CN112402365B - PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases - Google Patents
PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases Download PDFInfo
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Abstract
The invention discloses a PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating intervertebral disc degeneration diseases, and particularly relates to the technical field of intervertebral disc degeneration diseases, wherein each 2mL of the composition at least comprises 5X 10-5 umbilical cord mesenchymal stem cells, PRP gel and 100 units of thrombin, and as a preferable technical scheme, each 2mL of the composition also comprises 0.08mg NGF. The preparation method of the composition is simple, the cell morphology in the composition is not changed, and the cell activity rate can reach 98.4% -99.5%. Has better effect on treating the common disc degeneration of the disc degeneration. In the specific use process, on the premise of not changing the original spinal mechanical structure and not replacing the original intervertebral disc, the function of regenerating the intervertebral disc is generated in situ, TDR is not needed, the risk, sequelae and complications are reduced, the application operation difficulty is low, the acceptable degree of a patient is higher, and the trauma is smaller.
Description
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
Lower Back Pain (LBP) is an important public health problem. About 6.5 hundred million people in the world are affected by the world, and the social and economic consumption of the people is continuously increasing as the population ages. Furthermore, lower back pain is the second leading cause of hospitalization, and Chris Maher professor 2017 called lower back pain in Lancet is the leading cause of disability in developed and developing countries and loss of healthy life years (Years Lived with Disability).
The conventional treatment modes such as medicines, operations and the like for treating the intervertebral disc-derived lumbago have relatively high treatment cost, relatively high pain and relatively high trauma to patients, and long-term researches show that the diseases also have a certain probability of causing accelerated degeneration of adjacent intervertebral discs. Currently, intervertebral disc-derived lumbago is generally a medication, and if failed, is selected as a final choice by invasive surgery (spinal fusion or disc replacement). The clinical success rate of spinal fusion is between 50% and 70%. Spinal fusion may lead to the development of degeneration of adjacent segments, a condition that often requires re-surgery. Furthermore, spinal fusion is costly and may incur additional costs due to long recovery time and lifetime injuries. As for disc replacement (TDR), meta analysis of a randomized controlled study indicated that TDR had similar safety and efficacy as spinal fusion in a 2 year follow-up study, and that TDR exhibited advantages in improving physiological function, alleviating pain and shortening hospitalization. However, an earlier systematic review indicates that: spinal surgeons should be careful enough when performing a large number of disc replacements because complications may occur in later years.
Given the limitations of these therapeutic regimens, the approach of supplementing the nucleus pulposus with cells and biological materials (cytotherapy) has now become an alternative to preventing disc degeneration, given the recent knowledge of the pathophysiological mechanisms of disc degeneration, in particular the consumption of nucleus pulposus cells during disc degeneration. A number of preclinical studies have been conducted and partially demonstrated the scientificity of this regenerative cell therapy. Meanwhile, the effectiveness of cell therapies has been evaluated in preliminary studies of the human body. But there is no composition for disc degeneration.
Disclosure of Invention
The technical problem to be solved by the invention is that in the prior art, the common TDR for treating disc degeneration is used for treating disc degeneration, but the risk is high, and complications are common, and the present researches mention that the common TDR can be realized by using cells and biological materials to carry out nucleus pulposus supplementation (cell therapy), so that how to prepare the PRP gel-loaded umbilical mesenchymal stem cell composition suitable for treating disc degeneration diseases is the 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 a specific use process, the PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration generates the function of regenerating the intervertebral disc in situ on the premise of not changing the original spinal mechanical structure and not replacing the original intervertebral disc.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating an intervertebral disc degeneration disease, comprising at least 5 x 10-5 umbilical cord mesenchymal stem cells per 2mL of the composition, PRP gel and 100 units of thrombin.
Further, 0.08mg NGF is also included per 2mL of the composition.
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:
5X 10-5 umbilical cord mesenchymal stem cells were resuspended with 2ml PRP, mixed well, 100 units of thrombin were added thereto, and mixed well.
Further, the composition is prepared according to the following steps:
2ml of PRP was taken, 0.08mg of NGF was added thereto, and mixed well, then 5X 10≡5 umbilical cord mesenchymal stem cells were resuspended in 2ml of PRP to which NGF was added, and 100 units of thrombin was added thereto, and mixed well.
NGF plays a role in nourishing nerve cells, PRP is mainly a repairing effect, and the two are combined to reduce the influence of degenerative diseases on a nerve system.
