Disclosure of Invention
The application aims to provide a novel method for obtaining maternal mesenchymal stem cells from placenta.
The application adopts the following technical scheme:
The application discloses a method for obtaining maternal mesenchymal stem cells from placenta, which comprises the steps of obtaining placenta tissue from the position, which is within 5cm from the radius of an umbilical cord and is more than 0.5cm from the fetal surface, of the placenta tissue, performing enzyme digestion treatment on the placenta tissue by adopting enzyme digestion solution, and then performing cell culture on the product of the enzyme digestion treatment to obtain the maternal mesenchymal stem cells.
The application creatively obtains the matrix-source mesenchymal stem cells from other placenta tissues except the decidua basal, solves the problems that the number of the matrix-source mesenchymal stem cells obtained by the decidua basal is small and the decidua basal is easy to lose, and provides a new scheme and approach for preparing or separating the matrix-source mesenchymal stem cells in the placenta.
The size of the placental tissue obtained by the present application is not limited, and the placental tissue can be obtained by culturing a large number of maternal-derived mesenchymal stem cells from the placental tissue obtained from the position range defined by the present application, and the present application can obtain more maternal-derived mesenchymal stem cells than the equivalent decidua, and does not cause the problem of loss of the similar decidua.
It will be understood that one of the key points of the present application is that research has found that the placenta tissue specifically defined in the present application can obtain the mesenchymal stem cells of the parent source, and that the following placenta tissue washing, shearing, enzyme digestion, cell obtaining, mesenchymal stem cell culturing, etc. can refer to the existing method for preparing the mesenchymal stem cells from the tissue, and is not specifically limited herein. However, in order to improve the purity of the maternal mesenchymal stem cells of the present application, the present application describes the following steps, particularly the step of obtaining cells by enzymatic digestion treatment, in detail, the following technical scheme is described.
Preferably, the method of the present application further comprises rejecting blood vessels or white connective tissue in the placental tissue that are directly connected to the fetal surface.
The placenta tissue obtained by the present application may contain, in part, blood vessels or white connective tissue directly connected to the fetal surface, and in order to avoid that these blood vessels or white connective tissue affect the purity of the maternal mesenchymal stem cells, it is removed in a preferred embodiment of the present application. It will be appreciated that this step is not used if the placenta tissue taken is itself free of these blood vessels or white connective tissue.
Preferably, the enzyme digestion solution contains collagenase, hyaluronidase, neutral protease and papain.
The research of the application shows that the special enzyme digestion solution compounded by a plurality of enzymes is adopted for enzyme digestion treatment, so that the purity of the prepared matrix-derived mesenchymal stem cells can be further improved, the interference of neonatal cells is reduced, and the application has important significance for clinical application. It will be appreciated that if the purity requirements for the maternal mesenchymal stem cells are low, other enzymatic digestion protocols or other means of obtaining the cells may be used, and are not particularly limited herein.
Preferably, the enzyme digestion solution is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution. Among these, PBS solution is a phosphate buffer solution conventionally used in laboratories.
Preferably, in the enzyme digestion solution, the concentration of collagenase is 0.3-0.5% by mass, the concentration of hyaluronidase is 0.1-0.3% by mass, the concentration of neutral protease is 100-300U/mL, and the concentration of papain is 0.03-0.09% by mass.
Preferably, in the method of the application, the enzyme digestion treatment specifically comprises adding at least 3mL of enzyme digestion solution to each gram of placenta tissue, performing constant-temperature shaking digestion for at least 20 minutes at 37 ℃, then removing the enzyme digestion solution, adding fresh enzyme digestion solution, and performing constant-temperature shaking digestion for at least 40 minutes at 37 ℃.
The placenta tissue block is digested by a double digestion method. Since a very small amount of impurities, such as vascular tissues remained on the surface, may be mixed in the process of sampling the placenta tissue, the impurity cells contained in the digestive juice are removed after the first short pre-digestion, and at this time, most of the maternal mesenchymal stem cells remain in the placenta tissue mass, and then the placenta tissue mass is digested again, so that the maternal mesenchymal stem cells are digested from the tissue mass. The twice digestion method can improve the purity of the obtained maternal mesenchymal stem cells.
Preferably, the enzyme digestion solution is removed by taking out the tissue mass using a cell screen with a pore size of 50-100 μm and discarding the remaining solution.
The cell screen used in the present application is basically a cell screen used in one embodiment of the present application, and the pore size of 50 to 100 μm is only a cell screen which can be used in one embodiment of the present application.
