CN112402365A - PRP gel loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases - Google Patents

Abstract

The invention discloses a composition of umbilical cord mesenchymal stem cells loaded with PRP gel for treating intervertebral disc degeneration diseases, in particular to the technical field of intervertebral disc degeneration diseases, wherein each 2mL of the composition at least comprises 5 multiplied by 10^5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin, and as a preferred technical scheme, each 2mL of the composition also comprises 0.08mg of NGF. The composition of the invention has simple preparation method, the cell shape in the composition is not changed, and the cell survival rate can reach 98.4-99.5%. Has better intervertebral disc degeneration effect for treating intervertebral disc degeneration. In the specific use process, under the premise of not changing the original spine mechanical structure and replacing the original intervertebral disc, the intervertebral disc regeneration effect is generated in situ, the TDR is not needed, the risks, sequelae and complications are reduced, the application and operation difficulty is low, the acceptable degree of a patient is larger, and the wound is smaller.

Description

PRP gel loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases

Technical Field

The invention belongs to the technical field of intervertebral disc degeneration diseases, and particularly relates to a PRP gel loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases.

Background

Low Back Pain (LBP) is an important public health problem. About 6.5 million people in the world today are affected by it, and as the population ages, its socioeconomic consumption is increasing. What is more, the lower back pain is the second leading cause of hospitalization, and in Lancet, professor Chris Maher 2017 said that lower back pain is the leading cause of Disability loss of healthy life Years (Years live with Disability) in developed and developing countries.

The conventional treatment modes of medicines, operations and the like for the discogenic lumbago have relatively high treatment cost, relatively large pain and relatively large wound brought to patients, and long-term research shows that the disease causes the accelerated degeneration of the adjacent intervertebral discs with a certain probability. Currently, lumbago of disc origin is generally treated with drugs, if failed, by invasive surgery (spinal fusion or disc replacement) as the final option. The post-operative clinical success rate of spinal fusion is between 50% and 70%. Spinal fusion can lead to the development of degeneration of adjacent segments, which often requires re-surgery. Furthermore, spinal fusion is costly and may incur additional costs due to long recovery times and life-long injuries. As for disc replacement surgery (TDR), meta analysis of a randomized controlled study indicated that TDR has similar safety and efficacy to spinal fusion in a 2 year follow-up study, and that TDR has advantages in improving physiological function, alleviating pain, and shortening hospital stay. However, an earlier systematic review indicated that: the spinal surgeon should be cautious enough to perform a number of disc replacements because complications may occur some years later.

In the face of these limitations of treatment regimens, given the recent knowledge of the pathophysiological mechanisms of disc degeneration, and in particular the depletion of nucleus pulposus cells during disc degeneration, the use of functional cells and biomaterials for nucleus pulposus augmentation (cell therapy) has now become an alternative to the prevention of disc degeneration. A number of preclinical studies have been conducted and have partially demonstrated the scientificity of this regenerative cell therapy. Meanwhile, the effectiveness of cell therapy has been evaluated in preliminary studies in humans. However, there is no composition for intervertebral disc degeneration.

Disclosure of Invention

The technical problem to be solved by the invention is that in the prior art, common TDR is used for treating intervertebral disc degeneration in common use, but the risk is high and complications are frequent, some current researches mention that nucleus pulposus augmentation (cell therapy) can be realized by using functional cells and biological materials, so how to prepare a PRP gel loaded umbilical cord mesenchymal stem cell composition suitable for treating intervertebral disc degeneration diseases is an urgent technical problem to be solved.

Therefore, the PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration is researched, and in the specific use process, the PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration has the effect of regenerating the intervertebral disc in situ on the premise of not changing the original spinal mechanics structure and replacing the original intervertebral disc.

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

a PRP gel loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease, comprising at least 5 x 10^5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin per 2mL of the composition.

Further, every 2mL of the composition also contains 0.08mg of NGF.

Further, in the composition, the average cell activity of the umbilical cord mesenchymal stem cells is more than or equal to 98%.

