CN111793598A

CN111793598A - Method for obtaining maternal-derived mesenchymal stem cells from placenta

Info

Publication number: CN111793598A
Application number: CN202010470035.7A
Authority: CN
Inventors: 万桦; 李国喜; 于航海
Original assignee: Shenzhen Huada Gene Cell Technology Co ltd
Current assignee: Shenzhen Huada Gene Cell Technology Co ltd
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2020-10-20

Abstract

The application discloses a method for obtaining a maternal-derived mesenchymal stem cell from a placenta. The method for obtaining the maternal-derived mesenchymal stem cells from the placenta comprises the steps of obtaining a placenta tissue from a position, within a distance of 5cm from the radius of an umbilical cord and more than 0.5cm from the surface of a fetus, of the placenta; and (3) carrying out enzyme digestion treatment on the placenta tissue by adopting enzyme digestion solution, and then carrying out cell culture on a product of the enzyme digestion treatment to obtain the maternal-source mesenchymal stem cells. The method solves the technical problem of separating the maternal-derived mesenchymal stem cells from the placenta, and the prepared maternal-derived mesenchymal stem cells have large quantity and high purity, are not easy to mix with neonatal cells, and can meet the clinical application requirement; the method solves the problems that the quantity of mesenchymal stem cells from maternal sources obtained by the decidua basalis is small and the decidua basalis is easy to lose, and provides a new scheme and way for obtaining the mesenchymal stem cells from the maternal sources from the placenta.

Description

Method for obtaining maternal-derived mesenchymal stem cells from placenta

Technical Field

The application relates to the technical field of mesenchymal stem cell preparation, in particular to a method for obtaining a maternal-derived mesenchymal stem cell from a placenta.

Background

Mesenchymal Stem Cells (MSCs) are a population of mesoderm-derived pluripotent stem cells with self-renewing and multipotent differentiation potential. Originally discovered by Friedenstein et al from murine hematopoietic organs and demonstrated that it can differentiate into osteoblasts and adipocytes in vitro. Research shows that mesenchymal stem cells are widely present in bone marrow, umbilical cord placenta, fat and other tissues.

The placenta is an important organ for material exchange between a fetus and a mother body, and is a tissue-bound organ between the mother and the son, which is formed by the combined growth of an embryonic germ membrane and a mother body endometrium during pregnancy of a human. The placenta is rich in mesenchymal stem cells, but because the placenta is formed by combining maternal and neonatal tissues, the placenta contains two kinds of mesenchymal stem cells of different sources, namely fetal-derived mesenchymal stem cells and maternal-derived mesenchymal stem cells. The use of cells from a mixture of multiple people in clinical applications is at great risk.

Currently, the method for obtaining mesenchymal stem cells of high purity maternal origin from placenta is to separate the mesenchymal stem cells of the maternal origin from the decidua basalis. The method has the defects that the number of the maternal mesenchymal stem cells obtained from the decidua basalis is very small; and the decidua basalis and the amnion are broken and broken during delivery, and are easy to lose, so that the cell source is lost, and the mesenchymal stem cells of the maternal source cannot be obtained.

Therefore, how to prepare or isolate mesenchymal stem cells of maternal origin from placenta other than the decidua basalis is one of the research focuses and difficulties in the field.

Disclosure of Invention

The purpose of the application is to provide a novel method for obtaining a maternal-derived mesenchymal stem cell from a placenta.

The application specifically adopts the following technical scheme:

the application discloses a method for obtaining a maternal-derived mesenchymal stem cell from a placenta, which comprises the steps of obtaining a placenta tissue from a position, within a radius of 5cm from an umbilical cord and more than 0.5cm from a fetal surface, of the placenta; and carrying out enzyme digestion treatment on the placenta tissue by adopting enzyme digestion solution, and then carrying out cell culture on a product of the enzyme digestion treatment to obtain the maternal-source mesenchymal stem cell.