Preferably, the method for obtaining the maternal mesenchymal stem cells from the placenta comprises the following steps of;
1) Placenta tissue which is within 5cm from the radius of the umbilical cord and is more than 0.5cm from the fetal surface is taken, and blood vessels or white connective tissues which are directly communicated with the fetal surface in the placenta tissue are removed;
2) Washing and removing blood clots and capillary vessel tissues in the placenta tissues obtained in the step 1) by using normal saline;
3) Cutting placenta tissue cleaned in the step 2), and performing enzyme digestion treatment by adopting enzyme digestion solution;
4) Removing fragments after enzyme digestion treatment, and centrifuging to remove supernatant;
5) Re-suspending the sediment obtained by the centrifugation in the step 4) by adopting a stem cell culture medium, and transferring the sediment into a culture bottle for culture;
6) Digesting the culture product of the step 5) by pancreatin, wherein the digested cells are maternal mesenchymal stem cells.
Preferably, in step 4), the fragments are removed, in particular by taking out the tissue mass using a cell screen with a pore size of 50-100 μm, and then centrifuging the liquid to collect the precipitated cells.
Preferably, step 4) further comprises subjecting the scooped tissue pieces to the enzymatic digestion of step 3), and then passing the mixture through a cell screen having a pore size of 50-100 μm, centrifuging the filtrate, and collecting the precipitated cells.
It should be noted that the enzyme digestion treatment of step 3) is performed on the scooped tissue block in order to obtain as many cells as possible from the tissue block and avoid waste, and of course, in principle, in one implementation of the present application, in the case that step 3) has been performed twice, most of the cells in the tissue block have already been digested, and if time cost and production efficiency are considered, it may not be necessary to perform the enzyme digestion treatment of step 3) on the scooped tissue block in step 4).
Preferably, in step 5), the amount of stem cell culture medium added to the centrifugal precipitation product of step 4) is such that at least 10mL of stem cell culture medium is added per gram of placental tissue.
Preferably, in step 5), the cells are transferred to a culture flask for culturing, specifically comprising culturing at constant temperature for 24-48 hours under the conditions of 37 ℃ and 5% concentration of CO2, then discarding supernatant and residual tissues, adding fresh stem cell culture medium, and continuously culturing under the same conditions until the cell confluency reaches 80% -90%.
Preferably, the method of the present application further comprises, prior to step 6), at least one washing of the culture product of step 5) with physiological saline and then a pancreatin digestion.
Preferably, in the step 6), the culture product of the step 5) is digested by pancreatin, specifically comprising adding pancreatin to the culture product of the step 5), digesting for at least 2min at room temperature, then adding stem cell culture medium with the volume of three times of pancreatin, stopping digestion, centrifuging the mixture, discarding the supernatant to obtain cell sediment, re-suspending the cell sediment by normal saline, then centrifuging, discarding the supernatant, and re-suspending by stem cell culture medium to obtain the maternal-source mesenchymal stem cells of the application.
The application has the beneficial effects that:
The method for obtaining the maternal mesenchymal stem cells from the placenta solves the technical problem of separating the maternal mesenchymal stem cells from the placenta, and the prepared maternal mesenchymal stem cells have large quantity, high purity and difficult mixing of neonatal cells, and can meet the clinical application requirements; the method solves the problems of small quantity of the mesenchymal stem cells of the parent source obtained by the decidua bottom and easy loss of the decidua bottom, and provides a new scheme and a new way for obtaining the mesenchymal stem cells of the parent source from the placenta.
Detailed Description
Currently, there is no other technique capable of separating maternal-derived mesenchymal stem cells from the rest of the placenta, other than separating maternal-derived mesenchymal stem cells of higher purity from the decidua basal of the placenta. That is, the prior art has all been to separate the maternal mesenchymal stem cells from the decidua of the placenta by enzymatic digestion, while the other parts of the placenta are not.
Although the other parts of the placenta contain a large amount of mesenchymal stem cells, most of the mesenchymal stem cells are a mixture of maternal and neonatal cells, and it is a very important and difficult technique to accurately distinguish maternal and neonatal cells in the placenta, and no effective solution is available at present to obtain maternal mesenchymal stem cells from other placenta parts than the decidua basal, which can meet the use requirements.
