CN112410293A

CN112410293A - Culture medium for separating and extracting human intervertebral disc stem cells, preparation method and culture method of intervertebral disc stem cells

Info

Publication number: CN112410293A
Application number: CN202011493206.4A
Authority: CN
Inventors: 刘萍; 张赞
Original assignee: Beijing Zhongrun Tianhong Biotechnology Co ltd
Current assignee: Beijing Zhongrun Tianhong Biotechnology Co ltd
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-02-26

Abstract

The invention provides a culture medium for separating and extracting human intervertebral disc stem cells, a preparation method and a culture method of the intervertebral disc stem cells, belonging to the technical field of cell culture, wherein the culture medium comprises the following components: low-sugar dartbuck modified eagle DMEM medium, DMEM/F12 medium, fetal bovine serum, antibiotics, recombinant fibroblast growth factor, epidermal growth factor, insulin, acetylcysteine, and thyronine. The extraction method and the special culture medium can rapidly obtain a large amount of human intervertebral disc stem cells under the condition of less damage to the stem cells. The extracted primary adipose-derived stem cells have strong activity before passage and high stem cell purity. After passage, the intervertebral disc stem cell still has higher proliferation and differentiation capacity.

Description

Culture medium for separating and extracting human intervertebral disc stem cells, preparation method and culture method of intervertebral disc stem cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture medium for separating and extracting human intervertebral disc stem cells, a preparation method and a culture method of the intervertebral disc stem cells.

Background

With lifestyle changes, herniation of intervertebral discs caused by sedentary or compulsive positions has become a significant problem worldwide affecting human health. The herniated disc is easy to cause a series of diseases such as nervous system and cardiovascular system. Many studies have demonstrated that herniated intervertebral discs can cause paresthesia, cerebral blood supply disorders, and even cause symptoms of high paraplegia or cauda equina. The wide worldwide prevalence of this disease caused by herniated intervertebral discs is now making more and more concerned with the mechanistic studies associated therewith. Therefore, the generation process of the intervertebral disc cells is clarified, the mechanism of the cell molecule level in the differentiation and regulation of the intervertebral disc cells is disclosed, a solid theoretical basis can be provided for the prevention and treatment of related diseases caused by the degeneration of the intervertebral disc cells, and particularly, the screening of the drugs at the cell molecule level aiming at the diseases has important practical significance.

As one of the research hotspots in the fields of regenerative medicine and tissue engineering, the disc tissue regeneration technology is an important technical means for reconstructing and repairing disc tissue loss and damage caused by aging, pathological factors, and the like. The mesenchymal stem cells are used as a seed cell source for intervertebral disc regeneration engineering, and can greatly drive the reconstruction of new tissues.

The existing extraction method of mesenchymal stem cells based on umbilical cords, bone marrow and the like cannot well provide mesenchymal cell sources which are the same as or similar to intervertebral discs, has low efficiency, requires several weeks to obtain mature directional differentiated cells, not only has long induction period, but also increases cost to a certain extent. Therefore, the application value of the traditional technical means in clinical treatment and tissue engineering is weakened to a certain extent.

Research shows that the intervertebral disc stem cells are easy to obtain and are a very promising cell source for treating intervertebral disc degeneration. However, the current culture system cannot ensure efficient amplification, thereby reducing the large-scale foundation, clinical trials and clinical application prospects.

Disclosure of Invention

The invention aims to provide a culture medium for separating and extracting human intervertebral disc stem cells, a preparation method and a culture method of the intervertebral disc stem cells, so as to solve at least one technical problem in the background technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a culture medium for separating and extracting human intervertebral disc mesenchymal stem cells, which comprises the following components: low-sugar dartbuck modified eagle DMEM medium, DMEM/F12 medium, fetal bovine serum, antibiotics, recombinant fibroblast growth factor, epidermal growth factor, insulin, acetylcysteine, and thyronine.

Preferably, the volume ratio of the low-sugar Darber modified eagle DMEM medium to the DMEM/F12 medium is 1:1, and the sum of the volumes of the low-sugar Darber modified eagle DMEM medium and the DMEM/F12 medium accounts for 89% of the total volume of the culture medium of the intervertebral disc mesenchymal stem cells.