Further, the composition is prepared according to the following steps:

resuspending 5 x 10^5 umbilical cord mesenchymal stem cells with 2ml PRP, mixing well, adding 100 units thrombin into the mixture, and mixing well.

Further, the composition is prepared according to the following steps:

taking 2ml of PRP, adding 0.08mg of NGF into the PRP, uniformly mixing, then resuspending 5 x 10^5 umbilical cord mesenchymal stem cells by 2ml of PRP added with NGF, uniformly mixing, adding 100 units of thrombin into the mixture, and uniformly mixing.

NGF plays a role in nourishing nerve cells, PRP mainly plays a role in repairing, and the NGF and the PRP are combined to reduce the influence of degenerative changes on a nervous system.

Further, the air conditioner is provided with a fan,

the preparation method of the PRP gel comprises the following steps:

(1) evenly subpackaging the blood sample into 15ml centrifuge tubes, wherein each tube does not exceed 15ml, and centrifuging for 8-10 min at 300g (the speed is increased by 9 and the speed is decreased by 7);

(2) dividing whole blood into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin light yellow interface, namely a PRP layer, can be seen at the joint of the two layers, a pipette is used for contacting the liquid level, all supernatant is carefully sucked to be 3mm below the cross interface, the supernatant is transferred into a 15ml centrifuge tube and is balanced, and 800g (the speed is increased by 9 and the speed is decreased by 7) is centrifuged for 6 min;

(3) liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred to another centrifuge tube by a liquid transfer machine, the residual serum is contacted with the liquid surface by the liquid transfer machine, the supernatant is carefully sucked to be 3mm below an interface, and the supernatant is transferred to a new centrifuge tube, namely PRP gel.

Further, the first and second electrodes are provided with a first and a second electrode, respectively,

the centrifugation time in step (1) is 8 min.

Further, the umbilical cord mesenchymal stem cells are cultured in the following way:

(1) horizontally placing the culture bottle to ensure that the umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture bottle in a carbon dioxide constant-temperature constant-humidity culture box; the culture conditions are as follows: at 37 ℃, the volume fraction of carbon dioxide is 5 percent;

(2) taking out the culture bottle after one week, observing whether stem cells grow, observing every day until stem cells grow, and starting to change the culture solution;

(3) half amount of liquid change: if the stem cells are observed to grow in the culture bottle, half of the culture solution is changed, the culture bottle is slightly inclined, the old culture medium is slightly sucked off by a pipette (the tissue block is not sucked out), an equal amount of fresh culture medium is replenished, a carbon dioxide constant temperature and humidity incubator is placed to start culture, and the culture conditions are as follows: the carbon dioxide volume fraction is 5% at 37 ℃, the growth condition of the cells is observed, and the local fusion degree of the concentrated cells reaches 80% -90%;

(4) removing the culture solution and the tissue blocks in the culture bottle by suction, and slightly washing the cell culture bottle for 1-2 times by using sodium chloride injection;

(5) digestion: digesting the cells until most of the cells are changed from fusiform to round and shed under an inverted microscope;

(6) collecting cells, adding 10-15 ml of sodium chloride injection into each bottle of an original culture bottle, and slightly blowing and beating the adherent cells;

(7) and (3) filtering: collecting the cell suspension to a 50ml centrifuge tube, and filtering by a screen;

(8) cell counting: gently blowing and beating, re-suspending cells, uniformly blowing and beating, sampling and counting;

(9) centrifugal washing: counting 5 multiplied by 10^5, centrifugally washing, pouring off centrifugal supernatant, and obtaining the stem cells at the bottom, namely the umbilical cord mesenchymal stem cells.

The invention has the advantages that:

(1) the preparation method of the PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease is simple, the cell form in the composition is not changed, and the cell survival rate can reach 98.4-99.5%. Has better intervertebral disc degeneration effect for treating intervertebral disc degeneration.

(2) According to the PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease, in the specific use process, the PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration generates the intervertebral disc regeneration effect in situ on the premise of not changing the original spine mechanical structure and replacing the original intervertebral disc, and does not need TDR, so that risks, sequelae and complications are reduced, the application and operation difficulty is low, the acceptable degree of a patient is higher, and the wound is smaller.