Further, the method comprises the steps of,
the preparation method of the PRP gel comprises the following steps:
(1) The blood sample is divided into 15ml centrifuge tubes, each tube does not exceed 15ml,300g (increasing speed 9 and decreasing speed 7) is centrifuged for 8min to 10min;
(2) The whole blood is divided into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin pale yellow interface layer, namely a PRP layer, is visible at the joint of the two layers, a liquid level is contacted by a liquid-transferring gun, the whole supernatant is carefully sucked to 3mm below the interface layer, transferred into a 15ml centrifuge tube, balanced, and centrifuged for 6min at 800g (rising speed 9 and falling speed 7);
(3) The liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred into another centrifuge tube by a liquid transfer device, the rest serum is contacted with the liquid level by the liquid transfer device, the supernatant is carefully sucked and transferred into a new centrifuge tube by 3mm below the intersection, namely PRP gel.
Further, the method comprises the steps of,
the centrifugation time in step (1) was 8min.
Further, the umbilical cord mesenchymal stem cells are cultured in the following manner:
(1) Horizontally placing the culture flask to ensure that umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture flask in a carbon dioxide constant temperature and humidity incubator; culture conditions: 37 ℃ and the volume fraction of carbon dioxide is 5%;
(2) After one week, taking out the culture flask, observing whether stem cells grow or not, observing every day until the stem cells grow, and starting to change the liquid;
(3) Half liquid change: if stem cells are observed to grow in the culture flask, half liquid exchange is carried out, the culture flask is slightly inclined, old culture medium is gently sucked by a pipette (note that tissue blocks are not sucked out), the fresh culture medium with the same amount is complemented, and a carbon dioxide constant temperature and humidity incubator is placed for starting culture, wherein the culture conditions are as follows: observing the growth condition of cells at 37 ℃ with the volume fraction of carbon dioxide of 5%, wherein the fusion degree of the concentrated cells reaches 80% -90%;
(4) Sucking and discarding the culture solution and tissue blocks in the culture flask, and gently flushing the cell culture flask with sodium chloride injection for 1-2 times;
(5) Digestion: digesting the cells until the cells are observed to be mostly deformed into a round shape by the shuttle and shed under an inverted microscope;
(6) Collecting cells, adding 10-15 ml of sodium chloride injection into each original culture flask, gently blowing, and blowing off adherent cells;
(7) And (3) filtering: collecting cell suspension to a 50ml centrifuge tube, and filtering by a screen;
(8) Cell count: gently beating, re-suspending cells, uniformly mixing by beating, sampling and counting;
(9) And (3) centrifugal washing: counting 5 x 10-5, centrifuging and washing, and pouring out the supernatant, wherein the bottom stem cells are umbilical cord mesenchymal stem cells.
The invention has the advantages that:
(1) The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease has a simple preparation method, the cell morphology in the composition is not changed, and the cell activity rate can reach 98.4% -99.5%. Has better effect on treating the common disc degeneration of the disc degeneration.
(2) The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease provided by the invention has the advantages that in a specific use process, the PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration has the advantages that on the premise of not changing the original spinal mechanical structure and not replacing the original intervertebral disc, the effect of regenerating the intervertebral disc is generated in situ, TDR (time domain reflectometer) is not needed, the risk and sequelae and complications are reduced, the application operation difficulty is low, the patient acceptability is higher, and the wound is smaller.
Drawings
FIG. 1 is a graph showing two key indexes of nucleus pulposus cells col2 and sox-9 after the PRP gel loaded with umbilical cord mesenchymal stem cells, the PRP gel without umbilical cord mesenchymal stem cells and normal saline are respectively injected in clinic.
Detailed Description
The invention is further illustrated by the following specific examples, which should be understood to those skilled in the art that variations and modifications can be made without departing from the principles of the invention, and these should also be considered to be within the scope of the invention.
A PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating an intervertebral disc degeneration disease, comprising at least 5 x 10-5 umbilical cord mesenchymal stem cells per 2mL of the composition, PRP gel and 100 units of thrombin.
In some embodiments, 0.08mg NGF is also included per 2mL of the composition.
In some embodiments, the composition has an average cell activity of umbilical cord mesenchymal stem cells of greater than or equal to 98%.
In some embodiments, the composition is made according to the following steps:
5X 10-5 umbilical cord mesenchymal stem cells were resuspended with 2ml PRP, mixed well, 100 units of thrombin were added thereto, and mixed well.
The composition is prepared according to the following steps:
2ml of PRP was taken, 0.08mg of NGF was added thereto, and mixed well, then 5X 10≡5 umbilical cord mesenchymal stem cells were resuspended in 2ml of PRP to which NGF was added, and 100 units of thrombin was added thereto, and mixed well.
