A method and application for promoting neural stem cell adhesion

Abstract

The present invention discloses a method and application for promoting neural stem cell adhesion. The culture method of the present invention enables neural stem cells to grow by adhesion without using matrix gel for plating, eliminates cumbersome operation steps, simplifies the production process, and provides a new direction for the adhesion culture of neural stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for promoting neural stem cell adhesion and application. Background Art

[0002] Neurological diseases include central nervous system diseases and peripheral nervous system diseases. For the treatment of such diseases, the conventional treatment strategy is surgery plus medication, but this treatment method can only delay the progression of the disease and cannot fundamentally solve the disease. Most patients with neurological diseases cannot achieve good therapeutic effects after treatment because the nerve cells in the damaged area are lost and cannot regenerate, resulting in the formation of scar tissue, which prevents the patient from recovering better. Neural stem cells are the seed cells of the nervous system and can differentiate into different neurons and glial cells. NSCs transplanted into the human body can migrate and aggregate to the site of neurological lesions, allowing damaged or missing nerve cells to regenerate, promoting the recovery of damaged functions of the host nervous system, and achieving better therapeutic effects.

[0003] Neural stem cells (NSC) are cells located in the subventricular zone and certain parts of the central nervous system, with the potential for self-replication and multi-differentiation. Neural stem cells have the following characteristics: 1) Self-renewal: Neural stem cells have two ways of division, symmetrical and asymmetrical; 2) Symmetric division maintains its own proliferation ability; asymmetrical division allows NSC to differentiate; 3) Multidirectional differentiation: can differentiate into neurons, astrocytes and oligodendrocytes; 4) Homing: during transplantation, it tends to originate or function and anatomical site; 5) Low immunogenicity: relatively less immune rejection occurs after transplantation; 6) Tissue fusion: can fuse well with host neural tissue and survive in vivo for a long time. Neural stem cells are seed cells of the central nervous system, and play a vital role in the development and functional maintenance of the central nervous system.

[0004] At present, the methods for culturing neural stem cells in vitro mainly include suspension culture and adherent culture. However, suspension culture has the following problems: first, the primary culture inoculation density is high and the cells are easy to differentiate; second, the neural stem cell spheres need to be digested before use, and the self-renewal ability of the neural stem cells after digestion is seriously reduced; third, the neural stem cell sphere is a heterogeneous structure, which is manifested in the heterogeneity between neurospheres and the heterogeneity of cells within a single neurosphere, making it impossible to quantify neural stem cells in suspension culture and the actual amount of neural stem cells after large-scale culture is far lower than the theoretical number. Therefore, in order to obtain neural stem cells with higher homogeneity, it is crucial to provide a method of adherent culture in this field. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a method for promoting the adhesion of neural stem cells.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a method for promoting adherent culture of neural stem cells, the method comprising adding exosomes and co-culturing the neural stem cells.

[0008] Furthermore, the method further comprises the step of replacing fresh culture medium after co-cultivation.

[0009] Furthermore, the method further comprises the step of removing old culture medium containing exosomes before replacing with fresh culture medium.

[0010] Furthermore, the concentration of the exosomes is 3000-9000 particles / cell.

[0011] Furthermore, the concentration of the exosomes is 3000 particles / cell.

[0012] Furthermore, the density of the neural stem cells is 2×10^4 cells / cm 2 .

[0013] Furthermore, the culture conditions are 37°C and 5% CO2.

[0014] Furthermore, the exosomes are stem cell-derived exosomes.

[0015] Furthermore, the stem cells are mesenchymal stem cells.

[0016] Furthermore, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

[0017] Furthermore, the components of the culture medium used for co-culture include: basal culture medium, nutrient additives, fibroblast growth factor, mitogen-activated factor, and heparin sodium.

[0018] Furthermore, the basal culture medium is selected from DMEM / F12 basal culture medium.

[0019] Furthermore, the nutritional additives include B-27 and GlutaMAX.

[0020] Furthermore, the B-27 is B-27 without vitamin A.

[0021] Furthermore, the fibroblast growth factor is selected from bFGF.

[0022] Furthermore, the mitogenic proliferation factor is selected from EGF.

[0023] Furthermore, the heparin sodium is selected from porcine heparin sodium.

[0024] Furthermore, the concentration of bFGF is 20-40 ng / ml.

[0025] Furthermore, the concentration of bFGF is 20 ng / ml.

[0026] Furthermore, the concentration of EGF is 20-40 ng / ml.