Drawings

Fig. 1 is two key index diagrams of observing nucleus pulposus cells col2 and sox-9 after PRP gel loaded umbilical cord mesenchymal stem cells and PRP gel not loaded umbilical cord mesenchymal stem cells and physiological saline are respectively injected clinically.

Detailed Description

The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.

A PRP gel loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease, comprising at least 5 x 10^5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin per 2mL of the composition.

In some embodiments, 0.08mg NGF is also included per 2mL of the composition.

In some embodiments, the average cell activity of the umbilical cord mesenchymal stem cells in the composition is greater than or equal to 98%.

In some embodiments, the composition is made according to the following steps:

resuspending 5 x 10^5 umbilical cord mesenchymal stem cells with 2ml PRP, mixing well, adding 100 units thrombin into the mixture, and mixing well.

The composition is prepared by the following steps:

taking 2ml of PRP, adding 0.08mg of NGF into the PRP, uniformly mixing, then resuspending 5 x 10^5 umbilical cord mesenchymal stem cells by 2ml of PRP added with NGF, uniformly mixing, adding 100 units of thrombin into the mixture, and uniformly mixing.

The preparation method of the PRP gel comprises the following steps:

(1) slightly unscrewing the injector plug, pulling out the tube cover, evenly subpackaging the blood sample into 15ml centrifuge tubes, wherein each tube does not exceed 15ml, and centrifuging for 8-10 min at 300g (the speed is increased by 9 and decreased by 7);

(2) dividing whole blood into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin light yellow interface, namely a PRP layer, can be seen at the joint of the two layers, a pipette is used for contacting the liquid level, all supernatant is carefully sucked to be 3mm below the cross interface, the supernatant is transferred into a 15ml centrifuge tube and is balanced, and 800g (the speed is increased by 9 and the speed is decreased by 7) is centrifuged for 6 min;

(3) liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred to another centrifuge tube by a liquid transfer machine, the residual serum is contacted with the liquid surface by the liquid transfer machine, the supernatant is carefully sucked to be 3mm below an interface, and the supernatant is transferred to a new centrifuge tube, namely PRP gel.

In some embodiments, the centrifugation time in step (1) is 8 min.

The umbilical cord mesenchymal stem cells are cultured in the following way (in the following steps, all the culture media are commercial Dayou stem cell culture media):

(1) horizontally placing the culture bottle to ensure that the umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture bottle in a carbon dioxide constant-temperature constant-humidity culture box; the culture conditions are as follows: at 37 ℃, the volume fraction of carbon dioxide is 5 percent;

(2) taking out the culture bottle after one week, observing whether stem cells grow, observing every day until stem cells grow, and starting to change the culture solution;

(3) half amount of liquid change: if the stem cells are observed to grow in the culture bottle, half of the culture solution is changed, the culture bottle is slightly inclined, the old culture medium is slightly sucked off by a pipette (the tissue block is not sucked out), an equal amount of fresh culture medium is replenished, a carbon dioxide constant temperature and humidity incubator is placed to start culture, and the culture conditions are as follows: the carbon dioxide volume fraction is 5% at 37 ℃, the growth condition of the cells is observed, and the local fusion degree of the concentrated cells reaches 80% -90%;

(4) removing the culture solution and the tissue blocks in the culture bottle by suction, and slightly washing the cell culture bottle for 1-2 times by using sodium chloride injection;

(5) digestion: digesting the cells until most of the cells are changed from fusiform to round and shed under an inverted microscope;

(6) collecting cells, adding 10-15 ml of sodium chloride injection into each bottle of an original culture bottle, and slightly blowing and beating the adherent cells;

(7) and (3) filtering: collecting the cell suspension to a 50ml centrifuge tube, and filtering by a screen;

(8) cell counting: gently blowing and beating, re-suspending cells, uniformly blowing and beating, sampling and counting;

(9) centrifugal washing: counting 5 multiplied by 10^5, centrifugally washing, pouring off centrifugal supernatant, and obtaining the stem cells at the bottom, namely the umbilical cord mesenchymal stem cells.