Preferably, the volume percent of the added fetal calf serum is 6 percent, the volume percent of the antibiotic is 1 percent, the concentration of the recombinant fibroblast growth factor is 2-10ng/ml, the concentration of the epidermal growth factor is 2-10ng/ml, the concentration of the insulin is 10ug/ml, the concentration of acetylcysteine is 2mM, and the concentration of the thyronine is 10 ng/ml.

Preferably, the antibiotic is a mixture of penicillin and streptomycin.

In a second aspect, the present invention provides a method for preparing a culture medium for isolation and extraction of human mesenchymal stem cells, comprising adding 50ml of fetal bovine serum, 5ml of antibiotic penicillin and streptomycin, 2.5ug of recombinant human fibroblast growth factor, 2.5ug of epidermal growth factor, 5mg of insulin, 2.5ml of acetylcysteine, 5ug of thyronine, and 0.22 μm filter to 445ml of low sugar darberg's modified eagle's medium, filtering and sterilizing the mixture, and storing the filtrate at 4 ℃ for later use.

In a third aspect, the present invention provides a method for culturing mesenchymal stem cells of intervertebral disc by using the culture medium for separating and extracting mesenchymal stem cells of human intervertebral disc, comprising the following steps:

step S110: shearing human intervertebral disc tissues obtained under an aseptic condition, mixing with ice phosphate buffer solution PBS, and centrifuging;

step S120: adding collagenase mixture I and II for digestion and centrifugation;

step S130: resuspending single cells in a water bath after using a culture medium for separating and extracting human intervertebral disc mesenchymal stem cells;

step S140: re-suspending with culture medium for separating and extracting human intervertebral disc mesenchymal stem cells, filtering, and transferring into culture flask;

step S150: after continuous culture for 5-7 days, the stem cells can be subcultured continuously by using a common culture medium after the stem cells grow into clones.

Preferably, the minced disc tissue is mixed with a 4 ℃ ice phosphate buffer solution and then centrifuged at a low speed in step S110.

Preferably, in step S120, a mixed collagenase type I and type II is used for digestion, with a collagenase type I and type II mass ratio of 1:1.

Preferably, the total volume fraction ratio of the mixed collagenase and phosphate buffer is 1:1000, 1ml of collagenase-phosphate buffer is used for digesting each gram of intervertebral disc tissue, the digestion time of the oscillating water bath is 0.5-2 hours, and the temperature of the water bath is 37 ℃ during digestion.

Preferably, in step S130, the digested single cells are resuspended in a culture medium for isolating and extracting human mesenchymal stem cells, and the resuspended cells are placed in a water bath at 37 ℃ for 15 minutes.

The invention has the beneficial effects that: the extraction method and the special culture medium can rapidly obtain a large amount of human intervertebral disc stem cells under the condition of less damage to the stem cells. The extracted primary adipose-derived stem cells have strong activity before passage and high stem cell purity. After passage, the intervertebral disc stem cell still has higher proliferation and differentiation capacity.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a diagram of the morphology of cells under an optical microscope 3 days after primary cell seeding according to an embodiment of the present invention.

FIG. 2 is a diagram of the morphology of cells under a light microscope 7 days after primary cell seeding, according to an embodiment of the present invention.

Fig. 3 is a schematic diagram of a positive result detected by the flow cytometer according to the embodiment of the present invention.

FIG. 4 is a schematic diagram showing the cell culture state of a control group in a cell migration experiment in an embodiment of the present invention.

FIG. 5 is a schematic diagram showing the cell culture state in the experimental group of cell migration experiments in the chamber of the present invention.

FIG. 6 is a graph showing the results of cell migration experiments in the control and experimental chambers of the present invention.

FIG. 7 is a schematic diagram showing the cell proliferation results of the control group and the experimental group in the cell proliferation assay according to the embodiment of the present invention.

FIG. 8 is a schematic diagram of the wound healing status of a control group in a wound healing experiment according to an embodiment of the present invention.