The method has the advantages that the maternal-source mesenchymal stem cells are creatively obtained from other placenta tissues except for the decidua basalis, so that the problems that the number of the maternal-source mesenchymal stem cells obtained from the decidua basalis is small, and the decidua basalis is easy to lose are solved; provides a new scheme and a new way for preparing or separating the mesenchymal stem cells of the maternal source in the placenta.

The size of the placenta tissue taken in the present application is not limited, and as long as the placenta tissue is taken from the position range defined in the present application, a considerable amount of maternal mesenchymal stem cells can be obtained by culturing; compared with the equivalent decidua basalis, the mesenchymal stem cells can be obtained from more maternal sources, and the problem of loss of the similar decidua basalis can be solved.

It can be understood that one of the keys of the present application is the research finding that the placenta tissue specifically defined by the present application can obtain the mesenchyma stem cell of maternal source; for the subsequent placenta tissue cleaning, shearing, enzyme digestion, cell obtaining, mesenchymal stem cell culture and the like, reference may be made to the existing method for preparing mesenchymal stem cells from tissues, and no specific limitation is made herein. However, in order to improve the purity of the mesenchymal stem cells of maternal origin of the present application, the present application describes in detail the subsequent steps, in particular the steps of obtaining cells by enzymatic digestion, as detailed in the following technical schemes.

Preferably, the method of the present application further comprises rejecting blood vessels or white connective tissue in the placental tissue that is directly connected to the fetal face.

It should be noted that, some of the placenta tissues taken in this application may contain blood vessels or white connective tissues directly connected to the fetal surface, and in order to avoid the blood vessels or white connective tissues affecting the purity of the mesenchymal stem cells of maternal origin, the placenta tissues are removed in the preferred embodiment of this application. It will be appreciated that this step is not used if the placental tissue taken does not have such blood vessels or white connective tissue itself.

Preferably, the enzymatic digest contains collagenase, hyaluronidase, neutral protease, and papain.

It should be noted that, the research of the present application finds that, when the specific enzyme digestion solution compounded by a plurality of enzymes defined in the present application is used for carrying out enzyme digestion treatment, the purity of the prepared maternal mesenchymal stem cells can be further improved, the interference of neonatal cells can be reduced, and the method has important significance for clinical application. It is understood that if the requirement for the purity of the mesenchymal stem cells of maternal origin is low, the cells may be obtained by other enzymatic digestion treatment protocols or by other means, and are not particularly limited herein.

Preferably, the enzyme digest is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution. The PBS solution is phosphate buffer solution which is conventionally used in laboratories.

Preferably, in the enzyme digestion solution, the concentration of collagenase is 0.3-0.5 percent by mass, the concentration of hyaluronidase is 0.1-0.3 percent by mass, the concentration of neutral protease is 300U/mL, and the concentration of papain is 0.03-0.09 percent by mass.

Preferably, in the method of the present application, the enzymatic digestion treatment specifically includes adding at least 3mL of enzymatic digestion solution per gram of placenta tissue, and digesting for at least 20 minutes at 37 ℃ under constant temperature shaking; then, the enzyme digestion solution is removed, fresh enzyme digestion solution is added, and the digestion is carried out for at least 40 minutes with shaking at the constant temperature of 37 ℃.

It should be noted that the present application employs a double digestion method to digest the placental tissue mass. Since a very small amount of impurities, such as blood vessel tissue remaining on the surface, may be mixed in the placenta tissue sampling process, the impurity cells contained in the digestive fluid are removed after the first short pre-digestion, while 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-derived mesenchymal stem cells.

Preferably, the enzymatic 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 liquid.

It should be noted that the cell screen used in the present application is basically only required to be able to fish out the tissue mass, and the 50-100 μm pore size is only a cell screen specifically used in one implementation manner of the present application.

