A method for obtaining maternal-derived mesenchymal stem cells from the placenta

Abstract

The present application discloses a method for obtaining maternal-derived mesenchymal stem cells from the placenta. The method for obtaining maternal-derived mesenchymal stem cells from the placenta in the present application includes obtaining placental tissue from a position within a radius of 5 cm from the umbilical cord in the placenta and more than 0.5 cm from the fetal surface; subjecting the placental tissue to enzymatic digestion treatment with an enzymatic digestion solution, and then culturing the product of the enzymatic digestion treatment to obtain maternal-derived mesenchymal stem cells. The method of the present application solves the technical problem of separating maternal-derived mesenchymal stem cells from the placenta. The prepared maternal-derived mesenchymal stem cells are large in quantity, high in purity, and not easily contaminated with neonatal cells, and can meet the clinical application requirements; the method of the present application solves the problems of low quantity of maternal-derived mesenchymal stem cells obtained from the decidua basalis and easy loss of the decidua basalis, and provides a new solution and approach for obtaining maternal-derived mesenchymal stem cells from the placenta.

Description

Technical Field

[0001] This application relates to the technical field of mesenchymal stem cell preparation, and particularly to a method for obtaining maternal-derived mesenchymal stem cells from the placenta. Background Art

[0002] Mesenchymal stem cells (MSCs) are a group of pluripotent stem cells derived from the mesoderm with the potential for self-renewal and multi-directional differentiation. They were initially discovered by Friedenstein et al. in the hematopoietic organs of mice, and it was demonstrated that they could differentiate into osteoblasts and adipocytes in vitro. Research shows that mesenchymal stem cells are widely present in tissues such as bone marrow, umbilical cord placenta, and adipose tissue.

[0003] The placenta is an important organ for material exchange between the fetus and the mother, and is an organ formed by the combination of the embryonic membranes and the maternal endometrium during human pregnancy. The placenta contains abundant mesenchymal stem cells. However, since the placenta is formed by both the maternal and neonatal tissues, it contains two different sources of mesenchymal stem cells, namely fetal-derived mesenchymal stem cells and maternal-derived mesenchymal stem cells. Using a mixture of cells from multiple people in clinical applications poses great risks.

[0004] Currently, the method for obtaining a relatively high-purity of maternal-derived mesenchymal stem cells from the placenta is to isolate the maternal mesenchymal stem cells from the decidua basalis. The drawback of this method is that the number of maternal-derived mesenchymal stem cells obtained from the decidua basalis is very small; moreover, the decidua basalis and the amnion will break and fracture during childbirth, and are easily lost, resulting in the loss of the cell source and the inability to obtain maternal-derived mesenchymal stem cells.

[0005] Therefore, how to prepare or isolate maternal-derived mesenchymal stem cells from the placenta other than the decidua basalis is one of the research focuses and difficulties in this field. Summary of the Invention

[0006] The purpose of this application is to provide a new method for obtaining maternal-derived mesenchymal stem cells from the placenta.

[0007] This application specifically adopts the following technical solutions:

[0008] This application discloses a method for obtaining maternal-derived mesenchymal stem cells from the placenta, including obtaining placental tissue from a position within a radius of 5 cm from the umbilical cord and more than 0.5 cm from the fetal surface of the placenta; subjecting the placental tissue to enzymatic digestion treatment with an enzyme digestion solution, and then culturing the product of the enzymatic digestion treatment to obtain the maternal-derived mesenchymal stem cells of this application.

[0009] It should be noted that the present application creatively obtains maternal-derived mesenchymal stem cells from other placental tissues outside the decidua basalis, solving the problems of the small quantity of maternal-derived mesenchymal stem cells obtained from the decidua basalis and the easy loss of the decidua basalis; it provides a new solution and approach for the preparation or isolation of maternal-derived mesenchymal stem cells in the placenta.

[0010] It should also be noted that there is no limit to the size of the placental tissue taken in the present application. As long as it is the placental tissue taken from the position range defined in the present application, a considerable quantity of maternal-derived mesenchymal stem cells can be obtained through cultivation; compared with an equal amount of decidua basalis, the present application can obtain more maternal-derived mesenchymal stem cells, and there will be no problem of loss similar to that of the decidua basalis.

[0011] It can be understood that one of the keys of the present application is the research and discovery that the maternal-derived mesenchymal stem cells can be obtained from the placental tissue specifically defined in the present application; as for subsequent steps such as placental tissue washing, shearing, enzymatic digestion, cell acquisition, and mesenchymal stem cell culture, the existing methods for preparing mesenchymal stem cells from tissues can be referred to and are not specifically limited herein. However, in order to improve the purity of the maternal-derived mesenchymal stem cells in the present application, the subsequent steps, especially the step of enzymatic digestion to obtain cells, are described in detail, as shown in the following technical solutions.