The preparation method of the PRP gel comprises the following steps:
(1) After the injector plug is slightly loosened, the tube cover is pulled out, the blood sample is averagely split into 15ml centrifuge tubes, each tube does not exceed 15ml,300g (speed-up 9, speed-down 7) is centrifuged for 8min to 10min;
(2) The whole blood is divided into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin pale yellow interface layer, namely a PRP layer, is visible at the joint of the two layers, a liquid level is contacted by a liquid-transferring gun, the whole supernatant is carefully sucked to 3mm below the interface layer, transferred into a 15ml centrifuge tube, balanced, and centrifuged for 6min at 800g (rising speed 9 and falling speed 7);
(3) The liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred into another centrifuge tube by a liquid transfer device, the rest serum is contacted with the liquid level by the liquid transfer device, the supernatant is carefully sucked and transferred into a new centrifuge tube by 3mm below the intersection, namely PRP gel.
In some embodiments, the centrifugation time in step (1) is 8min.
The umbilical cord mesenchymal stem cells are cultured in the following manner (in the following steps, the culture medium is the commercially available Dayou stem cell culture medium):
(1) Horizontally placing the culture flask to ensure that umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture flask in a carbon dioxide constant temperature and humidity incubator; culture conditions: 37 ℃ and the volume fraction of carbon dioxide is 5%;
(2) After one week, taking out the culture flask, observing whether stem cells grow or not, observing every day until the stem cells grow, and starting to change the liquid;
(3) Half liquid change: if stem cells are observed to grow in the culture flask, half liquid exchange is carried out, the culture flask is slightly inclined, old culture medium is gently sucked by a pipette (note that tissue blocks are not sucked out), the fresh culture medium with the same amount is complemented, and a carbon dioxide constant temperature and humidity incubator is placed for starting culture, wherein the culture conditions are as follows: observing the growth condition of cells at 37 ℃ with the volume fraction of carbon dioxide of 5%, wherein the fusion degree of the concentrated cells reaches 80% -90%;
(4) Sucking and discarding the culture solution and tissue blocks in the culture flask, and gently flushing the cell culture flask with sodium chloride injection for 1-2 times;
(5) Digestion: digesting the cells until the cells are observed to be mostly deformed into a round shape by the shuttle and shed under an inverted microscope;
(6) Collecting cells, adding 10-15 ml of sodium chloride injection into each original culture flask, gently blowing, and blowing off adherent cells;
(7) And (3) filtering: collecting cell suspension to a 50ml centrifuge tube, and filtering by a screen;
(8) Cell count: gently beating, re-suspending cells, uniformly mixing by beating, sampling and counting;
(9) And (3) centrifugal washing: counting 5 x 10-5, centrifuging and washing, and pouring out the supernatant, wherein the bottom stem cells are umbilical cord mesenchymal stem cells.
Example 1:
a PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating an intervertebral disc degeneration disease, comprising at least 5 x 10-5 umbilical cord mesenchymal stem cells per 2mL of the composition, PRP gel and 100 units of thrombin; 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 according to the following steps:
5X 10-5 umbilical cord mesenchymal stem cells were resuspended with 2ml PRP gel, mixed well, 100 units of thrombin were added thereto, and mixed well.
The preparation method of the PRP gel comprises the following steps:
(1) After the injector plug is slightly loosened, the tube cover is pulled out, the blood sample is averagely split into 15ml centrifuge tubes, and each tube is centrifuged for 8min with the speed of no more than 15ml and 300g (speed of 9 and speed of 7);
(2) The whole blood is divided into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin pale yellow interface layer, namely a PRP layer, is visible at the joint of the two layers, a liquid level is contacted by a liquid-transferring gun, the whole supernatant is carefully sucked to 3mm below the interface layer, transferred into a 15ml centrifuge tube, balanced, and centrifuged for 6min at 800g (rising speed 9 and falling speed 7);
(3) The liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred into another centrifuge tube by a liquid transfer device, the rest serum is contacted with the liquid level by the liquid transfer device, the supernatant is carefully sucked and transferred into a new centrifuge tube by 3mm below the intersection, namely PRP gel.