Preferably, the method for obtaining the maternal mesenchymal stem cells from the placenta specifically comprises the following steps;

1) collecting placenta tissue within 5cm of umbilical cord radius and 0.5cm above the fetal surface, and removing blood vessel or white connective tissue directly connected to the fetal surface;

2) cleaning with normal saline to remove blood clots and capillary vessel tissues in the placenta tissues obtained in the step 1);

3) cutting the placenta tissue cleaned in the step 2), and performing enzyme digestion treatment by adopting an enzyme digestion solution;

4) after the enzyme digestion treatment is finished, removing fragments, and centrifuging to remove supernatant;

5) resuspending the precipitate obtained by centrifugation in step 4) by using a stem cell culture medium, and transferring the precipitate into a culture bottle for culture;

6) digesting the culture product obtained in the step 5) by using pancreatin, wherein the digested cells are the mesenchymal stem cells of the maternal source.

Preferably, the removing of the fragments in step 4) comprises taking out the tissue blocks by using a cell screen with a pore size of 50-100 μm, centrifuging the liquid, and collecting the precipitated cells.

Preferably, the step 4) further comprises performing the enzyme digestion treatment of the step 3) on the fished tissue block, then passing the mixture through a cell screen with a pore size of 50-100 μm, centrifuging the filtrate, and collecting the precipitated cells.

The enzyme digestion treatment of the step 3) is carried out on the fished tissue blocks, so that cells are obtained from the tissue blocks as much as possible, and waste is avoided; of course, in principle, in one implementation of the present application, in the case where step 3) has been performed twice with enzymatic digestion, most of the cells in the tissue mass have already been digested, and if time cost and production efficiency are taken into consideration, it may not be necessary to perform step 3) with enzymatic digestion again on the fished tissue mass at step 4).

Preferably, in step 5), the amount of the stem cell culture medium added in the step of resuspending the centrifuged and precipitated product in step 4) is at least 10mL per gram of the placenta tissue.

Preferably, in step 5), the culture medium is transferred to a culture flask for culture, and specifically comprises culturing the medium at 37 ℃ and 5% CO concentration₂Culturing for 24-48 hours at constant temperature, then removing supernatant and residual tissues, adding fresh stem cell culture medium, and continuously culturing under the same condition until the cell confluence reaches 80-90%.

Preferably, the method of the present application further comprises washing the culture product of step 5) with physiological saline at least once before step 6), and then performing pancreatin digestion.

Preferably, in the step 6), the culture product in the step 5) is digested by pancreatin, and the method specifically comprises the steps of adding pancreatin into the culture product in the step 5) and digesting for at least 2min at room temperature; then adding a stem cell culture medium with the volume three times that of pancreatin, and stopping digestion; centrifuging the mixture, and removing the supernatant to obtain cell precipitate; resuspending the cell pellet with normal saline, then centrifuging and discarding the supernatant; and (4) carrying out heavy suspension by adopting a stem cell culture medium, namely obtaining the matrix-derived mesenchymal stem cells.

The beneficial effect of this application lies in:

the method for obtaining the maternal-source mesenchymal stem cells from the placenta solves the technical problem of separating the maternal-source mesenchymal stem cells from the placenta, and the prepared maternal-source mesenchymal stem cells have large quantity, high purity and difficult intermixing with neonatal cells, and can meet the clinical application requirements; the method solves the problems that the quantity of mesenchymal stem cells from maternal sources obtained by the decidua basalis is small and the decidua basalis is easy to lose, and provides a new scheme and way for obtaining the mesenchymal stem cells from the maternal sources from the placenta.

Drawings

FIG. 1 is a schematic cross-sectional view of a material-extraction site of placental tissue according to an embodiment of the present application;

FIG. 2 is a graph of partial results of the identification of STR typing of maternal peripheral blood from a placenta delivered in accordance with the embodiments of the present application;

FIG. 3 is a partial result diagram of STR typing identification of mesenchymal stem cells harvested in test 1 according to the example of the present application;

FIG. 4 is a partial result diagram of STR typing identification of mesenchymal stem cells harvested in experiment 2 according to the example of the present application;

FIG. 5 is a partial result diagram of STR typing identification of mesenchymal stem cells harvested in experiment 3 according to the example of the present application;