[0012] Preferably, the method of the present application further includes removing the blood vessels or white connective tissues directly connected to the fetal surface in the placental tissue.

[0013] It should be noted that some of the placental tissues taken in the present application may contain blood vessels or white connective tissues directly connected to the fetal surface. In order to avoid the influence of these blood vessels or white connective tissues on the purity of the maternal-derived mesenchymal stem cells, they are removed in the preferred technical solution of the present application. It can be understood that if the taken placental tissue itself does not have these blood vessels or white connective tissues, this step is not required.

[0014] Preferably, the enzymatic digestion solution contains collagenase, hyaluronidase, neutral protease, and papain.

[0015] It should be noted that the present application has found through research that enzymatic digestion treatment with the special multi-enzyme composite enzymatic digestion solution defined in the present application can further improve the purity of the prepared maternal-derived mesenchymal stem cells, reduce neonatal cell interference, which is of great significance for clinical applications. It can be understood that if the requirement for the purity of the maternal-derived mesenchymal stem cells is relatively low, other enzymatic digestion treatment schemes or other methods for obtaining cells can also be adopted, which are not specifically limited herein.

[0016] Preferably, the enzyme digestion solution is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution, wherein the PBS solution is the phosphate buffer solution commonly used in laboratories.

[0017] 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 100-300 U / mL, and the concentration of papain is 0.03%-0.09% by mass.

[0018] Preferably, in the method of the present application, the enzyme digestion treatment specifically includes adding at least 3 mL of enzyme digestion solution per gram of placental tissue, and digesting at a constant temperature of 37°C with shaking for at least 20 minutes; then, removing the enzyme digestion solution, adding fresh enzyme digestion solution, and digesting at a constant temperature of 37°C with shaking for at least 40 minutes.

[0019] It should be noted that the present application uses a double digestion method to digest the placental tissue block. Since a very small amount of impurities may be mixed into the placental tissue sampling process, such as vascular tissue remaining on the surface, etc., after the first short pre-digestion, the impurity cells contained in the digestion fluid are removed. At this time, most of the maternal mesenchymal stem cells are still in the placental tissue block, and then the placental tissue block is digested again, so that the maternal mesenchymal stem cells are digested from the tissue block. The double digestion method of the present application can improve the purity of the obtained maternal mesenchymal stem cells.

[0020] Preferably, the enzymatic digestion solution is removed by using a cell sieve with a pore size of 50-100 μm to fish out the tissue blocks and discard the remaining liquid.

[0021] It should be noted that, in principle, the cell sieve used in the present application can be used as long as it can remove the tissue blocks, and the pore size of 50-100 μm is only a specific cell sieve used in one implementation of the present application.

[0022] Preferably, the method of obtaining maternal mesenchymal stem cells from the placenta of the present application specifically comprises the following steps:

[0023] 1) Take the placenta tissue within 5 cm radius from the umbilical cord and more than 0.5 cm from the fetal surface, and remove the blood vessels or white connective tissue directly connected to the fetal surface;

[0024] 2) washing with physiological saline to remove blood clots and capillary tissue in the placenta tissue obtained in step 1);

[0025] 3) chopping the cleaned placental tissue in step 2) and performing enzyme digestion treatment with an enzyme digestion solution;

[0026] 4) After the enzymatic digestion is completed, remove the debris and centrifuge to remove the supernatant.

[0027] 5) Resuspend the precipitate obtained by centrifugation in step 4) with a stem cell medium and transfer it to a culture flask for culturing.

[0028] 6) Digest the culture product in step 5) with trypsin, and the digested cells are mesenchymal stem cells derived from the mother body.

[0029] Preferably, in step 4), to remove the debris, specifically, use a cell sieve with a pore size of 50 - 100 μm to fish out the tissue mass, then centrifuge the liquid and collect the precipitated cells.

[0030] Preferably, step 4) further includes subjecting the fished-out tissue mass to the enzymatic digestion treatment in step 3), then passing the mixture through a cell sieve with a pore size of 50 - 100 μm, centrifuging the filtrate, and collecting the precipitated cells.

[0031] It should be noted that the purpose of subjecting the fished-out tissue mass to the enzymatic digestion treatment in step 3) is to obtain as many cells as possible from the tissue mass to avoid waste; of course, in principle, in one implementation manner of the present application, when the enzymatic digestion treatment has been carried out twice in step 3), most of the cells in the tissue mass have been digested. If considering the time cost and production efficiency, it may not be necessary to subject the fished-out tissue mass to the enzymatic digestion treatment in step 3) again in step 4).