The application researches that the placenta can obtain the maternal mesenchymal stem cells except the maternal mesenchymal stem cells from the decidua, and the placenta tissue which is within 5cm from the radius of an umbilical cord and more than 0.5cm from the fetal surface can also obtain the maternal mesenchymal stem cells, particularly, the maternal mesenchymal stem cells with higher purity can be obtained by matching with the improved enzyme digestion solution and enzyme digestion treatment of the application, the clinical application requirements can be met, and compared with the equal amount of the decidua, the application can obtain the maternal mesenchymal stem cells with large quantity and high purity, and solves the problems that the number of the maternal mesenchymal stem cells obtained from the decidua is small and the decidua is easy to lose.
The application is further illustrated by the following examples. The following examples are merely illustrative of the present application and should not be construed as limiting the application.
Examples
1. Primary reagents and materials
Stem cell culture medium was purchased from Stempro, sodium chloride injection from Guizhou heaven and earth, 50mL centrifuge tubes, T75 flask and T175 flask were purchased from Corning, collagenase, hyaluronidase, neutral protease and papain were purchased from Gibco.
The compound enzyme digestion solution is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution, wherein the concentration of collagenase in the compound enzyme digestion solution is 0.4% by mass, the concentration of hyaluronidase is 0.1% by mass, the concentration of neutral protease is 200U/mL, and the concentration of papain is 0.05% by mass.
And (3) dissolving collagenase in PBS (phosphate buffer solution) to prepare a control enzyme digestion solution, wherein the concentration of the collagenase is 0.5% by mass.
2. Mesenchymal stem cell preparation
The mesenchymal stem cells are prepared by taking the same placenta according to different parts. The placenta of this example was provided by the Guangdong praying hospital. The preparation method comprises the following steps:
test 1
1) Opening a sample collection bag filled with placenta, shearing placenta tissue which is in a circle with the placenta being 5cm away from the umbilical cord radius and more than 0.5cm away from the fetal surface by scissors, wherein the material drawing part is shown in figure 1, figure 1 is a schematic cross section of the material drawing part, the upper surface is a maternal surface, the lower surface is a fetal surface, and the black square frame position is the material drawing part.
2) Clamping the tissue with tissue forceps, removing capillary blood vessel tissue rich in the maternal surface as much as possible, leaving a part containing interstitial tissue, placing the tissue into a culture dish after separation, adopting normal saline, specifically adopting sodium chloride injection in the example, washing to remove residual blood, and repeatedly adopting the sodium chloride injection for washing until the washing liquid is clear;
3) 3g of the washed villus cluster is put into a 50mL centrifuge tube, and the tissue is further sheared into the size of 1-3mm3 by using surgical scissors;
4) Adding 10mL of prepared complex enzyme digestion solution, namely collagenase, hyaluronidase, neutral protease and papain into a centrifuge tube of the sheared tissue blocks, dissolving in PBS solution to prepare complex enzyme digestion solution, covering a centrifuge tube cover, uniformly mixing the materials upside down, putting the mixture into a constant temperature oscillator, setting the temperature to be 37 ℃, rotating at 190RPM, and digesting for 20 minutes;
5) After the digestion of the step 4), the content in the centrifuge tube passes through a cell screen with the aperture of 50 mu m, the filtrate is discarded, the substances on the cell screen are put into a 50mL centrifuge tube, 10mL of complex enzyme digestion solution is added, and the digestion is continued for 40 minutes;
6) Adding 30mL of PBS solution into a centrifuge tube containing digested tissue suspension after the digestion is completed in the step 5), uniformly mixing, then, passing the mixture through a cell screen with the size of 50 mu m, removing fragments, collecting filtrate, centrifuging for 5min at 800g, and removing supernatant;
7) Adding 30mL of stem cell culture medium into the centrifugal sediment in the step 6), blowing a pipette to resuspend the sediment, transferring the sediment into 1T 75 culture bottles, and culturing the sediment in a carbon dioxide incubator under the same culture conditions, wherein the parameters are set to be 37 ℃, the carbon dioxide concentration is 5%, and the relative humidity is 95%;
8) After culturing for 24 hours, the culture medium in the culture flask is sucked out by a pipette and discarded, then 30mL of stem cell culture medium is added, and the mixture is put back into a carbon dioxide incubator for continuous culture;
9) Pouring out the culture medium in the culture flask when the cell grows to reach 80% -90% of confluence, adding 10mL of sodium chloride injection by using a pipette, gently shaking and washing, pouring out, repeatedly using 10mL of sodium chloride injection for washing, and washing for 2 times;