FIG. 6 is a graph showing the results of the flow cytometry obtained from test 1 of example of the present application for the surface marker CD 73;

FIG. 7 is a graph showing the results of the flow cytometry obtained from test 1 of example of the present application for the surface marker CD 90;

FIG. 8 is a graph showing the results of the flow cytometry detection of the surface marker CD105 obtained in test 1 of example of the present application;

FIG. 9 is a graph showing the results of detecting a negative indicator for a surface marker by a flow cytometry in example 1 of the present application;

FIG. 10 is a graph showing the results of the flow cytometry detection of the surface marker CD73 obtained in example 2 of the present application;

FIG. 11 is a graph showing the results of the flow cytometry detection of the surface marker CD90 obtained in example 2 of the present application;

FIG. 12 is a graph showing the results of the flow assay of the surface marker CD105 in experiment 2 of the present application;

FIG. 13 is a graph showing the results of detecting a negative indicator for a surface marker by a flow cytometry in example 2 of the present application;

FIG. 14 is a graph showing the results of the flow cytometry obtained from test 3 of example of the present application for the surface marker CD 73;

FIG. 15 is a graph showing the results of the flow cytometry obtained from test 3 of example of the present application for the surface marker CD 90;

FIG. 16 is a graph showing the results of the flow assay of the surface marker CD105 in example 3 of the present application;

FIG. 17 is a graph showing the results of detecting a negative indicator for a surface marker by a flow cytometry in example 3 of the present application.

Detailed Description

At present, no other technology is available for separating maternal-derived mesenchymal stem cells from the rest of the placenta, except that the maternal-derived mesenchymal stem cells with higher purity can be separated from the decidua basalis of the placenta. That is, in the conventional technical solutions, the mesenchymal stem cells derived from the mother body are separated from the decidua basalis of the placenta by an enzymatic digestion method, but the other parts of the placenta are not.

Although other parts of the placenta contain a large number of mesenchymal stem cells, most of these mesenchymal stem cells are a mixture of maternal and neonatal cells, and it is an important and difficult technique to accurately distinguish maternal from neonatal cells in the placenta; there is no effective solution to obtain mesenchymal stem cells of maternal origin from parts of the placenta other than the decidua basalis, which can meet the demands of use.

The research of the application finds that in the placenta, besides the decidua basalis can obtain the maternal mesenchymal stem cells, the placenta tissues within 5cm of the umbilical cord radius and more than 0.5cm of the fetal surface can also obtain the maternal mesenchymal stem cells; particularly, by matching with the improved enzyme digestion solution and enzyme digestion treatment, the mesenchymal stem cells with high purity can be obtained, and the clinical application requirements can be met; and, for the equal amount of decidua basalis, this application can obtain the mesenchymal stem cell of maternal source that quantity is big, purity is high, has solved that the decidua basalis obtains that maternal source mesenchymal stem cell quantity is little and the problem that the decidua basalis is lost easily.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

First, primary reagents and materials

Stem cell culture medium was purchased from Stempro, sodium chloride injection from Guizhou Tiandi, 50mL centrifuge tube, T75 flask and T175 flask from Corning, collagenase, hyaluronidase, neutrase and papain from Gibco.

Compound enzyme digestive juice: dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution to obtain complex enzyme digestive juice; and the concentration of collagenase, hyaluronidase, neutral protease and papain in the compound enzyme digestive juice is 0.4 percent by mass, 0.1 percent by mass, 200U/mL by mass and 0.05 percent by mass respectively.

Control enzyme digest: and (3) dissolving collagenase in the PBS solution to prepare a control enzyme digestive juice, wherein the concentration of the collagenase is 0.5 percent by mass.