[0032] Preferably, in step 5), the amount of stem cell medium added to resuspend the centrifuged precipitate in step 4) is at least 10 mL of stem cell medium per gram of placental tissue.

[0033] Preferably, in step 5), transferring to a culture flask for culturing specifically includes incubating at 37 °C and 5% CO 2 under the condition of constant temperature for 24 - 48 hours, then discarding the supernatant and residual tissue, and adding fresh stem cell medium, and continuing to culture under the same conditions until the cell confluence reaches 80% - 90%.

[0034] Preferably, the method of the present application further includes washing the culture product in step 5) with physiological saline at least once before step 6), and then performing trypsin digestion.

[0035] Preferably, in step 6), digesting the culture product in step 5) with trypsin specifically includes adding trypsin to the culture product in step 5) and digesting at room temperature for at least 2 min; then adding stem cell medium with a volume three times that of trypsin to terminate the digestion; centrifuging the mixture, discarding the supernatant, and obtaining a cell precipitate; resuspending the cell precipitate with physiological saline, and then centrifuging and discarding the supernatant; resuspending with stem cell medium to obtain the mesenchymal stem cells derived from the mother body of the present application.

[0036] The beneficial effects of the present application are as follows:

[0037] The method for obtaining maternal-derived mesenchymal stem cells from the placenta in the present application solves the technical problem of separating maternal-derived mesenchymal stem cells from the placenta. The prepared maternal-derived mesenchymal stem cells are large in quantity, high in purity, and not easily contaminated with neonatal cells, and can meet the clinical application requirements; the method of the present application solves the problems of small quantity of maternal-derived mesenchymal stem cells obtained from the decidua basalis and easy loss of the decidua basalis, and provides a new solution and approach for obtaining maternal-derived mesenchymal stem cells from the placenta. Brief Description of the Drawings

[0038] Figure 1 is a schematic cross-sectional view of the sampling site of the placental tissue in the embodiment of the present application;

[0039] Figure 2 is a partial result diagram of the STR typing identification of the peripheral blood of the mother of the delivered placenta in the embodiment of the present application;

[0040] Figure 3 is a partial result diagram of the STR typing identification of the mesenchymal stem cells harvested in Experiment 1 in the embodiment of the present application;

[0041] Figure 4 is a partial result diagram of the STR typing identification of the mesenchymal stem cells harvested in Experiment 2 in the embodiment of the present application;

[0042] Figure 5 is a partial result diagram of the STR typing identification of the mesenchymal stem cells harvested in Experiment 3 in the embodiment of the present application;

[0043] Figure 6 is a result diagram of the flow cytometry detection of the surface marker CD73 of the cells obtained in Experiment 1 in the embodiment of the present application;

[0044] Figure 7 is a result diagram of the flow cytometry detection of the surface marker CD90 of the cells obtained in Experiment 1 in the embodiment of the present application;

[0045] Figure 8 is a result diagram of the flow cytometry detection of the surface marker CD105 of the cells obtained in Experiment 1 in the embodiment of the present application;

[0046] Figure 9 is a result diagram of the flow cytometry detection of the negative index of the surface marker of the cells obtained in Experiment 1 in the embodiment of the present application;

[0047] Figure 10 is a result diagram of the flow cytometry detection of the surface marker CD73 of the cells obtained in Experiment 2 in the embodiment of the present application;

[0048] Figure 11It is the result graph of flow cytometry detection of surface marker CD90 in Test 2 of the embodiments of the present application;

[0049] Figure 12 It is the result graph of flow cytometry detection of surface marker CD105 in Test 2 of the embodiments of the present application;

[0050] Figure 13 It is the result graph of flow cytometry detection of negative index of surface marker in Test 2 of the embodiments of the present application;

[0051] Figure 14 It is the result graph of flow cytometry detection of surface marker CD73 in Test 3 of the embodiments of the present application;

[0052] Figure 15 It is the result graph of flow cytometry detection of surface marker CD90 in Test 3 of the embodiments of the present application;

[0053] Figure 16 It is the result graph of flow cytometry detection of surface marker CD105 in Test 3 of the embodiments of the present application;

[0054] Figure 17 It is the result graph of flow cytometry detection of negative index of surface marker in Test 3 of the embodiments of the present application. Detailed implementation manners

[0055] At present, in addition to being able to isolate mesenchymal stem cells of maternal origin with relatively high purity from the basal decidua of the placenta, there is no other technology that can isolate mesenchymal stem cells of maternal origin from the rest of the placenta. That is to say, the existing technical solutions are all to isolate mesenchymal stem cells of maternal origin from the basal decidua of the placenta by enzymatic digestion method, while the other parts of the placenta cannot.

