting 20 th generation umbilical cord mesenchymal stem cells with good growth state according to 8.0X10 3 cells/cm 2 Inoculating into 6-hole culture plate, adding lipogenic inducer dexamethasone 1 μmol/L, IBMX 0.5.5 mmol/L and indomethacin 100 μmol/L when cell wall growth reaches 60% fusion, standing at 37deg.C and 5% CO 2 An incubator; the liquid was changed 1 time at 3-day intervals. Cells were identified on day 14 using 2% oil red O staining. The staining results are shown in fig. 4.
2) Osteoblastic differentiation of human umbilical cord mesenchymal stem cells
Taking umbilical cord mesenchymal stem cells of 20 th generation with good growth state according to 7.0X10 3 cells/cm 2 Inoculating into 6-hole culture plate, adding into dexamethasone 10 as osteogenesis inducer when cell wall-adhering growth reaches 60% fusion -7 mol/L, 10mmol/L of beta-sodium glycerophosphate and 50 mug/mL of ascorbic acid; placing at 37deg.C and 5% CO 2 Incubator, change liquid 1 time every 3 days. 4% paraformaldehyde fixation at osteoinduction to 14dIdentification of osteoblasts by von kusa staining. The staining results are shown in fig. 5.
3) Chondrogenic induced differentiation of human umbilical cord mesenchymal stem cells
Taking amplified 20 th generation umbilical cord mesenchymal stem cells at 2.0X10 × 5 Inoculating the total number of cells into 15mL centrifuge tube, centrifuging at 1500r/min for 10min to precipitate cells at the bottom of the culture tube, adding cartilage induction culture medium, adding dexamethasone 10 into DMEM/Ham's F12 culture medium -7 mol/L, ascorbic acid 50 mug/mL, bovine serum albumin 1.25 mug/mL, transferrin 6.25 mug/mL, sodium pyruvate 1mmol/L, linoleic acid 5.35 mug/mL; placing at 37deg.C and 5% CO 2 Incubator, 1 time at 3-day intervals. On day 21 of culture, chondrocyte pellet was fixed with 4% paraformaldehyde, frozen sections were performed, and cells were identified by staining with alpha blue. The staining results are shown in fig. 6.
(6) Experimental results
From Table 3, there is no obvious difference in the content of exosomes in the supernatant of the cells of the generation P3 and the generation P20, which indicates that the exosomes can be stably promoted to be released by the cells; table 4 and FIGS. 2-3 show that after 20 passages of stem cells, the cell phenotype is not significantly changed after the exosome releasing agent is used, which indicates that the cells are still undifferentiated and have higher purity; figures 4-6 show that umbilical cord mesenchymal stem cells passaged to 20 passages still have the potential to differentiate into osteogenic, adipose, cartilage.
TABLE 3NTA detection results
TABLE 4 flow phenotype after 20 passages of Stem cells
| Cell phenotype | P3(％) | P20(％) |
| CD90 | 99.6 | 99.6 |
| CD44 | 98.5 | 98.7 |
| CD105 | 99.8 | 99.5 |
| CD73 | 99.4 | 99.5 |
| HLA-DR | 0.2 | 0.1 |
| CD34 | 0.2 | 0.5 |
| CD45 | 0.1 | 0.1 |
| CD19 | 0.1 | 0.1 |
| CD11b | 0.1 | 0.1 |
As can be seen from tables 3 to 4 and fig. 2 to 3, the umbilical cord mesenchymal stem cells are cultured by using the steady-state mesenchymal stem cell exosome-promoting high-efficiency releasing agent, and the NTA result shows that the secretion amounts of the P3 and P20 generation cell exosomes have no obvious difference; the expression levels of the CD44, the CD73, the CD90 and the CD105 are all over 95 percent, and the expression levels of the CD34, the CD45, the CD19, the CD11b and the HLA-DR are all less than 2.0 percent; the exosome form is tea tray-shaped under the transmission electron microscope, and the grain diameter accords with the exosome range; differentiation experiments show that umbilical mesenchymal stem cells passaged to 20 generations still have the potential to differentiate into osteogenesis, fat and cartilage. The result shows that when the steady-state mesenchymal stem cell exosome high-efficiency releasing agent is used for culturing umbilical cord mesenchymal stem cells until the generation of P20, the cells are still not differentiated, and the purity is high.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (10)
1. The steady-state mesenchymal stem cell exosome high-efficiency release agent is characterized by comprising the following components: sodium monensin, gentamicin, ESCRT related proteins, alix, HSP90, sphingomyelin, glycosphingolipids, cholesterol, phosphatidylinositol phosphates, ceramides, phosphatidylserine, curcumin, EGCG, resveratrol, sulforaphane, β -catenin, rabs family proteins, integrins.