10 Adding 2mL pancreatin into the culture flask for digestion for 2 minutes, gently beating the bottom of the culture flask to enable cells to fall off, and shaking left and right for a plurality of times to mix uniformly;
11 Adding 6mL of stem cell culture medium to stop digestion, sucking out the cell suspension in all culture bottles into a 50mL centrifuge tube, centrifuging at 500g and 20 ℃ for 5 minutes, and discarding the supernatant after centrifuging to obtain a cell precipitate;
12 Adding 15mL of sodium chloride injection into the cell sediment, re-suspending the cell sediment, setting parameters of 500g and 20 ℃ and centrifuging for 5 minutes, and pouring supernatant after centrifuging to obtain the cell sediment;
13 Adding 10mL of stem cell complete culture medium into a centrifugal tube of cell precipitation, and gently blowing and uniformly mixing by a pipette to obtain a culture medium cell suspension;
14 Adding the cell suspension in the step 13) into a T175 culture flask according to the density of 8000 cells/cm2, adding a stem cell culture medium to a constant volume of 25mL, placing the culture flask into a carbon dioxide incubator for culturing, and setting the parameters to be 37 ℃ and the carbon dioxide concentration of 5% and the relative humidity of 95%;
15 When the cell grows to reach 80% -90% of confluence, harvesting the cell according to the steps 9-12;
16 Flow-through the harvested cells and STR typing was tested according to the French science DNA laboratory test Specification (GA/T383-2002) standard to determine the genotyping of the harvested cells. Meanwhile, STR typing detection is carried out on blood of a parturient delivering the placenta by adopting the same method so as to determine the genotyping of the placenta maternal system.
Test 2
The mesenchymal stem cells of the test were prepared by performing enzyme digestion treatment of the placenta tissue obtained in the test 1 with different enzyme digests. The method comprises the following steps:
1) Placing 3g of the washed villus cluster into a 50mL centrifuge tube, and further shearing the tissue to a size of 1-3mm3 by using surgical scissors, wherein the washed villus cluster is the same as the washed villus cluster in the step 3) in the test 1;
2) Adding 10mL of prepared control enzyme digestive juice, namely control enzyme digestive juice prepared by dissolving collagenase in PBS solution, covering a centrifugal tube cover, uniformly mixing the materials upside down, putting the materials into a constant temperature shaker, setting the temperature to 37 ℃, rotating at 190RPM, and digesting for 60 minutes;
3) Adding 30mL of PBS solution into a centrifuge tube containing digested tissue suspension after the digestion is completed in the step 2), uniformly mixing, then, passing the mixture through a cell screen with the size of 50 mu m, removing fragments, collecting filtrate, centrifuging for 5min at 800g, and removing supernatant;
4) Adding 30mL of stem cell culture medium into the centrifugal sediment in the step 3), blowing a pipette to resuspend the sediment, transferring the sediment into 1T 75 culture bottles, and culturing the sediment in a carbon dioxide incubator under the same culture conditions, wherein the parameters are set to be 37 ℃, the carbon dioxide concentration is 5%, and the relative humidity is 95%;
5) After culturing for 24 hours, the culture medium in the culture flask is sucked out by a pipette and discarded, then 30mL of stem cell culture medium is added, and the mixture is put back into a carbon dioxide incubator for continuous culture;
6) Pouring out the culture medium in the culture flask when the cell grows to reach 80% -90% of confluence, adding 10mL of sodium chloride injection by using a pipette, gently shaking and washing, pouring out, repeatedly using 10mL of sodium chloride injection for washing, and washing for 2 times;
7) Adding 2mL pancreatin into the culture flask for digestion for 2 minutes, gently beating the bottom of the culture flask to enable cells to fall off, and shaking left and right for a plurality of times to mix uniformly;
8) Adding 6mL of culture medium to stop digestion, sucking out the cell suspension in all culture flasks into a 50mL centrifuge tube, centrifuging at 500g and 20 ℃ for 5 minutes, and discarding the supernatant after centrifuging to obtain a cell precipitate;
9) Adding 15mL of sodium chloride injection into the cell sediment, re-suspending the cell sediment, setting parameters of 500g and 20 ℃ and centrifuging for 5 minutes, and pouring supernatant after centrifuging to obtain the cell sediment;
10 Adding 10mL of stem cell complete culture medium into a centrifugal tube of cell precipitation, and gently blowing and uniformly mixing by a pipette to obtain a culture medium cell suspension;
11 Adding the cell suspension obtained in the step 10) into a T175 culture flask according to the density of 8000 cells/cm2, adding a stem cell culture medium to a constant volume of 25mL, placing the culture flask into a carbon dioxide incubator for culturing, and setting the parameters to be 37 ℃ and the carbon dioxide concentration of 5% and the relative humidity of 95%;
12 After the cells grow to 80% -90% confluence, harvesting the cells according to steps 6-9, detecting the flow of the harvested cells, and detecting STR typing according to the standard of the French science DNA laboratory test Specification (GA/T383-2002) to determine the genotyping of the harvested cells.