Preparation of mesenchymal stem cells

In this example, the same placenta is used, and the mesenchymal stem cells are prepared by taking materials from 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 provided with a placenta, and shearing placenta tissues which are in a circle with a placenta distance of 5cm from the radius of an umbilical cord and are more than 0.5cm from the surface of a fetus by using scissors; the material-drawing part is shown in fig. 1, and fig. 1 is a schematic cross-sectional view of the material-drawing part, wherein the upper surface is the maternal surface, the lower surface is the fetal surface, and the black square position is the material-drawing part.

2) Clamping the tissue by using tissue forceps, removing capillary vessel tissue rich in the surface of the mother body as much as possible to leave a part containing interstitial tissue, putting the tissue into a culture dish after separation, washing with normal saline, specifically sodium chloride injection in the embodiment to remove residual blood, and repeatedly washing with the sodium chloride injection until the washing liquid is clear;

3) 3g of the washed villus are put into a 50mL centrifuge tube, and the tissue is further cut into 1-3mm by a surgical scissors³Size;

4) adding 10mL of prepared compound enzyme digestive juice, namely collagenase, hyaluronidase, neutral protease and papain into a centrifuge tube of the cut tissue blocks to be dissolved in a PBS solution to prepare the compound enzyme digestive juice, covering a centrifuge tube cover, turning upside down and uniformly mixing, putting into a constant-temperature shaking instrument, setting the temperature at 37 ℃, rotating speed at 190RPM, and digesting for 20 minutes;

5) step 4), after digestion is finished, passing the contents in the centrifugal tube through a cell screen with the aperture of 50 microns, discarding filtrate, putting the substances on the cell screen into a 50mL centrifugal tube, adding 10mL of complex enzyme digestive juice, and continuing digestion for 40 minutes;

6) step 5), after digestion is finished, adding 30mL of PBS solution into a centrifuge tube containing the digested tissue suspension, and uniformly mixing; then, the mixture was passed through a 50 μm cell sieve to remove fragments, the filtrate was collected, centrifuged at 800g for 5min, and the supernatant was removed;

7) adding 30mL of stem cell culture medium into the centrifugal sediment obtained in the step 6), blowing and pumping the heavy suspension sediment by using a pipette, transferring the heavy suspension sediment into 1T 75 culture bottle, and placing the culture bottle into a carbon dioxide incubator for culture, wherein the parameters are set to 37 ℃, the carbon dioxide concentration is 5%, the relative humidity is 95%, and the culture conditions are the same below;

8) after culturing for 24 hours, sucking out the culture medium in the culture bottle by using a pipette, discarding the culture medium, then adding 30mL of stem cell culture medium, putting the stem cell culture medium back into the carbon dioxide incubator, and continuing culturing;

9) pouring out the culture medium in the culture bottle when the cells grow until the confluence reaches 80% -90%, adding 10mL of sodium chloride injection by a pipette, slightly shaking and washing, then pouring out, repeatedly adopting 10mL of sodium chloride injection to wash, and washing for 2 times;

10) adding 2mL of pancreatin into the culture bottle for digestion for 2 minutes, slightly beating the bottom of the culture bottle 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 terminate digestion, sucking out all cell suspension in the culture bottle into a 50mL centrifuge tube, centrifuging for 5 minutes at 500g and 20 ℃, and removing supernatant after centrifugation to obtain cell precipitate;

12) adding 15mL of sodium chloride injection into the cell sediment, re-suspending the cell sediment, setting the parameter of 500g, centrifuging at 20 ℃ for 5 minutes, and pouring supernatant after centrifugation to obtain the cell sediment;

13) adding 10mL of a stem cell complete culture medium into a centrifugal tube of the cell sediment, and gently blowing and beating the stem cell complete culture medium and the stem cell complete culture medium uniformly by using a pipette to obtain a culture medium cell suspension;

14) according to 8000 pieces/cm²Adding the cell suspension obtained in the step 13) into a T175 culture bottle, adding a stem cell culture medium to a constant volume of 25mL, and putting the culture bottle into a carbon dioxide incubator for culture, wherein the parameters are set to 37 ℃ of temperature, 5% of carbon dioxide concentration and 95% of relative humidity;

15) when the cells grow until the confluence reaches 80% -90%, harvesting the cells according to the steps 9-12;

16) the harvested cells were flow-tested and STR-typed according to the standards of the "forensic science DNA laboratory test Specification" (GA/T383-2002) to determine the genotyping of the harvested cells. At the same time, STR typing detection is carried out on the blood of the lying-in woman giving birth to the placenta by adopting the same method so as to determine the genotyping of the maternal line of the placenta.