Example 1:

a PRP gel loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease, comprising at least 5 x 10^5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin per 2mL of the composition; in the composition, the average cell activity of the umbilical cord mesenchymal stem cells is more than or equal to 98 percent.

The composition is prepared by the following steps:

resuspending 5X 10^5 umbilical cord mesenchymal stem cells with 2ml PRP gel, mixing well, adding 100 units thrombin into the mixture, and mixing well.

The preparation method of the PRP gel comprises the following steps:

(1) slightly unscrewing the injector plug, pulling out the tube cover, evenly subpackaging the blood sample into 15ml centrifuge tubes, wherein each tube does not exceed 15ml, and centrifuging for 8min at 300g (the speed is increased by 9 and the speed is decreased by 7);

(2) dividing whole blood into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin light yellow interface, namely a PRP layer, can be seen at the joint of the two layers, a pipette is used for contacting the liquid level, all supernatant is carefully sucked to be 3mm below the cross interface, the supernatant is transferred into a 15ml centrifuge tube and is balanced, and 800g (the speed is increased by 9 and the speed is decreased by 7) is centrifuged for 6 min;

(3) liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred to another centrifuge tube by a liquid transfer machine, the residual serum is contacted with the liquid surface by the liquid transfer machine, the supernatant is carefully sucked to be 3mm below an interface, and the supernatant is transferred to a new centrifuge tube, namely PRP gel.

The umbilical cord mesenchymal stem cells are cultured in the following way (in the following steps, all the culture media are commercial Dayou stem cell culture media):

(1) horizontally placing the culture bottle to ensure that the umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture bottle in a carbon dioxide constant-temperature constant-humidity culture box; the culture conditions are as follows: at 37 ℃, the volume fraction of carbon dioxide is 5 percent;

(2) taking out the culture bottle after one week, observing whether stem cells grow, observing every day until stem cells grow, and starting to change the culture solution;

(3) half amount of liquid change: if the stem cells are observed to grow in the culture bottle, half of the culture solution is changed, the culture bottle is slightly inclined, the old culture medium is slightly sucked off by a pipette (the tissue block is not sucked out), an equal amount of fresh culture medium is replenished, a carbon dioxide constant temperature and humidity incubator is placed to start culture, and the culture conditions are as follows: the carbon dioxide volume fraction is 5% at 37 ℃, the growth condition of the cells is observed, and the local fusion degree of the concentrated cells reaches 80% -90%;

(4) removing the culture solution and the tissue blocks in the culture bottle by suction, and slightly washing the cell culture bottle for 1-2 times by using sodium chloride injection;

(5) digestion: digesting the cells until most of the cells are changed from fusiform to round and shed under an inverted microscope;

(6) collecting cells, adding 10-15 ml of sodium chloride injection into each bottle of an original culture bottle, and slightly blowing and beating the adherent cells;

(7) and (3) filtering: collecting the cell suspension to a 50ml centrifuge tube, and filtering by a screen;

(8) cell counting: gently blowing and beating, re-suspending cells, uniformly blowing and beating, sampling and counting;

(9) centrifugal washing: counting 5 multiplied by 10^5, centrifugally washing, pouring off centrifugal supernatant, and obtaining the stem cells at the bottom, namely the umbilical cord mesenchymal stem cells.

The PRP gel loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease obtained in the above example 1 is detected by a flow cytometer, and the detection result is shown in the following table 1, the cell morphology is not changed, and the cell viability can reach 98.4% -99.3%.

Table 1 results of cell assay of the composition of example 1

Number of times	CD105+(％)	CD73+(％)	CD90+(％)	CD45+(％)	CD34+(％)	HLA-DR(％)	Cell viability (%)
								For the first time	100％	99.4	99.9	0.01	0.07	0.04	99.1
For the second time	99.6	99.8	99.1	0.04	0.56	0.07	98.4
								The third time	99.5	99.6	99.4	0.08	0.27	0.89	99.3
Fourth time	99.2	99.7	99.7	0.24	0.68	0.21	99.2
								Fifth time	99.1	99.4	99.6	0.81	0.09	0.31	98.5
The sixth time	99.5	99.8	100	0.36	0.06	0.03	98.8

Example 2:

a PRP gel loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease, comprising at least 5 x 10^5 umbilical cord mesenchymal stem cells, PRP gel, 0.08mg NGF and 100 units thrombin per 2mL of the composition.