[0056] 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. To accurately distinguish between maternal and neonatal cells in the placenta is a very important and difficult technology; at present, there is no effective solution to obtain mesenchymal stem cells of maternal origin that can meet the usage requirements from other parts of the placenta other than the basal decidua.

[0057] The present application has found that in addition to the decidua basalis, maternal mesenchymal stem cells can be obtained from the placenta. The placental tissue within a radius of 5 cm from the umbilical cord and more than 0.5 cm from the fetal surface can also obtain maternal mesenchymal stem cells. In particular, with the improved enzyme digestion solution and enzyme digestion treatment of the present application, maternal mesenchymal stem cells with higher purity can be obtained, which can meet the needs of clinical applications. Moreover, compared with the same amount of decidua basalis, the present application can obtain a large number of high-purity maternal mesenchymal stem cells, which solves the problem of small number of maternal mesenchymal stem cells obtained from the decidua basalis and easy loss of the decidua basalis.

[0058] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0059] Example

[0060] 1. Main reagents and materials

[0061] Stem cell culture medium was purchased from Stempro, sodium chloride injection was purchased from Guizhou Tiandi, 50 mL centrifuge tubes, T75 culture flasks, and T175 culture flasks were purchased from Corning, and collagenase, hyaluronidase, neutral protease, and papain were purchased from Gibco.

[0062] Compound enzyme digestion solution: collagenase, hyaluronidase, neutral protease and papain are dissolved in PBS solution to prepare compound enzyme digestion solution; and 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 200 U / mL, and the concentration of papain is 0.05% by mass.

[0063] Control enzyme digestion solution: Collagenase was dissolved in PBS solution to prepare control enzyme digestion solution, wherein the concentration of collagenase was 0.5% by mass.

[0064] 2. Preparation of Mesenchymal Stem Cells

[0065] In this case, the same placenta was used to prepare mesenchymal stem cells from different parts of the placenta. The placenta in this case was provided by Guangdong Qifu Hospital. The specific preparation method is as follows:

[0066] Test 1

[0067] 1) Open the sample collection bag containing the placenta, and use scissors to cut the placenta tissue within a circle with a radius of 5 cm from the umbilical cord and more than 0.5 cm from the fetal surface; Figure 1 As shown, Figure 1 It is a schematic diagram of the cross-section of the sampling site, where the upper surface is the maternal surface, the lower surface is the fetal surface, and the black square position is the sampling site.

[0068] 2) Use tissue forceps to hold the tissue, and try to remove the capillary-rich tissue on the maternal surface as much as possible, leaving the part containing interstitial tissue. After separation, place the tissue in a culture dish, and use normal saline. In this case, sodium chloride injection is specifically used to wash away the residual blood. Repeat the washing with sodium chloride injection until the washing solution is clear;

[0069] 3) Take 3 g of the washed villi and put them into a 50 mL centrifuge tube. Further cut the tissue into pieces with a surgical scissors to a size of 1 - 3 mm 3 in size;

[0070] 4) Add 10 mL of the prepared composite enzyme digestion solution to the centrifuge tube with the cut tissue pieces. That is, collagenase, hyaluronidase, neutral protease, and papain are dissolved in PBS solution to make the composite enzyme digestion solution. Cover the centrifuge tube lid, invert and mix well, then put it into a constant temperature shaker, set the temperature at 37 °C, the rotation speed at 190 RPM, and digest for 20 minutes;

[0071] 5) After the digestion in step 4) is completed, pass the content in the centrifuge tube through a cell sieve with a pore size of 50 μm, discard the filtrate, put the substance on the cell sieve into a 50 mL centrifuge tube, and then add 10 mL of the composite enzyme digestion solution and continue to digest for 40 minutes;

[0072] 6) After the digestion in step 5) is completed, add 30 mL of PBS solution to the centrifuge tube containing the digested tissue suspension and mix well; then, pass the mixture through a 50 μm cell sieve to remove the debris, collect the filtrate, centrifuge at 800 g for 5 min, and discard the supernatant;

[0073] 7) Add 30 mL of stem cell medium to the centrifugal precipitate in step 6), pipette and resuspend the precipitate, then transfer it to 1 T75 culture flask, and place it in a carbon dioxide incubator for culture. The parameters are set as temperature 37 °C, carbon dioxide concentration 5%, and relative humidity 95%. The following culture conditions are the same;