FIG. 9 is a schematic representation of the wound healing status of the experimental group in a wound healing experiment in accordance with an embodiment of the present invention.

FIG. 10 is a graph showing the results of wound healing in the control group and the experimental group in the wound healing experiment according to the example of the present invention.

Detailed Description

The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or modules, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, modules, and/or groups thereof.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

For the convenience of understanding of the embodiments of the present invention, the following description will be further explained by taking specific embodiments as examples with reference to the drawings, and the embodiments are not to be construed as limiting the embodiments of the present invention.

It will be understood by those of ordinary skill in the art that the figures are merely schematic representations of one embodiment and that the elements or devices in the figures are not necessarily required to practice the present invention.

Example 1

Embodiment 1 of the present invention provides a method for separating and extracting human intervertebral disc stem cells, which comprises the following steps:

step 1, taking human intervertebral discs through surgical operation under aseptic condition, cutting the human intervertebral discs into 3 fragments with the size of 1-3mm, mixing the fragments with ice Phosphate Buffer Solution (PBS), shaking and cleaning, standing and absorbing supernatant, repeating the step for 3-5 times until the supernatant has no blood color, and precipitating the supernatant into yellow-white particles;

step 2, digesting the mixture of collagenase I and collagenase II for 1 hour in a water bath at 37 ℃, and manually shaking and resuspending every 10-20 minutes;

and 3, after enough time, centrifuging 1000g of the upper suspension to obtain the upper suspension. Resuspending single cells by using a special extraction culture medium, and then carrying out water bath, wherein the special extraction culture medium contains specific chemical components and growth factors;

step 4, re-suspending with the special culture medium for extraction, filtering, and transferring into a culture flask;

after continuous culture for 5-7 days, after stem cells grow into clones, the stem cells can be continuously cultured by passage with a common culture medium;

in step 1, the minced disc tissue is mixed with 4 ℃ ice phosphate buffer and centrifuged at low speed.

In the step 2, the mixed collagenase of type I and type II is used for digestion, the mass ratio of the collagenase of type I to the collagenase of type II is 1:1, the total volume fraction ratio of the mixed collagenase to the phosphate buffer solution is 1:1000, 1ml of collagenase-phosphate buffer solution is used for digestion of each gram of intervertebral disc tissue, the digestion time of the oscillating water bath is 0.5 to 2 hours, and the temperature of the water bath during digestion is 37 ℃.

In step 3, the digested single cells are resuspended in a special extraction medium and placed in a 37 ℃ water bath for 15 minutes.

Example 2

In embodiment 2 of the present invention, a special medium for extraction is provided, which is used for separating and extracting a special medium for human intervertebral disc stem cells, and the special medium for extraction includes a low-sugar dartbucker modified eagle medium, fetal bovine serum, recombinant fibroblast growth factor, epidermal growth factor, insulin, acetylcysteine, and thyronine. The specific component proportion is that the volume percentage of the low-sugar Darber modified eagle culture medium is 89%, the volume percentage of the added fetal calf serum is 10%, the volume percentage of antibiotics (penicillin and streptomycin) is 1%, the concentration of recombinant fibroblast growth factor is 5ng/ml, the concentration of epidermal growth factor is 5ng/ml, the concentration of insulin is 10ug/ml, the concentration of acetylcysteine is 2mM, and the concentration of thyronine is 10 ng/ml.

In this example 2, the extraction and culture steps of human intervertebral disc stem cells include:

preparing a special culture medium for extracting human mesenchymal stem cells from intervertebral disc tissues: 50ml of fetal bovine serum, 5ml of antibiotics (penicillin and streptomycin), 2.5ug of recombinant human fibroblast growth factor, 2.5ug of epidermal growth factor, 5mg of insulin, 2.5ml of acetylcysteine (400nM stock solution), 5ug of thyronine, 0.22 μm filter were added to 445ml of low-sugar Darber modified eagle's medium, and the mixture was filtered and sterilized at 4 ℃ for further use.