[0027] Furthermore, the concentration of EGF is 20 ng / ml.

[0028] Furthermore, the concentration of the FBS is 1%-10%.

[0029] Furthermore, the concentration of the FBS is 1%.

[0030] Furthermore, the concentration of B-27 is 2%.

[0031] Furthermore, the concentration of the GlutaMAX is 1%.

[0032] Furthermore, the concentration of the porcine heparin sodium is 2 μg / ml.

[0033] The second aspect of the present invention provides a method for inducing differentiation of neural stem cells, the method comprising the step of replacing the differentiation medium after the step of adding exosomes and co-culturing neural stem cells as described in the first aspect of the present invention.

[0034] Furthermore, the neural stem cells differentiate into neurons and / or astrocytes.

[0035] Furthermore, the differentiation medium includes basal medium and FBS.

[0036] Furthermore, the method further comprises the step of removing old culture medium containing exosomes before replacing the differentiation culture medium.

[0037] Furthermore, the basal culture medium is selected from DMEM / F12 basal culture medium.

[0038] Furthermore, the concentration of the FBS is 1%-10%.

[0039] Furthermore, the concentration of the FBS is 1%.

[0040] The third aspect of the present invention provides a culture composition for promoting the adhesion of neural stem cells, the composition comprising the culture medium described in the first aspect of the present invention and exosomes.

[0041] A fourth aspect of the present invention provides the use of exosomes in promoting the adhesion of neural stem cells.

[0042] Furthermore, the exosomes are stem cell-derived exosomes.

[0043] Furthermore, the stem cells are mesenchymal stem cells.

[0044] Furthermore, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

[0045] Advantages and beneficial effects of the present invention:

[0046] The culture method of the present invention can make neural stem cells grow by adherence without using matrix gel for plating, thus eliminating cumbersome operation steps, simplifying the production process, and providing a new direction for the adherence culture of neural stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the morphology of neural stem cells cultured without exosomes;

[0048] Figure 2 This is a morphological picture of neural stem cells cultured with exosomes;

[0049] Figure 3 This is the flow cytometry result of neural stem cells cultured with exosomes;

[0050] Figure 4 Figure 4A shows neural stem cells cultured with exosomes that differentiate into astrocytes and neurons, and Figure 4B shows neural stem cells cultured with exosomes that differentiate into neurons.

[0051] Figure 5 This is the flow cytometry result of neural stem cells cultured with exosomes and differentiating into astrocytes and neurons. DETAILED DESCRIPTION

[0052] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0053] The present invention provides a method for promoting adherent culture of neural stem cells, which comprises adding exosomes and co-culturing the neural stem cells.

[0054] In one embodiment of the present invention, co-culture generally refers to culturing cells of two or more different groups with a certain degree of contact between them. In addition to conventionally recognized cells, the cells also include substances secreted by cells, such as exosomes, etc. The contact of two or more different groups of cells of the present application can be through direct contact in one embodiment, that is, direct physical contact between cells of one group and cells of another group. Or in one embodiment, it can be indirect contact mediated by a shared culture medium. The shared culture medium of the present application may contain metabolites produced and released by at least one group of co-cultured cells, and is used to culture cells of another group.

[0055] In a specific embodiment of the present invention, the co-culture condition is 37° C., 5% CO 2 .

[0056] In one embodiment of the present invention, exosomes are vesicular vesicles secreted by living cells, present in the body fluids of almost all eukaryotic organisms, and are endoplasmic reticulum with a diameter of about 30-100 nm, larger than low-density lipoprotein (LDL) protein, but much smaller than red blood cells. It is known that exosomes can be released from cells when multivesicular bodies fuse with cell membranes, or directly released from cell membranes, and they play important specialized functions, such as coagulation and intercellular signaling.

[0057] In one embodiment of the present invention, the exosomes may be exosomes from any source.

[0058] In a preferred embodiment of the present invention, the exosomes are stem cell-derived exosomes.

[0059] Stem cells, as an undifferentiated cell, refer to cells that have the ability to self-replicate and differentiate into two or more different types of cells.

[0060] The stem cells may be stem cells derived from autologous or homologous tissues, may be derived from any type of animal including humans and non-human mammals, may be stem cells derived from adults, or may be stem cells derived from embryos. For example, the stem cells may be selected from embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells, BxC stem cells, BxC-A1 stem cells, BxC-I10 stem cells, BxC-G63 stem cells, BxC-R11 stem cells, and BxC-R56 stem cells, but are not limited thereto.