Test 2

The placenta tissue obtained in test 1 is subjected to enzyme digestion treatment by using different enzyme digestive juices to prepare the mesenchymal stem cells of the test. The method comprises the following specific steps:

1) 3g of the washed villus are put into a 50mL centrifuge tube, and the tissue is further cut into 1-3mm by a surgical scissors³Size; this "washed villus" was the same as the "washed villus" of step 3) of test 1;

2) adding 10mL of prepared control enzyme digestive juice, namely the control enzyme digestive juice prepared by dissolving collagenase in a PBS solution, into a centrifuge tube containing the tissue mass, covering a centrifuge tube cover, turning upside down and uniformly mixing, putting into a constant temperature shaking instrument, setting the temperature at 37 ℃, the rotating speed at 190RPM, and digesting for 60 minutes;

3) step 2), after digestion is finished, adding 30mL of PBS solution into a centrifuge tube containing the digested tissue suspension, and uniformly mixing; then, the mixture was passed through a 50 μm cell sieve to remove fragments, the filtrate was collected, centrifuged at 800g for 5min, and the supernatant was removed;

4) adding 30mL of stem cell culture medium into the centrifugal sediment obtained in the step 3), blowing and pumping the heavy suspension sediment by using a pipette, transferring the heavy suspension sediment into 1T 75 culture bottle, and placing the culture bottle into a carbon dioxide incubator for culture, wherein the parameters are set to 37 ℃, the carbon dioxide concentration is 5%, the relative humidity is 95%, and the culture conditions are the same below;

5) after culturing for 24 hours, sucking out the culture medium in the culture bottle by using a pipette, discarding the culture medium, then adding 30mL of stem cell culture medium, putting the stem cell culture medium back into the carbon dioxide incubator, and continuing culturing;

6) pouring out the culture medium in the culture bottle when the cells grow until the confluence reaches 80% -90%, adding 10mL of sodium chloride injection by a pipette, slightly shaking and washing, then pouring out, repeatedly adopting 10mL of sodium chloride injection to wash, and washing for 2 times;

7) adding 2mL of pancreatin into the culture bottle for digestion for 2 minutes, slightly beating the bottom of the culture bottle 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 terminate digestion, sucking out all cell suspension in the culture bottle into a 50mL centrifuge tube, centrifuging for 5 minutes at 500g and 20 ℃, and removing supernatant after centrifugation to obtain cell precipitate;

9) adding 15mL of sodium chloride injection into the cell sediment, re-suspending the cell sediment, setting the parameter of 500g, centrifuging at 20 ℃ for 5 minutes, and pouring supernatant after centrifugation to obtain the cell sediment;

10) adding 10mL of a stem cell complete culture medium into a centrifugal tube of the cell sediment, and gently blowing and beating the stem cell complete culture medium and the stem cell complete culture medium uniformly by using a pipette to obtain a culture medium cell suspension;

11) according to 8000 pieces/cm²Adding the cell suspension obtained in the step 10) into a T175 culture bottle, adding a stem cell culture medium to a constant volume of 25mL, and putting the culture bottle into a carbon dioxide incubator for culture, wherein the parameters are set to 37 ℃ of temperature, 5% of carbon dioxide concentration and 95% of relative humidity;

12) when the cells grow until the confluence reaches 80% -90%, harvesting the cells according to the steps 6-9; the harvested cells were flow-tested and STR-typed according to the standards of the "forensic science DNA laboratory test Specification" (GA/T383-2002) to determine the genotyping of the harvested cells.