The extraction and culture steps of the human intervertebral disc stem cells are as follows:

step 1, taking the intervertebral disc tissue taken out by a surgical operation, putting the intervertebral disc tissue into precooled PBS (phosphate buffer solution) with the mass of about 10g, and using the intervertebral disc tissue within 24 h.

Step 2, shearing the intervertebral disc tissues to l-3mm³Small fragments of (a); the minced disc tissue was transferred to a 50ml sterile centrifuge tube, and the tissue was washed several times with PBS to remove residual blood, and centrifuged at 1000rmp for 5 minutes.

And 3, adding 25ml of collagenase I and collagenase II and a PBS mixture, sealing, putting into a shaking water bath box at 37 ℃ for digesting lh, and continuously shaking the tissue block (once every 10-20 minutes) in the digestion process to promote digestion.

Step 4, centrifuging at 1000rmp for 5 minutes after digestion to remove upper undigested intervertebral disc tissues, adding 40ml of PBS for resuspension, and centrifuging at 800rmp for 5 minutes; the remaining tissue mass, if still large, can be collected and then digested to improve extraction efficiency.

Step 5, resuspending the extract by using 40ml of special culture medium for extraction, standing the extract for 15 minutes in a water bath at 37 ℃,

step 6, centrifuging for 5 minutes at 1000rmp to remove supernatant, then re-suspending with a special extraction culture medium, filtering with a sterile nylon net with the aperture of 100 mu m, counting the single-cell suspension after filtering, transferring the single-cell suspension into a T25 culture bottle according to the concentration of 105 cells/ml, putting the single-cell suspension into an incubator, controlling the temperature at 37 ℃ and 5% CO₂Culturing under the condition of humidity of 95%;

after steps 7 and 12h, replacing half of the volume of the special culture medium for extraction, completely replacing the culture medium after 1 day, and completely replacing the culture medium on the 4 th day. Observing the growth condition of the cells, wherein the cells grow into clones after 7 days generally; intervertebral disc cells are short spindle cells, which often grow in concentric circles;

after digestion with 0.25% trypsin, the cells were subcultured normally at a ratio of 1:3 in a conventional medium (DMEM: F12-1: 1 basal medium supplemented with 6% fetal bovine serum).

As shown in figures 1 and 2, after 12 hours, the human intervertebral disc stem cells are attached, and the stem cells grow into clones after 7 days of culture.

Experiment one: cell purity identification of human primary mesenchymal stem cells derived from intervertebral disc:

step 1, digesting primary cells with 0.25% pancreatin for 2 minutes, transferring the cells into a 2ml centrifuge tube, and centrifuging the cells for 5 minutes at 800 rmp; removing the supernatant;

step 2, adding 2ml PBS into each tube for resuspension, and centrifuging for 5 minutes at 800rmp to remove supernatant;

step 3, adding Iml PBS to resuspend the cells, counting, and adding PBS to adjust the number of the cells to be 1 × 106/ml;

step 4, dividing the cell suspension into multiple tubes, wherein each tube is 500 mu l, and selecting one tube as a flow control group;

step 5, adding CD29, CD44, CD73, CD90, CD105, CD34 and CD45 flow antibodies with FITC markers according to the components, incubating for 30 minutes at 4 ℃ in a dark place, and centrifuging for 5 minutes at 800rmp to remove supernatant;

step 6, add 1ml PBS heavy suspension, 800rmp centrifugation for 5 minutes to remove supernatant.

Step 7, adding 500 mu l of PBS to resuspend the cells;

and 8, detecting the sample by using a flow cytometer.

As shown in fig. 3, the flow cytometry detection shows that the positive expression rates of several markers CD29, CD44 and CD73 of the primary adipose-derived mesenchymal stem cells extracted by the method and the special extraction medium of the present invention are high. The extraction method and the extraction culture medium can separate stem cells with higher purity from the intervertebral disc.

And (2) test II: cell migration capacity assay various media proliferation capacities were tested:

step 1, co-incubation of different groups of cells in culture flasks for 24 hours.

Step 2, removing the original culture medium, and washing for several times by using a serum-free culture medium; trypsinized and cells collected in serum-free medium.