2. The steady-state mesenchymal stem cell exosome high-efficiency release agent according to claim 1, wherein the concentrations of each component in the steady-state mesenchymal stem cell exosome high-efficiency release agent are respectively: 2.0 to 8.0 mu mol/mL of monensin sodium, 1 to 100 mu g/mL of gentamicin, 10 to 150ng/mL of ESCRT related protein, 10 to 60pg/mL of Alix, 5 to 30ng/mL of HSP, 20 to 600 mu g/mL of sphingomyelin, 20 to 300 mu g/mL of glycosphingolipid, 1 to 100mg/mL of cholesterol, 5 to 300 mu g/mL of phosphatidylinositol, 5 to 300mg/mL of ceramide, 10 to 100mg/mL of phosphatidylserine, 5 to 50 mu g/mL of curcumin, 10 to 150mg/mL of EGCG, 10 to 100 mu g/mL of resveratrol, 1 to 10 mu mol/mL of glucoraphanin, 5 to 100IU/mL of beta-catenin, 5 to 100IU/mL of Rabs family protein, and 0.5 to 8.0 mu g/mL of integrin.
3. The steady-state mesenchymal stem cell exosome high-efficiency release agent according to claim 1, wherein the concentrations of each component in the steady-state mesenchymal stem cell exosome high-efficiency release agent are respectively: 1.0 to 5.0 mu mol/mL of monensin sodium, 10 to 50 mu g/mL of gentamicin, 50 to 100 mu g/mL of ESCRT related protein, 20 to 50 mu mol/mL of Alix, 10 to 15 mu g/mL of HSP902.5, 50 to 400 mu g/mL of sphingomyelin, 10 to 200 mu g/mL of glycosphingolipids, 10 to 50mg/mL of cholesterol, 10 to 200 mu g/mL of phosphatidylinositol, 10 to 400mg/mL of ceramide, 20 to 50mg/mL of phosphatidylserine, 10 to 30 mu g/mL of curcumin, 20 to 125mg/mL of EGCG, 20 to 40 mu g/mL of resveratrol, 1.5 to 3.5 mu mol/mL of glucoraphanin, 10 to 60IU/mL of beta-catenin, 20 to 50IU/mL of Rabs family protein, and 1.0 to 4.0 mu g/mL of integrin.
4. The steady state, mesenchymal stem cell-promoting exosome high-potency delivery agent of claim 1, wherein the mesenchymal stem cell comprises: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells and dental pulp mesenchymal stem cells.
5. Use of the steady-state, mesenchymal stem cell exosome-promoting, high-efficiency release agent according to any one of claims 1-4 for maintaining the release function of mesenchymal stem cell exosome.
6. Use of a steady-state mesenchymal stem cell exosome high-efficiency release agent according to any one of claims 1-4 for promoting release of exosome by mesenchymal stem cells.
7. Use of a steady-state, mesenchymal stem cell-promoting exosome high-efficiency release agent according to any one of claims 1-4 for preserving mesenchymal stem cell proliferation capacity.
8. Use of a steady-state, mesenchymal stem cell-promoting exosome high-efficiency release agent according to any one of claims 1-4 for maintaining the multipotent differentiation potential of mesenchymal stem cells.
9. A mesenchymal stem cell culture medium characterized by comprising the steady-state mesenchymal stem cell exosome high-efficiency releasing agent according to any one of claims 1 to 4.
10. The culture medium according to claim 9, wherein the culture medium comprises a steady-state mesenchymal stem cell exosome high-efficiency releasing agent and a mesenchymal stem cell serum-free culture medium in a volume ratio of 1:50.
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