Test 3

1) Using the same placenta sample of the test 1, shearing the placenta tissue which is marked by the sample 1 and is 5cm away from the umbilical cord radius and close to the fetal face by using scissors;

2) clamping the tissue by using tissue forceps, removing the capillary vessel tissue rich in the tissue as much as possible to leave a part containing interstitial tissue, putting the tissue into a culture dish after the separation is finished, washing with normal saline, specifically sodium chloride injection in the embodiment to remove residual blood, and repeatedly washing with the sodium chloride injection until the washing liquid is clear;

5) step 4), after digestion is finished, passing the contents in the centrifugal tube through a cell screen with the aperture of 50 microns, discarding filtrate, putting tissues on the cell screen into a 50mL centrifugal tube, adding 10mL of complex enzyme digestive juice, and continuing digestion for 40 minutes;

15) when the cells grow until the confluence reaches 80% -90%, harvesting the cells according to the steps 9-12; the harvested cells were flow-tested and STR-typed according to the standards of the "forensic science DNA laboratory test Specification" (GA/T383-2002) to determine the genotyping of the harvested cells.

Third, results and analysis

1. Flow assay results for cells

In this example, the results of flow cytometry on the mesenchymal stem cells harvested in test 1, test 2 and test 3 are shown in table 1 and fig. 6 to 17.

TABLE 1 flow assay results of cells obtained by different methods for the three assays

Surface marker	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 results of the surface marker CD73 obtained in test 1, FIG. 7 is a graph showing the results of the surface marker CD90 obtained in test 1, FIG. 8 is a graph showing the results of the surface marker CD105 obtained in test 1, FIG. 9 is a graph showing the results of the surface marker negative index obtained in test 1, FIG. 10 is a graph showing the results of the surface marker CD73 obtained in test 2, FIG. 11 is a graph showing the results of the surface marker CD90 obtained in test 2, FIG. 12 is a graph showing the results of the surface marker CD105 obtained in test 2, FIG. 13 is a graph showing the results of the surface marker negative index obtained in test 2, FIG. 14 is a graph showing the results of the surface marker CD73 obtained in test 3, FIG. 15 is a graph showing the results of the surface marker CD90 obtained in test 3, and FIG. 16 is a graph showing the results of the surface marker CD105 obtained in test 3, FIG. 17 is a graph showing the results of the negative indicators of surface markers in the flow cytometry obtained in experiment 3.

The results shown in table 1, fig. 6 to fig. 17 show that the surface markers of the mesenchymal stem cells obtained in test 1, test 2 and test 3 were all qualified; three ways are demonstrated to be able to effectively obtain mesenchymal stem cells.

STR typing detection results

In this example, STR typing tests were performed on the mesenchymal stem cells harvested in test 1, test 2 and test 3, and also on the blood of the parturient who delivered the placenta, and the test results are shown in fig. 2 to 5.

Fig. 2 shows the result of STR typing identification of maternal peripheral blood from placenta delivered, fig. 3 shows the result of STR typing identification of mesenchymal stem cells harvested in test 1, fig. 4 shows the result of STR typing identification of mesenchymal stem cells harvested in test 2, and fig. 5 shows the result of STR typing identification of mesenchymal stem cells harvested in test 3.

Comparing the results of fig. 2 and fig. 3, it can be seen that the loci of the two result maps have the same phenotype, indicating that the two result maps are the same person, which indicates that the mesenchymal stem cells obtained in the test 1 are derived from the placenta, i.e. the high-purity maternal 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 individual's DNA is contaminating the sample, i.e., due to the contamination of fetal cells in maternal cells, indicating that the mesenchymal stem cells obtained in experiment 2 include cells from both maternal and fetal sources.

The results in FIG. 5 show that there are three peaks at some of the sites, indicating that the second individual's DNA is contaminating the sample, i.e., the fetal cells are contaminating the maternal cells, indicating that the mesenchymal stem cells obtained in experiment 3 also include maternal-derived cells and fetal-derived cells.