Step 3, adding 600 microliters of culture medium containing 10% FBS into a lower chamber of a Transwell (8-micron pore size), and adding 200 microliters of cell suspension into an upper chamber; incubate for 24 hours.

And 4, removing the liquid in the upper chamber and the lower chamber.

Step 5, 600 microliters of 4% paraformaldehyde is added into the lower chamber to fix the cells for half an hour.

And 6, removing paraformaldehyde, and erasing cells which do not penetrate through the membrane by using a cotton swab.

And 7, adding 600 microliters of 0.1% crystal violet into a lower chamber, and dyeing for 10 minutes.

Step 8, using PBS washing chamber.

Step 9, cells were observed under a microscope and counted.

As shown in fig. 4 to fig. 6, the intervertebral disc cells cultured by the medium provided in this example 2 have higher proliferation capacity than the common medium, as verified by the chamber migration experiment.

And (3) test III: the scar repair test verifies the difference of cell proliferation capacity.

Step 1, firstly, marking pens are used at the back of the 6-hole plate, transverse lines are uniformly marked by using a ruler, and the transverse lines cross the through holes approximately every 0.5-1 cm. Each hole passes through at least 5 lines.

Step 2, about 5 × 105 cells are added into the air, the specific number is different according to different cells, and the cells can be fully filled overnight.

And 3, comparing the gun head with the ruler on the next day, making the gun head perpendicular to the transverse line scratch on the back as much as possible, and making the gun head perpendicular and not capable of inclining.

And 4, washing the cells for 3 times by using PBS, removing the scratched cells, and adding a serum-free culture medium.

And 5, placing the mixture into a 37-degree 5% CO2 incubator for culture. Samples were taken at 24 hours and photographed.

As shown in fig. 8 to fig. 10, the intervertebral disc cells cultured by the medium provided in this example have higher migration ability than the common medium, as verified by the chamber migration experiment.

And (4) testing: cell count CCK-8 assay cells cultured on different media were tested for proliferative capacity:

the number of cells in the prepared cell suspension was counted using a cell counting plate, and then the cells were seeded into a culture plate.

According to the proportion of 1/2, the cell concentration gradient is formed by sequentially diluting the culture medium in equal proportion, 3-5 cell concentration gradients are generally made, and 3-6 multiple wells are recommended for each concentration.

Culturing for 2-4 hours after inoculation to allow the cells to adhere to the wall, adding CCK reagent to culture for a certain time, and measuring OD value to prepare a standard curve with the number of the cells as the abscissa (X axis) and the OD value as the ordinate (Y axis). The number of cells in the unknown sample can be determined from the standard curve (using the standard curve is based on the consistency of the experimental conditions, which facilitates the determination of the number of cells to be inoculated and the incubation time after the addition of CCK.)

Cell suspensions (100. mu.L/well) were seeded in 96-well plates. The plates were pre-incubated in an incubator for a period of time (37 ℃, 5% CO2), 10 μ L of CCK solution was added to each well (taking care not to generate bubbles in the wells which would affect the OD reading), and the plates were incubated in the incubator for 1-4 hours.

Absorbance at 450nm was measured with a microplate reader. If OD is not to be measured temporarily, 10. mu.L of 0.1M HCl solution or 1% w/v SDS solution may be added to each well, and the plate may be covered and kept at room temperature under protection from light. 1. The absorbance was changed by measurement for 3, 5 and 7 days.

As shown in FIG. 7, the cell count CCK-8 demonstrates that the intervertebral disc cells cultured by the culture medium provided in example 2 have higher proliferation capacity than the common culture medium.

In summary, the extraction method of the human intervertebral disc-derived stem cells and the special culture medium for extraction provided by the embodiments of the present invention can rapidly extract mesenchymal stem cells from intervertebral disc tissues, and the extracted stem cells have high purity and high viability. Can effectively enrich the variety of the adipose-derived stem cells, improve the primary extraction efficiency of the stem cells and provide relevant basis for the further scientific research and clinical application of the stem cells.