[0074] 8) After culturing for 24 hours, use a pipette to suck out the medium in the culture flask and discard it, then add 30 mL of stem cell medium, put it back into the carbon dioxide incubator, and continue to culture;

[0075] 9) When the cell confluence reaches 80% - 90%, pour out the medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, gently shake and wash, then pour out, and repeat the washing with 10 mL of sodium chloride injection for a total of 2 times;

[0076] 10) Add 2 mL of trypsin to the culture flask and digest for 2 minutes. Gently pat the bottom of the culture flask to make the cells detach, and shake it left and right several times to mix evenly;

[0077] 11) Add 6 mL of stem cell culture medium to terminate digestion. Aspirate all the cell suspension in the culture flasks into a 50 mL centrifuge tube, centrifuge at 500 g and 20 °C for 5 minutes. After centrifugation, discard the supernatant to obtain cell pellet.

[0078] 12) Add 15 mL of sodium chloride injection to the cell pellet to resuspend it. Set the parameters to centrifuge at 500 g and 20 °C for 5 minutes. After centrifugation, pour out the supernatant to obtain cell pellet.

[0079] 13) Add 10 mL of complete stem cell culture medium to the centrifuge tube with the cell pellet, and gently pipette and mix to obtain a cell suspension in the culture medium.

[0080] 14) Add the cell suspension from step 13) to a T175 culture flask at a density of 8000 cells / cm 2 , add stem cell culture medium to make the volume up to 25 mL. Place the culture flask in a carbon dioxide incubator for culture. Set the parameters as temperature 37 °C, carbon dioxide concentration 5%, and relative humidity 95%.

[0081] 15) When the cell confluence reaches 80% - 90%, harvest the cells according to steps 9 - 12.

[0082] 16) Detect the harvested cells by flow cytometry, and detect the STR typing according to the standard of "Forensic Science - DNA Laboratory Testing Specification" (GA / T 383 - 2002) to determine the genotyping of the harvested cells. At the same time, use the same method to detect the STR typing of the blood of the parturient who delivered the placenta to determine the genotyping of the maternal line of the placenta.

[0083] Experiment 2

[0084] In this experiment, the placental tissue obtained in Experiment 1 was used and digested with different enzyme digestion solutions to prepare mesenchymal stem cells for this experiment. The specific steps are as follows:

[0085] 1) Take 3 g of the washed chorionic tuft and put it into a 50 mL centrifuge tube. Further cut the tissue into pieces with a surgical scissors to a size of 1 - 3 mm 3 ; the "washed chorionic tuft" is the same as the "washed chorionic tuft" in step 3) of Experiment 1.

[0086] 2) Add 10 mL of the prepared control enzyme digestion solution, that is, the control enzyme digestion solution made by dissolving collagenase in PBS solution, to the centrifuge tube containing the tissue pieces. Cover the centrifuge tube lid, invert and mix well, then put it into a thermostatic shaker. Set the temperature to 37 °C and the rotation speed to 190 RPM, and digest for 60 minutes.

[0087] 3) After the digestion in step 2) is completed, add 30 mL of PBS solution to the centrifuge tube containing the digested tissue suspension, and mix well; then, pass the mixture through a 50-μm cell sieve to remove debris, collect the filtrate, centrifuge at 800 g for 5 min, and remove the supernatant;

[0088] 4) Add 30 mL of stem cell medium to the centrifugal precipitate in step 3), pipette to resuspend the precipitate, and then transfer it to one T75 culture flask, and place it in a carbon dioxide incubator for culture. The parameters are set as temperature 37 °C, carbon dioxide concentration 5%, and relative humidity 95%. The following culture conditions are the same;

[0089] 5) After culturing for 24 hours, pipette out the medium in the culture flask and discard it, then add 30 mL of stem cell medium, put it back into the carbon dioxide incubator, and continue culturing;

[0090] 6) When the cell confluence reaches 80%-90%, pour out the medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, gently shake and wash, then pour out, and repeat the washing with 10 mL of sodium chloride injection for a total of 2 times;

[0091] 7) Add 2 mL of trypsin to the culture flask and digest for 2 minutes, gently pat the bottom of the culture flask to make the cells detach, and shake it left and right several times to mix evenly;

[0092] 8) Add 6 mL of medium to terminate the digestion, aspirate all the cell suspension in the culture flask into a 50-mL centrifuge tube, centrifuge at 500 g and 20 °C for 5 minutes. After centrifugation, discard the supernatant to obtain cell precipitate;