The comparison and analysis of the above results show that the placenta tissue with the sample-taking part of the present example, i.e. the placenta within 5cm of the umbilical cord radius and more than 0.5cm from the fetal face, can be combined with the special complex enzyme digestion solution and enzyme digestion treatment of the present example to obtain the high-purity maternal mesenchymal stem cells, i.e. test 1. The material-taking parts of the experiment 2 are the same as those of the experiment 1, but the specific conditions of the adopted enzyme digestion solution and the enzyme digestion treatment are different, so that certain newborn cells are mixed, and the purity of the maternal mesenchymal stem cells is influenced. Experiment 3 although the same complex enzyme digest and enzyme digestion treatment as in experiment 1 were used; however, the sampling position of experiment 3 is incorrect, the mesenchymal stem cells of maternal origin cannot be separated and obtained, and the obtained mesenchymal stem cells contain both maternal-origin cells and neonatal cells.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

Claims

1. A method for obtaining a maternal-derived mesenchymal stem cell from a placenta, comprising the steps of: the method comprises the following steps of,

obtaining a placenta tissue from a position, within a radius of 5cm from an umbilical cord and above 0.5cm from a fetal surface, of the placenta;

and carrying out enzyme digestion treatment on the placenta tissue by adopting enzyme digestion liquid, and then carrying out cell culture on a product of the enzyme digestion treatment to obtain the maternal-source mesenchymal stem cell.

2. The method of claim 1, wherein: removing blood vessels or white connective tissues in the placenta tissue which are directly communicated with the surface of a fetus before carrying out enzyme digestion treatment on the placenta tissue.

3. The method of claim 1, wherein: the enzyme digestive juice contains collagenase, hyaluronidase, neutral protease and papain;

preferably, the enzyme digestive juice is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in a PBS solution;

4. The method of claim 3, wherein: the enzyme digestion treatment specifically comprises adding at least 3mL of enzyme digestion solution into each gram of the placenta tissue, and performing constant-temperature shaking digestion at 37 ℃ for at least 20 minutes; then, removing the enzyme digestive juice, adding fresh enzyme digestive juice, and carrying out constant-temperature shaking digestion at 37 ℃ for at least 40 minutes;

5. The method according to any one of claims 1-4, wherein: the method specifically comprises the following steps;

6) digesting the culture product of the step 5) by using pancreatin, wherein the digested cells are the mesenchymal stem cells of the maternal source.

6. The method of claim 5, wherein: removing fragments in the step 4), specifically comprising fishing out the tissue blocks by using a cell screen with the aperture of 50-100 mu m, centrifuging the liquid, and collecting precipitated cells;

7. The method of claim 5, wherein: in the step 5), the stem cell culture medium added in the centrifugal precipitation product in the step 4) is resuspended in an amount that at least 10mL of the stem cell culture medium is added per gram of the placenta tissue.

8. The method of claim 5, wherein: in step 5), the cells were transferred to a culture flask for culture, specifically comprising culturing at 37 ℃ with 5% CO concentration₂Culturing for 24-48 hours at constant temperature, then removing supernatant and residual tissues, adding fresh stem cell culture medium, and continuously culturing under the same condition until the cell confluence reaches 80-90%.

9. The method of claim 5, wherein: further comprises washing the culture product of step 5) with physiological saline at least once before step 6), and then performing pancreatin digestion.

10. The method of claim 9, wherein: in the step 6), the culture product in the step 5) is digested by pancreatin, and the method specifically comprises the steps of adding pancreatin into the culture product in the step 5) and digesting for at least 2min at room temperature; then adding a stem cell culture medium with the volume three times that of pancreatin, and stopping digestion; centrifuging the mixture, and removing the supernatant to obtain cell precipitate; resuspending the cell pellet with normal saline, then centrifuging and discarding the supernatant; and (4) carrying out heavy suspension by adopting a stem cell culture medium to obtain the maternal-source mesenchymal stem cells.