Test 3
1) Using placenta samples with the same test 1, and shearing placenta tissues marked by the test 1 and close to the fetal surface outside circles with the distance of 5cm from the radius of the umbilical cord by using scissors;
2) Clamping the tissue with tissue forceps, removing the capillary tissue rich in the tissue as much as possible, leaving a part containing interstitial tissue, placing the tissue into a culture dish after separation, adopting normal saline, specifically adopting sodium chloride injection, washing to remove residual blood, and repeatedly adopting the sodium chloride injection for washing until the washing liquid is clear;
3) 3g of the washed villus cluster is put into a 50mL centrifuge tube, and the tissue is further sheared into the size of 1-3mm3 by using surgical scissors;
4) Adding 10mL of prepared complex enzyme digestion solution, namely collagenase, hyaluronidase, neutral protease and papain into a centrifuge tube of the sheared tissue blocks, dissolving in PBS solution to prepare complex enzyme digestion solution, covering a centrifuge tube cover, uniformly mixing the materials upside down, putting the mixture into a constant temperature oscillator, setting the temperature to be 37 ℃, rotating at 190RPM, and digesting for 20 minutes;
5) After the digestion of the step 4), the content in the centrifuge tube passes through a cell screen with the aperture of 50 mu m, the filtrate is removed, the tissue on the cell screen is put into a 50mL centrifuge tube, 10mL of complex enzyme digestion solution is added, and the digestion is continued for 40 minutes;
6) Adding 30mL of PBS solution into a centrifuge tube containing digested tissue suspension after the digestion is completed in the step 5), uniformly mixing, then, passing the mixture through a cell screen with the size of 50 mu m, removing fragments, collecting filtrate, centrifuging for 5min at 800g, and removing supernatant;
7) Adding 30mL of stem cell culture medium into the centrifugal sediment in the step 6), blowing a pipette to resuspend the sediment, transferring the sediment into 1T 75 culture bottles, and culturing the sediment in a carbon dioxide incubator under the same culture conditions, wherein the parameters are set to be 37 ℃, the carbon dioxide concentration is 5%, and the relative humidity is 95%;
8) After culturing for 24 hours, the culture medium in the culture flask is sucked out by a pipette and discarded, then 30mL of stem cell culture medium is added, and the mixture is put back into a carbon dioxide incubator for continuous culture;
9) Pouring out the culture medium in the culture flask when the cell grows to reach 80% -90% of confluence, adding 10mL of sodium chloride injection by using a pipette, gently shaking and washing, pouring out, repeatedly using 10mL of sodium chloride injection for washing, and washing for 2 times;
10 Adding 2mL pancreatin into the culture flask for digestion for 2 minutes, gently beating the bottom of the culture flask to enable cells to fall off, and shaking left and right for a plurality of times to mix uniformly;
11 Adding 6mL of stem cell culture medium to stop digestion, sucking out the cell suspension in all culture bottles into a 50mL centrifuge tube, centrifuging at 500g and 20 ℃ for 5 minutes, and discarding the supernatant after centrifuging to obtain a cell precipitate;
12 Adding 15mL of sodium chloride injection into the cell sediment, re-suspending the cell sediment, setting parameters of 500g and 20 ℃ and centrifuging for 5 minutes, and pouring supernatant after centrifuging to obtain the cell sediment;
13 Adding 10mL of stem cell complete culture medium into a centrifugal tube of cell precipitation, and gently blowing and uniformly mixing by a pipette to obtain a culture medium cell suspension;
14 Adding the cell suspension in the step 13) into a T175 culture flask according to the density of 8000 cells/cm2, adding a stem cell culture medium to a constant volume of 25mL, placing the culture flask into a carbon dioxide incubator for culturing, and setting the parameters to be 37 ℃ and the carbon dioxide concentration of 5% and the relative humidity of 95%;
15 After the cells grow to 80% -90% confluence, harvesting the cells according to steps 9-12, detecting the flow of the harvested cells, and detecting STR typing according to the standard of the French science DNA laboratory test Specification (GA/T383-2002) to determine the genotyping of the harvested cells.