[0093] 9) Add 15 mL of sodium chloride injection to the cell precipitate to resuspend the cell precipitate, set the parameters to centrifuge at 500 g and 20 °C for 5 minutes. After centrifugation, pour out the supernatant to obtain cell precipitate;

[0094] 10) Add 10 mL of complete stem cell medium to the centrifuge tube with the cell precipitate, and gently pipette to mix well to obtain a medium-cell suspension;

[0095] 11) Add the cell suspension in step 10) to a T175 culture flask at a density of 8000 cells / cm 2 , add stem cell medium to make the volume up to 25 mL, put the culture flask into a carbon dioxide incubator for culture, and set the parameters as temperature 37 °C, carbon dioxide concentration 5%, and relative humidity 95%;

[0096] 12) When the cell confluence reaches 80%-90%, harvest the cells according to steps 6-9; detect the harvested cells by flow cytometry, and detect the STR typing according to the standard of "Forensic Science - DNA Laboratory Testing Specifications" (GA / T 383-2002) to determine the genotyping of the harvested cells.

[0097] Experiment 3

[0098] 1) Use the same placental sample as in Experiment 1. Cut the placental tissue marked with Specimen 1 outside the circle with a radius of 5 cm from the umbilical cord and close to the fetal surface with scissors.

[0099] 2) Hold the tissue with tissue forceps and try to remove the capillary-rich tissue as much as possible, leaving the part containing interstitial tissue. After separation, put the tissue into a culture dish, and use normal saline, specifically sodium chloride injection in this example, to wash away the residual blood. Repeat the washing with sodium chloride injection until the washing liquid is clear.

[0100] 3) Take 3 g of the washed villi and put them into a 50 mL centrifuge tube. Further cut the tissue into pieces with a size of 1-3 mm with surgical scissors. 3 size

[0101] 4) Add 10 mL of the prepared composite enzyme digestion solution to the centrifuge tube with the cut tissue pieces. The composite enzyme digestion solution is made by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution. Cover the centrifuge tube lid, mix well by inverting up and down, and then put it into a constant temperature shaker. Set the temperature at 37°C and the rotation speed at 190 RPM for digestion for 20 minutes.

[0102] 5) After the digestion in step 4) is completed, pass the content in the centrifuge tube through a cell sieve with a pore size of 50 μm, discard the filtrate, put the tissue on the cell sieve into a 50 mL centrifuge tube, and then add 10 mL of the composite enzyme digestion solution to continue digestion for 40 minutes.

[0103] 6) After the digestion in step 5) is completed, add 30 mL of PBS solution to the centrifuge tube containing the digested tissue suspension and mix well; then, pass the mixture through a 50 μm cell sieve to remove the fragments, collect the filtrate, centrifuge at 800 g for 5 min, and remove the supernatant.

[0104] 7) Add 30 mL of stem cell medium to the centrifugal precipitate in step 6), resuspend the precipitate by pipetting, and then transfer it to 1 T75 culture flask and place it in a carbon dioxide incubator for culture. The parameters are set as temperature 37°C, carbon dioxide concentration 5%, and relative humidity 95%. The following culture conditions are the same.

[0105] 8) After culturing for 24 hours, aspirate and discard the culture medium in the culture flask with a pipette, then add 30 mL of stem cell culture medium, place it back in the carbon dioxide incubator, and continue culturing;

[0106] 9) When the cell confluence reaches 80%-90%, pour out the culture medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, gently shake and wash, then pour out, and repeat the washing with 10 mL of sodium chloride injection for a total of 2 times;

[0107] 10) Add 2 mL of trypsin to the culture flask and digest for 2 minutes. Gently tap the bottom of the culture flask to make the cells detach, and shake it several times from side to side to mix evenly;

[0108] 11) Add 6 mL of stem cell culture medium to terminate the digestion. Aspirate all the cell suspension in the culture flask into a 50 mL centrifuge tube, centrifuge at 500 g and 20 °C for 5 minutes. After centrifugation, discard the supernatant to obtain cell pellets;

[0109] 12) Add 15 mL of sodium chloride injection to the cell pellets, resuspend the cell pellets, set the parameters at 500 g and 20 °C for centrifugation for 5 minutes. After centrifugation, pour out the supernatant to obtain cell pellets;

[0110] 13) Add 10 mL of complete stem cell culture medium to the centrifuge tube with cell pellets, gently pipette and mix to obtain a cell suspension in the culture medium;

[0111] 14) According to the density of 8000 cells / cm 2 , add the cell suspension in step 13) to a T175 culture flask, add stem cell culture medium to make the volume up to 25 mL, place the culture flask in the carbon dioxide incubator for culture, and set the parameters as temperature 37 °C, carbon dioxide concentration 5%, and relative humidity 95%;

[0112] 15) When the cell confluence reaches 80%-90%, harvest the cells according to steps 9-12; Detect the harvested cells by flow cytometry, and detect the STR typing according to the standard of "Code of Practice for DNA Laboratory in Forensic Science" (GA / T 383-2002) to determine the gene typing of the harvested cells.