The composition is prepared by the following steps:

taking 2ml of PRP, adding 0.08mg of NGF into the PRP, uniformly mixing, then resuspending 5 x 10^5 umbilical cord mesenchymal stem cells by 2ml of PRP added with NGF, uniformly mixing, adding 100 units of thrombin into the mixture, and uniformly mixing.

The preparation method of the PRP gel is the same as that of example 1.

The umbilical cord mesenchymal stem cells are cultured in the same manner as in example 1.

Table 2 results of cell assay for the composition of example 2

Number of times	CD105+(％)	CD73+(％)	CD90+(％)	CD45+(％)	CD34+(％)	HLA-DR(％)	Cell viability (%)
								For the first time	99.4％	99.5	100	0.02	0.13	0.65	99.3
For the second time	99.6	99.6	99.4	0.12	0.33	0.27	99.1
								The third time	99.7	99.4	99.8	0.04	0.08	0.31	98.7
Fourth time	99.6	99.7	99.6	0.02	0.08	0.08	98.8
								Fifth time	100	99.8	99.5	0.13	0.21	0.12	99.5
The sixth time	99.3	99.6	99.8	0.24	0.37	0.52	99.2

Application example 1

The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease obtained in the example 1 is respectively used for treating patients with intervertebral disc degeneration clinically, patients with lumbar intervertebral disc protrusion and pseudohypodiscectomy clinically, and patients with lumbar pain caused by lumbar vertebra degeneration clinically by a way of transforaminal injection.

Application example 2

The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease obtained in the example 2 is respectively used for treating patients with intervertebral disc degeneration clinically, patients with lumbar intervertebral disc protrusion and pseudohypodiscectomy clinically, and patients with lumbar pain caused by lumbar vertebra degeneration clinically by a way of transforaminal injection.

Application comparative example 1

The PRP gel without loading the umbilical cord mesenchymal stem cells is respectively used for treating patients with intervertebral disc degeneration clinically, patients with lumbar intervertebral disc protrusion and pseudohypodiscectomy of intervertebral disc prolapse clinically and patients with lumbar pain caused by lumbar vertebra degeneration clinically in an intervertebral disc degeneration injection mode.

Comparative application example 2

The normal saline is respectively used for treating patients with intervertebral disc degeneration clinically, patients with lumbar intervertebral disc protrusion and simulated hypodiscectomy of intervertebral disc prolapse clinically and patients with lumbar pain caused by lumbar vertebra degeneration clinically by an intervertebral foramen injection mode.

Specific results are shown in table 3 below:

table 3: results of the above application examples 1-2 and comparative examples 1-2

Numbering	Height of intervertebral disc	Water content of intervertebral disc	VAS score
				Application example 1	+9％	+11％	7 fen → 4 fen
Application example 2	+10％	+11.5％	7 fen → 3 fen
				Application comparative example 1	+1％	+3％	Is not changed
Comparative application example 2	Is not changed	Is not changed	Is not changed

FIG. 1 shows two key indicators of the nucleus pulposus cells col2 and sox-9 observed after injecting PRP gel loaded with the umbilical cord mesenchymal stem cells, PRP gel not loaded with the umbilical cord mesenchymal stem cells and normal saline respectively in application example 1, application comparative example 1 and application comparative example 2, and it can be concluded that the effect of application example 1 is better than that of other comparative examples 1 and 2 by increasing the relative density of the nucleus pulposus cells loaded with the umbilical cord mesenchymal stem cells by the PRP gel.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Variations or modifications in other variations may occur to those skilled in the art based upon the foregoing description. Not all embodiments need be illustrated or described herein. And obvious variations or modifications of this embodiment may be made without departing from the spirit or scope of the invention.