By using the method for extracting the intervertebral disc stem cells and the special culture medium for extracting the intervertebral disc stem cells, disclosed by the embodiment of the invention, a large amount of adipose-derived mesenchymal stem cells can be extracted from human intervertebral discs, and the purity of the stem cells in primary cultured cells is higher and the activity of the stem cells is stronger. The stem cell has strong stem cell activity and differentiation capacity after being subcultured for multiple times by using a common culture medium. The mesenchymal stem cells from the intervertebral disc are extracted through a specific extraction step and a special extraction culture medium in the human intervertebral disc, the damage of the stem cells can be reduced in the extraction method, and meanwhile, the problems that the primary cells cultured in vitro by the stem cells are low in culture purity, the stem cells are easy to age and differentiate after passage, cannot be continuously cultured for a long time and the like can be solved by using the special extraction culture medium (adding specific chemical components and growth factors).

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the above embodiment, but equivalent modifications or changes made by those skilled in the art according to the present disclosure should be included in the scope of the present invention as set forth in the appended claims.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of this invention and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the invention will fall within the scope of the invention as claimed.

The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.

Claims

1. A culture medium for separating and extracting human intervertebral disc mesenchymal stem cells is characterized by comprising the following components: low-sugar dartbuck modified eagle DMEM medium, DMEM/F12 medium, fetal bovine serum, antibiotics, recombinant fibroblast growth factor, epidermal growth factor, insulin, acetylcysteine, and thyronine.

2. The medium for isolating and extracting human mesenchymal stem cells according to claim 1, wherein: the volume ratio of the low-sugar Darber modified eagle DMEM medium to the DMEM/F12 medium is 1:1, and the sum of the volumes of the low-sugar Darber modified eagle DMEM medium and the DMEM/F12 medium accounts for 89% of the total volume of the culture medium of the intervertebral disc mesenchymal stem cells.

3. The medium for isolating human mesenchymal stem cells according to claim 1, wherein: the volume percent of the added fetal calf serum is 6 percent, the volume percent of the antibiotic is 1 percent, the concentration of the recombinant fibroblast growth factor is 2-10ng/ml, the concentration of the epidermal growth factor is 2-10ng/ml, the concentration of the insulin is 10ug/ml, the concentration of acetylcysteine is 2mM, and the concentration of the thyronine is 10 ng/ml.

4. The medium for isolating and extracting human mesenchymal stem cells according to claim 1, wherein: the antibiotic is a mixture of penicillin and streptomycin.

5. A method for preparing a culture medium for isolating and extracting human mesenchymal stem cells according to any one of claims 1 to 4, wherein the culture medium comprises: 50ml of fetal calf serum, 5ml of antibiotic penicillin and streptomycin, 2.5ug of recombinant human fibroblast growth factor, 2.5ug of epidermal growth factor, 5mg of insulin, 2.5ml of acetylcysteine and 5ug of thyronine are added into 445ml of low-sugar Darber modified eagle culture medium, and the mixture is filtered and sterilized by a 0.22 mu m filter and stored at 4 ℃ for later use.

6. A method for culturing the mesenchymal stem cells of the intervertebral disc by using the culture medium for separating and extracting the mesenchymal stem cells of the human intervertebral disc as claimed in any one of claims 1 to 5, which is characterized by comprising the following steps:

7. The method of claim 6, wherein: in step S110, the minced intervertebral disc tissue is mixed with a 4 ℃ ice phosphate buffer and then centrifuged at a low speed.

8. The method of claim 6, wherein: in step S120, a mixed collagenase of type I and type II is used for digestion, and the quality ratio of the collagenase of type I to type II is 1:1.

9. The method of claim 8, wherein: the total volume fraction ratio of the mixed collagenase and the phosphate buffer solution is 1:1000, 1ml of collagenase-phosphate buffer solution is used for digesting each gram of intervertebral disc tissue, the digestion time of the oscillating water bath is 0.5-2 hours, and the temperature of the water bath is 37 ℃ during digestion.

10. The method of claim 6, wherein: in the step S130, the digested single cells are resuspended in a medium for separating and extracting human mesenchymal stem cells, and the resuspended single cells are placed in a water bath at 37 ℃ and kept still for 15 minutes.