[0113] III. Results and Analysis

[0114] 1. Flow cytometry detection results of cells

[0115] In this example, the mesenchymal stem cells harvested from Experiment 1, Experiment 2, and Experiment 3 were detected by flow cytometry, and the results are shown in Table 1, Figures 6 to 17 as shown.

[0116] Table 1 Flow cytometry detection results of cells obtained by different methods in three experiments

[0117] 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％

[0118] Figure 6 It is the result graph of flow cytometry detection of surface marker CD73 for the cells obtained in Experiment 1. Figure 7 It is the result graph of flow cytometry detection of surface marker CD90 for the cells obtained in Experiment 1. Figure 8 It is the result graph of flow cytometry detection of surface marker CD105 for the cells obtained in Experiment 1. Figure 9 It is the result graph of flow cytometry detection of negative index of surface markers for the cells obtained in Experiment 1. Figure 10 It is the result graph of flow cytometry detection of surface marker CD73 for the cells obtained in Experiment 2. Figure 11 It is the result graph of flow cytometry detection of surface marker CD90 for the cells obtained in Experiment 2. Figure 12 It is the result graph of flow cytometry detection of surface marker CD105 for the cells obtained in Experiment 2. Figure 13 It is the result graph of flow cytometry detection of negative index of surface markers for the cells obtained in Experiment 2. Figure 14 It is the result graph of flow cytometry detection of surface marker CD73 for the cells obtained in Experiment 3. Figure 15 It is the result graph of flow cytometry detection of surface marker CD90 for the cells obtained in Experiment 3. Figure 16 It is the result graph of flow cytometry detection of surface marker CD105 for the cells obtained in Experiment 3. Figure 17 It is the result graph of flow cytometry detection of negative index of surface markers for the cells obtained in Experiment 3.

[0119] Table 1 Figures 6 to 17 The results shown in Table 1 indicate that all the surface markers of the mesenchymal stem cells obtained in Experiment 1, Experiment 2, and Experiment 3 are qualified, indicating that all three methods can effectively obtain mesenchymal stem cells.

[0120] 2. STR Typing Detection Results

[0121] In this example, STR typing detection was performed on the mesenchymal stem cells harvested in Experiment 1, Experiment 2, and Experiment 3. At the same time, STR typing detection was also performed on the blood of the parturient who delivered the placenta. The detection results are as Figures 2 to 5 shown.

[0122] Figure 2 It is the result of STR typing identification of the peripheral blood of the mother who delivered the placenta. Figure 3 It is the result of STR typing identification of the mesenchymal stem cells harvested in Experiment 1. Figure 4 It is the result of STR typing identification of the mesenchymal stem cells harvested in Experiment 2. Figure 5 It is the result of STR typing identification of the mesenchymal stem cells harvested in Experiment 3.

[0123] Comparison Figure 2 and Figure 3As can be seen from the results, the phenotypes of the gene loci in the two result graphs are the same, indicating that the two are from the same person, which means that the mesenchymal stem cells obtained in Experiment 1 are derived from the placenta mother, that is, high-purity maternal-derived mesenchymal stem cells are obtained.

[0124] Figure 4 The results showed that some sites had three peaks, indicating that the sample was mixed with the DNA of a second person, that is, fetal cells were mixed with maternal cells, indicating that the mesenchymal stem cells obtained in Experiment 2 included cells from the mother and cells from the fetus.

[0125] Figure 5 The results showed that some sites had three peaks, indicating that the sample was mixed with the DNA of a second person, that is, fetal cells mixed with maternal cells, indicating that the mesenchymal stem cells obtained in Experiment 3 also included cells from the mother and cells from the fetus.

[0126] Comparative analysis of the above results shows that the sampling site of this example, that is, the placenta within 5 cm of the umbilical cord radius and the placenta tissue more than 0.5 cm away from the fetal surface, combined with the special compound enzyme digestion solution and enzyme digestion treatment of this example, can obtain high-purity maternal-derived mesenchymal stem cells, that is, Experiment 1. Experiment 2 has the same sampling site as Experiment 1, but due to the different specific conditions of the enzyme digestion solution and enzyme digestion treatment, it will cause a certain amount of neonatal cell mixing, affecting the purity of maternal-derived mesenchymal stem cells. Although Experiment 3 used the same compound enzyme digestion solution and enzyme digestion treatment as Experiment 1; however, the sampling site of Experiment 3 was incorrect, and it was impossible to separate and obtain maternal-derived mesenchymal stem cells. The mesenchymal stem cells obtained contained both maternal-derived cells and neonatal cells.