Claims (8)

1. A PRP gel loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases is characterized by comprising at least 5 x 10^5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin in every 2mL of the composition.

2. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for the treatment of a degenerative disc disease according to claim 1, further comprising 0.08mg NGF per 2mL of the composition.

3. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration disease according to claim 1 or 2, wherein the average cell activity of umbilical cord mesenchymal stem cells in the composition is greater than or equal to 98%.

4. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for the treatment of a degenerative disc disease according to claim 1 or 3, wherein the composition is prepared according to the following steps:

resuspending 5 x 10^5 umbilical cord mesenchymal stem cells with 2ml PRP, mixing well, adding 100 units thrombin into the mixture, and mixing well.

5. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease according to claim 2 or 3, wherein the composition is prepared by the steps of:

taking 2ml of PRP, adding 0.08mg of NGF into the PRP, uniformly mixing, then resuspending 5 x 10^5 umbilical cord mesenchymal stem cells by 2ml of PRP added with NGF, uniformly mixing, adding 100 units of thrombin into the mixture, and uniformly mixing.

6. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease according to claim 1, which is characterized in that,

the preparation method of the PRP gel comprises the following steps:

(1) evenly subpackaging the blood sample into 15ml centrifuge tubes, wherein each tube does not exceed 15ml, and centrifuging for 8-10 min at 300g (the speed is increased by 9 and the speed is decreased by 7);

(2) dividing whole blood into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin light yellow interface, namely a PRP layer, can be seen at the joint of the two layers, a pipette is used for contacting the liquid level, all supernatant is carefully sucked to be 3mm below the cross interface, the supernatant is transferred into a 15ml centrifuge tube and is balanced, and 800g (the speed is increased by 9 and the speed is decreased by 7) is centrifuged for 6 min;

(3) liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred to another centrifuge tube by a liquid transfer machine, the residual serum is contacted with the liquid surface by the liquid transfer machine, the supernatant is carefully sucked to be 3mm below an interface, and the supernatant is transferred to a new centrifuge tube, namely PRP gel.

7. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease according to claim 6, wherein,

the centrifugation time in step (1) is 8 min.

8. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating a degenerative disc disease according to claim 1, which is characterized in that,

the umbilical cord mesenchymal stem cells are cultured in the following way:

(1) horizontally placing the culture bottle to ensure that the umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture bottle in a carbon dioxide constant-temperature constant-humidity culture box; the culture conditions are as follows: at 37 ℃, the volume fraction of carbon dioxide is 5 percent;

(2) taking out the culture bottle after one week, observing whether stem cells grow, observing every day until stem cells grow, and starting to change the culture solution;

(3) half amount of liquid change: if the stem cells are observed to grow in the culture bottle, half of the culture solution is changed, the culture bottle is slightly inclined, the old culture medium is slightly sucked off by a pipette (the tissue block is not sucked out), an equal amount of fresh culture medium is replenished, a carbon dioxide constant temperature and humidity incubator is placed to start culture, and the culture conditions are as follows: the carbon dioxide volume fraction is 5% at 37 ℃, the growth condition of the cells is observed, and the local fusion degree of the concentrated cells reaches 80% -90%;

(4) removing the culture solution and the tissue blocks in the culture bottle by suction, and slightly washing the cell culture bottle for 1-2 times by using sodium chloride injection;

(5) digestion: digesting the cells until most of the cells are changed from fusiform to round and shed under an inverted microscope;

(6) collecting cells, adding 10-15 ml of sodium chloride injection into each bottle of an original culture bottle, and slightly blowing and beating the adherent cells;

(7) and (3) filtering: collecting the cell suspension to a 50ml centrifuge tube, and filtering by a screen;

(8) cell counting: gently blowing and beating, re-suspending cells, uniformly blowing and beating, sampling and counting;

(9) centrifugal washing: counting 5 multiplied by 10^5, centrifugally washing, pouring off centrifugal supernatant, and obtaining the stem cells at the bottom, namely the umbilical cord mesenchymal stem cells.

Images