The umbilical cord mesenchymal stem cells are cultured in the following manner (in the following steps, the culture medium is the commercially available Dayou stem cell culture medium):
(1) Horizontally placing the culture flask to ensure that umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture flask in a carbon dioxide constant temperature and humidity incubator; culture conditions: 37 ℃ and the volume fraction of carbon dioxide is 5%;
(2) After one week, taking out the culture flask, observing whether stem cells grow or not, observing every day until the stem cells grow, and starting to change the liquid;
(3) Half liquid change: if stem cells are observed to grow in the culture flask, half liquid exchange is carried out, the culture flask is slightly inclined, old culture medium is gently sucked by a pipette (note that tissue blocks are not sucked out), the fresh culture medium with the same amount is complemented, and a carbon dioxide constant temperature and humidity incubator is placed for starting culture, wherein the culture conditions are as follows: observing the growth condition of cells at 37 ℃ with the volume fraction of carbon dioxide of 5%, wherein the fusion degree of the concentrated cells reaches 80% -90%;
(4) Sucking and discarding the culture solution and tissue blocks in the culture flask, and gently flushing the cell culture flask with sodium chloride injection for 1-2 times;
(5) Digestion: digesting the cells until the cells are observed to be mostly deformed into a round shape by the shuttle and shed under an inverted microscope;
(6) Collecting cells, adding 10-15 ml of sodium chloride injection into each original culture flask, gently blowing, and blowing off adherent cells;
(7) And (3) filtering: collecting cell suspension to a 50ml centrifuge tube, and filtering by a screen;
(8) Cell count: gently beating, re-suspending cells, uniformly mixing by beating, sampling and counting;
(9) And (3) centrifugal washing: counting 5 x 10-5, centrifuging and washing, and pouring out the supernatant, wherein the bottom stem cells are 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 was detected, and the detection result was shown in the following table 1, the cell morphology was not changed, and the cell viability rate was 98.4% -99.3%.
TABLE 1 results of cell detection for the composition of example 1
| Number of times | CD105+(％) | CD73+(％) | CD90+(％) | CD45+(％) | CD34+(％) | HLA-DR(％) | Cell viability (%) |
| First time | 100％ | 99.4 | 99.9 | 0.01 | 0.07 | 0.04 | 99.1 |
| Second time | 99.6 | 99.8 | 99.1 | 0.04 | 0.56 | 0.07 | 98.4 |
| 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 |
| 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 an intervertebral disc degeneration disease, comprising at least 5×10ζ5 umbilical cord mesenchymal stem cells per 2mL of the composition, PRP gel, 0.08mg NGF and 100 units of thrombin.
The composition is prepared according to the following steps:
2ml of PRP was taken, 0.08mg of NGF was added thereto, and mixed well, then 5X 10≡5 umbilical cord mesenchymal stem cells were resuspended in 2ml of PRP to which NGF was added, and 100 units of thrombin was added thereto, and mixed well.
Wherein, the PRP gel was prepared in the same manner as in example 1.
The umbilical cord mesenchymal stem cells were cultured in the same manner as in example 1.
TABLE 2 results of cell detection for the composition of example 2
| Number of times | CD105+(％) | CD73+(％) | CD90+(％) | CD45+(％) | CD34+(％) | HLA-DR(％) | Cell viability (%) |
| First time | 99.4％ | 99.5 | 100 | 0.02 | 0.13 | 0.65 | 99.3 |
| Second time | 99.6 | 99.6 | 99.4 | 0.12 | 0.33 | 0.27 | 99.1 |
| 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 |
| 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 above example 1 is respectively used for treating a clinical intervertebral disc degeneration patient, a clinical patient with lumbar disc herniation, a clinical patient with a subvertebroscopic nuclectomy, and a clinical patient with lumbar pain caused by lumbar disc degeneration by an intervertebral foramen mirror injection method.
Application example 2
The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating the intervertebral disc degeneration disease obtained in the above example 2 is respectively used for treating a clinical intervertebral disc degeneration patient, a clinical patient with lumbar disc herniation, a clinical patient with a subvertebroscopic nuclectomy, and a clinical patient with lumbar pain caused by lumbar disc degeneration by an intervertebral foramen mirror injection method.
Comparative example 1 was used
The PRP gel without the umbilical cord mesenchymal stem cells is respectively used for treating clinical patients suffering from intervertebral disc degeneration, clinical patients with lumbar disc herniation imitating the operation of nuclectomy under the intervertebral foramen mirror and patients suffering from lumbar vertebra pain caused by lumbar vertebra degeneration in a clinical mode through an intervertebral foramen mirror injection mode.
Comparative example 2 was used
Physiological saline is respectively used for treating clinical patients suffering from intervertebral disc degeneration, clinical patients with lumbar disc herniation, which are subjected to the operation of performing the nuclectomy under the intervertebral foramen mirror, and clinical patients suffering from lumbar vertebra pain caused by lumbar vertebra degeneration in an injection mode of the intervertebral foramen mirror.