[0127] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for obtaining maternal-derived mesenchymal stem cells from the placenta, characterized in that: the method includes, obtaining placental tissue from a position within a radius of 5 cm from the umbilical cord of the placenta and more than 0.5 cm from the fetal surface; performing enzymatic digestion on the placental tissue with an enzymatic digestion solution, and then culturing the product of the enzymatic digestion treatment to obtain the maternal-derived mesenchymal stem cells; the enzymatic digestion solution contains collagenase, hyaluronidase, neutral protease and papain; wherein, the concentration of collagenase is 0.3%-0.5% by mass fraction, the concentration of hyaluronidase is 0.1%-0.3% by mass fraction, the concentration of neutral protease is 100-300 U / mL, and the concentration of papain is 0.03%-0.09% by mass fraction.

2. The method according to claim 1, characterized in that: before performing enzymatic digestion on the placental tissue, the blood vessels or white connective tissues directly connected to the fetal surface in the placental tissue are removed first.

3. The method according to claim 1, characterized in that: the enzymatic digestion solution is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution.

4. The method according to claim 3, characterized in that: the enzymatic digestion treatment specifically includes adding at least 3 mL of enzymatic digestion solution to each gram of the placental tissue, and performing constant-temperature shaking digestion at 37°C for at least 20 minutes; then, removing the enzymatic digestion solution, adding fresh enzymatic digestion solution again, and performing constant-temperature shaking digestion at 37°C for at least 40 minutes.

5. The method according to claim 4, characterized in that: the way of removing the enzymatic digestion solution is to fish out the tissue blocks with a cell sieve with a pore size of 50-100 μm and discard the remaining liquid.

6. The method according to any one of claims 1-5, characterized in that: the method specifically includes the following steps; 1) Take placental tissue within a radius of 5 cm from the umbilical cord of the placenta and more than 0.5 cm from the fetal surface, and remove the blood vessels or white connective tissues directly connected to the fetal surface in the taken placental tissue; 2) Wash and remove blood clots and capillary tissues in the placental tissue obtained in step 1) with physiological saline; 3) Cut the placental tissue washed in step 2) into pieces and perform enzymatic digestion treatment with an enzymatic digestion solution; 4) After the enzymatic digestion treatment is completed, remove the fragments and centrifuge to remove the supernatant; 5) Resuspend the precipitate obtained by centrifugation in step 4) with a stem cell culture medium and transfer it to a culture flask for culturing; 6) Digest the culture product in step 5) with trypsin, and the digested cells are the maternal-derived mesenchymal stem cells.

7. The method according to claim 6, characterized in that: in step 4), removing the fragments specifically includes fishing out the tissue blocks with a cell sieve with a pore size of 50-100 μm, and then centrifuging the liquid to collect the precipitated cells.

8. The method according to claim 7, characterized in that: step 4) further includes performing the enzymatic digestion treatment in step 3) on the fished-out tissue blocks, then passing the mixture through a cell sieve with a pore size of 50-100 μm, centrifuging the filtrate, and collecting the precipitated cells.

9. The method according to claim 6, wherein: in step 5), the amount of stem cell culture medium added to resuspend the centrifuged precipitate of step 4) is at least 10 mL of stem cell culture medium per gram of placental tissue.

10. The method according to claim 6, wherein: In step 5), transfer to a culture flask for culturing, specifically including culturing at a constant temperature of 37 °C and a CO concentration of 5% 2 for 24 - 48 hours, then discard the supernatant and residual tissue, and then add fresh stem cell medium and continue to culture under the same conditions until the cell confluence reaches 80% - 90%.

11. The method according to claim 6, wherein: it further includes, before step 6), washing the culture product of step 5) with physiological saline at least once, and then performing trypsin digestion.

12. The method according to claim 11, wherein: in step 6), performing trypsin digestion on the culture product of step 5) specifically includes adding trypsin to the culture product of step 5) and digesting at room temperature for at least 2 min; then adding stem cell culture medium with a volume three times that of trypsin to terminate digestion; centrifuging the mixture, discarding the supernatant to obtain cell precipitate; resuspending the cell precipitate with physiological saline, and then centrifuging and discarding the supernatant; resuspending with stem cell culture medium to obtain the maternal-derived mesenchymal stem cells.

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