[0061] In a more preferred embodiment of the present invention, the stem cells are selected from mesenchymal stem cells.

[0062] Mesenchymal stem cells (MSC) refer to stem cells derived from the mesenchyme. Mesenchymal stem cells can differentiate into one or more cells selected from the group consisting of osteoblasts, chondrocytes, adipocytes or muscle cells. Mesenchymal stem cells can be isolated from all types of adult tissues, for example, they can be isolated from bone marrow adipose tissue, umbilical cord, peripheral blood or adipose tissue. A mesenchymal stem cell population (MSC population) can be defined as exhibiting a specific phenotype.

[0063] In a specific embodiment of the present invention, the mesenchymal stem cells are selected from umbilical cord-derived mesenchymal stem cells.

[0064] In one embodiment of the present invention, the concentration of the exosomes is 3000-9000 particles / cell, for example, it can be 3000 particles / cell, 4000 particles / cell, 5000 particles / cell, 6000 particles / cell, 7000 particles / cell, 8000 particles / cell, 9000 particles / cell, and the concentration between any two of the above points.

[0065] In a specific embodiment of the present invention, the concentration of the exosomes is 3000 particles / cell.

[0066] In one embodiment of the present invention, the culture medium used for co-culture can be any culture medium suitable for the growth of neural stem cells.

[0067] In a preferred embodiment of the present invention, the components of the culture medium used for co-culture include: basal culture medium, nutrient additives, fibroblast growth factor, mitogen-activated proliferation factor, and heparin sodium salt.

[0068] In one embodiment of the invention, culture medium is generally recognized in the art and refers to any substance or preparation for culturing living cells. Culture medium as used to refer to cell culture includes components of the cell surrounding environment. Culture medium includes liquid growth medium, gas culture medium. Culture medium also includes gelatinous culture medium, such as agar, agarose, gelatin and collagen matrix. Exemplary gas culture medium includes a gas phase, and cells grown on a culture dish or other solid or semi-solid support are exposed to the gas phase. Culture medium also refers to a material intended for cell culture, even if the material has not yet contacted with cells. In other words, a nutrient-rich liquid prepared for cultivation is also a culture medium.

[0069] In one embodiment of the present invention, basal medium refers to a medium that promotes the growth of many types of cells that do not require any special nutritional supplements. Most basal mediums generally contain four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. Basal medium is generally used as the basis of more complex culture media, to which supplements such as serum or serum substitutes, buffers, growth factors, antibiotics, etc. are added. Examples of basal medium include, but are not limited to, Eagle basal medium (BME), minimum essential medium (MEM), DuIbecco's modified Eagle medium (DMEM), culture medium 199, nutrient mixture HamsF10 (F10) and HamsF12 (F12), McCoy's 5A, DuIbecco's MEM / F-12 (DMEM / F12), RPMI 1640, Iscove's modified DuIbecco medium (IMDM), L-15 culture medium, neurobasal one or more.

[0070] In a specific embodiment of the present invention, the basal culture medium is selected from DMEM / F12 culture medium.

[0071] In one embodiment of the present invention, the nutritional supplement includes Gluta Max, B-27 and / or N2.

[0072] In a particular embodiment of the present invention, the nutritional additive is selected from Gluta Max and B-27.

[0073] In one embodiment of the present invention, examples of fibroblast growth factors (FGF) in the culture medium include, but are not limited to, one or more of FGF-1, bFGF (FGF-2), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23.

[0074] In a specific embodiment of the present invention, the fibroblast growth factor is selected from bFGF (FGF-2).

[0075] In one embodiment of the present invention, the concentration of bFGF is 20-40ng / ml, for example, it can be 20ng / ml, 23ng / ml, 25ng / ml, 28ng / ml, 30ng / ml, 32ng / ml, 35ng / ml, 37ng / ml, 40ng / ml, and the concentration between any two of the above points.

[0076] In a specific embodiment of the present invention, the concentration of bFGF is 20 ng / ml.

[0077] In one embodiment of the present invention, the mitogenic growth factors include, but are not limited to, a family of growth factors such as EGF (epidermal growth factor), BDNF (brain derived neurotrophic factor), and KGF (keratinocyte growth factor). These mitogenic growth factors can be used in a variety of combinations.

[0078] In a specific embodiment of the present invention, the mitogenic growth factor is selected from EGF (Epidermal Growth Factor).