3. Results and analysis
1. Flow assay results for cells
In this example, mesenchymal stem cells harvested in test 1, test 2 and test 3 were examined by flow cytometry, and the results are shown in table 1, fig. 6 to fig. 17.
Table 1 results of flow assays on cells obtained by different methods for three assays
| Surface markers | CD73 | CD90 | CD105 | Negative index |
| Test 1 | 99.80% | 99.68% | 99.73% | 1.21% |
| Test 2 | 99.78% | 99.60% | 99.72% | 1.15% |
| Test 3 | 99.76% | 99.60% | 99.46% | 1.54% |
Fig. 6 is a graph showing the result of cell flow assay surface marker CD73 obtained in test 1, fig. 7 is a graph showing the result of cell flow assay surface marker CD90 obtained in test 1, fig. 8 is a graph showing the result of cell flow assay surface marker CD105 obtained in test 1, fig. 9 is a graph showing the result of cell flow assay surface marker CD73 obtained in test 1, fig. 10 is a graph showing the result of cell flow assay surface marker CD73 obtained in test 2, fig. 11 is a graph showing the result of cell flow assay surface marker CD90 obtained in test 2, fig. 12 is a graph showing the result of cell flow assay surface marker CD105 obtained in test 2, fig. 13 is a graph showing the result of cell flow assay surface marker CD73 obtained in test 2, fig. 14 is a graph showing the result of cell flow assay surface marker CD90 obtained in test 3, fig. 16 is a graph showing the result of cell flow assay surface marker CD105 obtained in test 3, and fig. 17 is a graph showing the result of cell flow assay surface marker negative index obtained in test 3.
The results shown in Table 1 and FIGS. 6 to 17 show that the surface markers of the mesenchymal stem cells obtained in test 1, test 2 and test 3 are all qualified, and demonstrate that the mesenchymal stem cells can be obtained effectively in all three ways.
STR typing detection results
In this example, the mesenchymal stem cells harvested in test 1, test 2 and test 3 were subjected to STR typing test, and at the same time, the maternal blood from which the placenta was delivered was also subjected to STR typing test, and the test results are shown in fig. 2 to 5.
Fig. 2 shows the results of STR typing identification of maternal peripheral blood from which the placenta was delivered, fig. 3 shows the results of STR typing identification of mesenchymal stem cells harvested in test 1, fig. 4 shows the results of STR typing identification of mesenchymal stem cells harvested in test 2, and fig. 5 shows the results of STR typing identification of mesenchymal stem cells harvested in test 3.
Comparing the results of fig. 2 and 3, the locus phenotypes of the two results are identical, indicating that the two are identical, indicating that the mesenchymal stem cells obtained in experiment 1 are derived from placenta precursors, i.e. the high-purity maternal-derived mesenchymal stem cells are obtained.
The results in FIG. 4 show that there are three peaks at some of the sites, indicating that the second person's DNA is present in the sample as a result of the fetal cells being present in the maternal cells, indicating that the mesenchymal stem cells obtained in test 2 include cells from the maternal and cells from the fetal.
The results in FIG. 5 show that there are three peaks at some of the sites, indicating that the second person's DNA is present in the sample as a result of the fetal cells being present in the maternal cells, indicating that the mesenchymal stem cells obtained in test 3 also include cells from the maternal and cells from the fetal.
The comparison analysis of the above results shows that the high purity mesenchymal stem cells of maternal origin, i.e. test 1, can be obtained by adopting the placenta tissue of the example, i.e. the placenta within 5cm from the radius of the umbilical cord and more than 0.5cm from the fetal surface, and combining the special complex enzyme digestion solution and enzyme digestion treatment of the example. The sample of test 2 is the same as that of test 1, but the specific conditions of the enzyme digestion solution and the enzyme digestion treatment adopted are different, so that a certain amount of neonatal cells are mixed, and the purity of the maternal mesenchymal stem cells is affected. In test 3, the same complex enzyme digestion solution and enzyme digestion treatment as in test 1 were used, but the obtained mesenchymal stem cells of maternal origin could not be isolated because of incorrect material taking position in test 3, and the obtained mesenchymal stem cells contained both maternal cells and neonatal cells.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.