The specific results are shown in Table 3 below:
table 3: the results of the above application examples 1-2 and comparative examples 1-2
| Numbering device | Disc height | Intervertebral disc water content | VAS scoring |
| Application example 1 | +9％ | +11％ | 7 min to 4 min |
| Application example 2 | +10％ | +11.5％ | 7 min to 3 min |
| Comparative example 1 was used | +1％ | +3％ | Unchanged |
| Comparative example 2 was used | Unchanged | Unchanged | Unchanged |
FIG. 1 shows two key indexes of nucleus pulposus cell col2 and sox-9 after PRP gel is respectively injected into the PRP gel loaded umbilical cord mesenchymal stem cells, the PRP gel not loaded umbilical cord mesenchymal stem cells and normal saline, and the relative density of the nucleus pulposus cells of the umbilical cord mesenchymal stem cells is increased by the PRP gel, so that the effect of the application example 1 is superior to that of other comparative examples 1 and 2.
The foregoing is merely a preferred embodiment of the present invention and is not limited thereto. Other variations or modifications of the above description will be apparent to those of skill in the art. It is not necessary or nor practical to exemplify all embodiments herein. While obvious variations or modifications of the solution are still within the scope of the invention.
Claims (2)
1. A PRP gel-loaded umbilical cord mesenchymal stem cell composition for use in the treatment of an intervertebral disc degeneration disease, characterized in that at least 5 x 10-5 PRP gel, 100 units of thrombin and 0.08mg NGF are included per 2mL of the composition; in the composition, the average cell activity of umbilical cord mesenchymal stem cells is more than or equal to 98%;
the preparation method of the PRP gel comprises the following steps:
(1) The blood sample is divided into 15ml centrifuge tubes in average, each tube is not more than 15ml,300g is centrifuged for 8-10 min, the speed is increased by 9, and the speed is reduced by 7;
(2) The whole blood is divided into three layers, wherein the upper layer is supernatant, the lower layer is red blood cells, a thin pale yellow interface layer, namely a PRP layer, is visible at the joint of the two layers, a liquid level is contacted by a liquid-transferring gun, the whole supernatant is carefully sucked to 3mm below the interface layer, and is transferred into a 15ml centrifuge tube for balancing, and 800g is centrifuged for 6min, and the speed is increased by 9 and reduced by 7;
(3) The liquid in the centrifuge tube is divided into two layers, 3/4 supernatant is sucked and transferred into another centrifuge tube by a liquid transfer device, the rest serum is contacted with the liquid level by the liquid transfer device, the supernatant is carefully sucked and transferred into a new centrifuge tube by 3mm below the intersection, namely PRP gel.
2. The PRP gel-loaded umbilical cord mesenchymal stem cell composition for treating an intervertebral disc degeneration disease of claim 1, wherein,
the umbilical cord mesenchymal stem cells are cultured in the following manner:
(1) Horizontally placing the culture flask to ensure that umbilical cord tissue blocks are uniformly distributed on the whole bottom surface as much as possible, and placing the culture flask in a carbon dioxide constant temperature and humidity incubator; culture conditions: 37 ℃ and the volume fraction of carbon dioxide is 5%;
(2) After one week, taking out the culture flask, observing whether stem cells grow or not, observing every day until the stem cells grow, and starting to change the liquid;
(3) Half liquid change: if stem cells are observed to grow in the culture flask, half liquid exchange is carried out, the culture flask is slightly inclined, the old culture medium is gently sucked by a pipette, tissue blocks are not required to be sucked out, the fresh culture medium with the same amount is complemented, and a carbon dioxide constant temperature and humidity incubator is placed for starting culture under the conditions of: observing the growth condition of cells at 37 ℃ with the volume fraction of carbon dioxide of 5%, wherein the fusion degree of the concentrated cells reaches 80% -90%;
(4) Sucking and discarding the culture solution and tissue blocks in the culture flask, and gently flushing the cell culture flask with sodium chloride injection for 1-2 times;
(5) Digestion: digesting the cells until the cells are observed to be mostly deformed into a round shape by the shuttle and shed under an inverted microscope;
(6) Collecting cells, adding 10-15 ml of sodium chloride injection into each original culture bottle, gently blowing, and blowing off adherent cells;
(7) And (3) filtering: collecting cell suspension to a 50ml centrifuge tube, and filtering by a screen;
(8) Cell count: gently beating, re-suspending cells, uniformly mixing by beating, sampling and counting;
(9) And (3) centrifugal washing: counting 5 x 10-5, centrifuging and washing, and pouring out the supernatant, wherein the bottom stem cells are umbilical cord mesenchymal stem cells.
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