[0079] In one embodiment of the present invention, the concentration of EGF is 20-40ng / ml, for example, it can be 20ng / ml, 23ng / ml, 25ng / ml, 28ng / ml, 30ng / ml, 32ng / ml, 35ng / ml, 37ng / ml, 40ng / ml, and the concentration between any two of the above points.

[0080] In a specific embodiment of the present invention, the concentration of EGF is 20 ng / ml.

[0081] The differentiation medium includes a basal medium and FBS (fetal bovine serum).

[0082] In one embodiment of the present invention, the concentration of FBS is 1%-10%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and the concentration between any two of the above points.

[0083] In a specific embodiment of the present invention, the concentration of FBS is 1%.

[0084] The culture medium of the present invention may further comprise at least one amino acid. The amino acid comprises L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and a combination thereof.

[0085] The culture medium of the present invention may further comprise at least one vitamin. The vitamins include, but are not limited to, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0086] The culture medium of the present invention may further contain at least one inorganic salt. The inorganic salt includes, but is not limited to, salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are generally used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, salts may include CaCl2, CuSO4·5H2O, Fe(NO3)·9H2O, FeSO4·7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4·H2O, and ZnSO4·7H2O.

[0087] The culture medium of the present invention may further contain at least one sugar that can serve as a carbon energy source, including glucose, galactose, mannose, fructose, and the like.

[0088] The culture medium of the present invention may further comprise at least one trace element. The trace element comprises barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum or ions thereof.

[0089] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0090] Example

[0091] 1 Experimental Materials

[0092] The experimental instruments are shown in Table 1, and the experimental reagents are shown in Table 2.

[0093] Table 1 Experimental instruments

[0094]

[0095] Table 2 Experimental reagents

[0096]

[0097] Neural stem cell culture medium preparation: DMEM / F12 basal medium, 2% B-27 Supplement (50X) without vitamin A, 20ng / ml bFGF, 20ng / ml EGF, 1% GlutaMAX TM Additives, 2μg / ml Heparin sodium salt from porcine.

[0098] 2 Experimental methods

[0099] 1. Extraction and purification of exosomes from umbilical cord-derived mesenchymal stem cells

[0100] Use 3D The vivaEXO exosome harvesting system is an automated device that achieves large-scale concentration and enrichment of exosomes based on the principle of multi-stage separation.

[0101] 2. Culture of neural stem cells in the control group

[0102] (1) NeuroEasy human neural stem cells at 2×10^4 cells / cm 2 Inoculate in ultra-low adsorption T25 bottles and place in a carbon dioxide incubator for culture at 37°C and 5% CO2;

[0103] (2) After 3 days of suspension culture of neural stem cells, the cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 200 g for 5 min at room temperature;

[0104] (3) Discard the supernatant, add 7 ml of fresh neural stem cell culture medium to resuspend the neurospheres and transfer to ultra-low attachment T25 medium for further culture.

[0105] 3. Cultivation of neural stem cells in the experimental group

[0106] (1) After NeuroEasy neural stem cells were digested into single cells, the density was 2×10^4 cells / cm 2 Inoculate in TC-treated T25 culture flasks, add exosomes at a concentration of 3000 particles / cell, mix well in an 8-shaped pattern, and place in a carbon dioxide incubator for culture at 37°C and 5% CO2;

[0107] (2) The next day, neural stem cells adhered to the wall, the old culture medium containing exosomes was removed, and fresh neural stem cell culture medium was replaced to continue culture;

[0108] (3) Fresh neural stem cell culture medium was replaced daily, and cells were collected on the sixth day for flow cytometry: After discarding the original culture medium, the cells were washed twice with DPBS, and then 2 ml of ACCUTASE was added to the cells and digested in a carbon dioxide incubator for 2-3 min. After adding 2 ml of culture medium to terminate digestion, the cells were blown away, and the cell suspension was collected and centrifuged (300 g / min, 5 min, room temperature). After centrifugation, the supernatant was discarded, 1 ml of culture medium was added to resuspend the cells, and 20 μl was taken for trypan blue technique and then flow cytometry (indicators: Nestin positive, Sox1Sox2 double positive).

[0109] 4. Neural stem cells differentiate downward

[0110] (1) After NeuroEasy neural stem cells were digested into single cells, the density was 2×10^4 cells / cm 2 Inoculate in a common T25 culture flask, add exosomes at a concentration of 3000 particles / cell, mix well in an 8-shaped pattern, and place in a carbon dioxide incubator for culture at 37°C and 5% CO2;

[0111] (2) The next day, neural stem cells adhered to the wall, the old culture medium containing exosomes was removed, and the culture medium that promotes neural stem cell differentiation (DMEM / F12 + 1% FBS) was replaced;

[0112] (3) Fresh culture medium for promoting neural stem cell differentiation was replaced daily. After one week of culture, cells were collected for flow cytometry analysis (neurons: Tubulin-β3; astrocytes: GFAP).

[0113] 3 Experimental results

[0114] The results showed that the neural stem cells in the control group without exosomes did not adhere to the wall and were cultured in a neurosphere suspension culture ( Figure 1 ); In the experimental group, all the neural stem cells cultured with exosomes grew adherently ( Figure 2 , Figure 3 , Table 3); neural stem cells cultured with exosomes differentiated into astrocytes and neurons ( Figure 4 , Figure 5 , Table 4).

[0115] Table 3 Statistics of flow cytometry results of neural stem cells cultured with exosomes

[0116]

[0117] Table 4 Statistics of flow cytometry results of neural stem cells cultured with exosomes that differentiated into astrocytes and neurons

[0118]

[0119] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for promoting adherent culture of neural stem cells, characterized in that: The method comprises adding exosomes for co-culturing with neural stem cells, wherein the exosomes are stem cell-derived exosomes, and the stem cells are mesenchymal stem cells.

2. The method according to claim 1, characterized in that The method further comprises the step of replacing fresh culture medium after the co-cultivation.

3. The method according to claim 2, characterized in that The method further comprises the step of removing old culture medium containing exosomes before replacing with fresh culture medium.

4. The method according to claim 1, characterized in that: The concentration of the exosomes is 3000-9000 particles / cell.

5. The method according to claim 4, characterized in that The concentration of the exosomes is 3000 particles / cell.

6. The method according to claim 1, characterized in that The density of the neural stem cells is 2×10 4 cells / cm 2 .

7. The method according to claim 1, characterized in that The culture conditions are 37°C and 5% CO2.

8. The method according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

9. The method according to claim 1, characterized in that: The components of the culture medium used for co-culture include: basic culture medium, nutrient additives, fibroblast growth factor, mitogen-activated growth factor, and heparin sodium, wherein the mitogen-activated growth factor is EGF.

10. The method according to claim 9, characterized in that The basal culture medium is selected from DMEM / F12 basal culture medium.

11. The method according to claim 9, characterized in that The nutritional supplements include B-27 and GlutaMAX.

12. The method according to claim 11, characterized in that The B-27 is B-27 without vitamin A.

13. The method according to claim 9, characterized in that The fibroblast growth factor is selected from bFGF.

14. The method according to claim 9, characterized in that The heparin sodium is selected from porcine heparin sodium.

15. The method according to claim 13, characterized in that The concentration of bFGF is 20-40 ng / ml.

16. The method according to claim 15, characterized in that The concentration of bFGF is 20 ng / ml.

17. The method according to claim 9, characterized in that The concentration of EGF is 20-40 ng / ml.

18. The method according to claim 17, characterized in that The concentration of EGF is 20 ng / ml.

19. The method according to claim 12, characterized in that The concentration of B-27 is 2%.

20. The method according to claim 11, characterized in that The concentration of GlutaMAX is 1%.

21. The method according to claim 14, characterized in that The concentration of the heparin sodium is 2 μg / ml.

22. A method for inducing neural stem cell differentiation, characterized in that: The method comprises the step of replacing the differentiation medium after the step of adding exosomes and co-culturing neural stem cells according to claim 1, wherein the neural stem cells differentiate into astrocytes, The differentiation medium comprises a basal medium and FBS, The method further comprises the step of removing old culture medium containing exosomes before replacing the differentiation culture medium. The concentration of the FBS is 1%-10%.

23. The method according to claim 22, characterized in that The basal culture medium is selected from DMEM / F12 basal culture medium.

24. The method according to claim 22, characterized in that The concentration of the FBS is 1%.

25. A culture composition for promoting neural stem cell adhesion, characterized in that: The composition comprises the culture medium according to any one of claims 9 to 21 and exosomes, wherein the exosomes are stem cell-derived exosomes, and the stem cells are mesenchymal stem cells.

26. Use of exosomes in promoting neural stem cell adhesion, wherein the exosomes are stem cell-derived exosomes, and the stem cells are mesenchymal stem cells.

27. The use according to claim 26, characterized in that The mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

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