CN111793598A - A method for obtaining maternal-derived mesenchymal stem cells from the placenta - Google Patents

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 and more than 0.5 cm from the fetal surface; using an enzymatic digestion solution to enzymatically digest the placental tissue, The enzymatically digested product is then subjected to cell culture 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, and the prepared maternal-derived mesenchymal stem cells have a large quantity, high purity, and are not easily mixed with neonatal cells, which can meet the needs of clinical applications; The method of the application solves the problems that the number of maternal-derived mesenchymal stem cells obtained from the decidua basalis is small and the decidua basalis is easily lost, and provides a new solution and approach for obtaining the maternal-derived mesenchymal stem cells from the placenta.

Description

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

technical field

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

Background technique

Mesenchymal stem cells (MSCs) are a group of mesoderm-derived pluripotent stem cells with self-renewal and multi-directional differentiation potential. It was originally discovered from the murine hematopoietic organ by Friedenstein et al., and it was demonstrated that it can differentiate into osteoblasts and adipocytes in vitro. Studies have shown that mesenchymal stem cells are widely found in bone marrow, umbilical cord placenta, adipose and other tissues.

The placenta is an important organ for material exchange between the fetus and the mother, and is a tissue-binding organ between the mother and the child formed by the joint growth of the embryonic membrane and the maternal endometrium during human pregnancy. The placenta is rich in mesenchymal stem cells, but because the placenta is formed by 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. stem cells. The use of cells from mixed individuals in clinical applications is extremely risky.

At present, the method to obtain higher-purity maternal-derived mesenchymal stem cells from the placenta is to isolate the maternal-derived mesenchymal stem cells from the decidua basalis. The disadvantage of this method is that the number of maternal-derived mesenchymal stem cells obtained in the decidua basalis is very small; and the decidua basal membrane and the amniotic membrane together will be broken and broken during childbirth, and are easily lost, resulting in the loss of cell sources and the inability to obtain maternal source of mesenchymal stem cells.

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

SUMMARY OF THE INVENTION

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

This application specifically adopts the following technical solutions:

The present application discloses a method for obtaining maternal-derived mesenchymal stem cells from the placenta. The enzymatic digestion treatment is performed, and then the product of the enzymatic digestion treatment is subjected to cell culture to obtain the maternal-derived mesenchymal stem cells of the present application.

It should be noted that the present application creatively obtains maternal-derived mesenchymal stem cells from other placental tissues other than decidua basalis, which solves the problem that the number of maternal-derived mesenchymal stem cells obtained from decidua basalis is small and the decidua basal membrane is easily lost. ; provides a new scheme and approach for the preparation or isolation of maternal-derived mesenchymal stem cells from the placenta.

It should also be noted that the size of the placental tissue taken in this application is not limited, as long as it is the placental tissue taken from the position range defined in this application, a considerable amount of maternal-derived mesenchymal stem cells can be obtained by culture; In terms of the amount of decidua basalis, the present application can obtain more maternal-derived mesenchymal stem cells, and the problem of loss similar to the decidua basal membrane will not occur.

It can be understood that one of the key points of this application is that it is found that the placental tissue specifically defined in this application can obtain maternal-derived mesenchymal stem cells; as for the subsequent placental tissue cleaning, shearing, enzymatic digestion, cell acquisition, mesenchymal stem cell culture, etc. All can refer to existing methods for preparing mesenchymal stem cells from tissues, which are not specifically limited here. However, in order to improve the purity of the parent-derived mesenchymal stem cells of the present application, the present application provides detailed descriptions of the subsequent steps, especially the steps of obtaining cells by enzymatic digestion, and the details are described in the following technical solutions.

Preferably, the method of the present application further comprises removing blood vessels or white connective tissue directly connected to the fetal surface in the placental tissue.

It should be noted that part of the placental tissue taken in this application may contain blood vessels or white connective tissue directly connected to the fetal surface. In order to avoid these blood vessels or white connective tissue from affecting the purity of maternal-derived mesenchymal stem cells, this application preferably It has been eliminated from the technical scheme of . It can be understood that this step is not required if the placental tissue itself does not have these blood vessels or white connective tissue.

Preferably, the enzyme digestion solution contains collagenase, hyaluronidase, neutral protease and papain.

It should be noted that the research in this application has found that the use of the special enzymatic digestion solution defined in this application that is compounded by multiple enzymes for enzymatic digestion can further improve the purity of the prepared maternal-derived mesenchymal stem cells and reduce the number of newborns. Cell interference is of great significance for clinical applications. It can be understood that if the purity requirements of the maternal-derived mesenchymal stem cells are lower, other enzymatic digestion treatment schemes or other methods can also be used to obtain cells, which are not specifically limited here.

Preferably, the enzymatic digestion solution is prepared by dissolving collagenase, hyaluronidase, neutral protease and papain in PBS solution. Among them, the PBS solution is the phosphate buffer solution routinely used in the laboratory.

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, the concentration of papain is The concentration is 0.03%-0.09% by mass fraction.

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

It should be noted that, in the present application, the placental tissue block is digested by a double digestion method. Since a very small amount of impurities may be mixed in the placental tissue sampling process, such as residual vascular tissue on the surface, etc., after the first short pre-digestion, the impurity cells contained in the digestive juice are removed. At this time, most of the maternal sources The mesenchymal stem cells are still in the placental tissue block, and then the placental tissue block is digested again, so that the maternal-derived 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-derived mesenchymal stem cells.

Preferably, the method of removing the enzymatic digestion solution is to use a cell screen with a pore size of 50-100 μm to fish out the tissue block, and discard the remaining liquid.

It should be noted that, in principle, the cell screen used in this application only needs to be able to fish out the tissue block, and the pore size of 50-100 μm is only the cell screen specifically used in an implementation manner of the present application.

Preferably, the method for obtaining maternal-derived mesenchymal stem cells from the placenta of the present application specifically includes the following steps;

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

2) washing with normal saline to remove blood clots and capillary tissue in the placental tissue obtained in step 1);

3) shredding the cleaned placental tissue in step 2), and performing enzymatic digestion with an enzymatic digestion solution;

4) After the enzymatic digestion treatment is completed, remove debris and centrifuge to remove the supernatant;

5) use the stem cell medium to resuspend the pellet obtained in step 4) centrifugation, and transfer it to a culture flask for cultivation;

6) The cultured product of step 5) is digested with trypsin, and the digested cells are maternal-derived mesenchymal stem cells.

Preferably, in step 4), removing the fragments specifically includes using a cell screen with a pore size of 50-100 μm to fish out the tissue fragments, and then centrifuging the liquid to collect the precipitated cells.

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

It should be noted that the enzymatic digestion treatment of step 3) is performed on the removed tissue block, the purpose of which is to obtain as many cells as possible from the tissue block to avoid waste; of course, in principle, in an implementation manner of the present application , Step 3) Under the circumstance that two enzymatic digestion treatments have been performed, most of the cells in the tissue block have been digested. If time cost and production efficiency are considered, it is not necessary to re-dig out the tissue block in step 4). Carry out the enzymatic digestion treatment of step 3).

Preferably, in step 5), the amount of stem cell culture medium added to the resuspended product of step 4) centrifugation precipitation is at least 10 mL of stem cell culture medium corresponding to each gram of placental tissue.

Preferably, in step 5), transfer to a culture flask for cultivation, which specifically includes culturing at a constant temperature for 24-48 hours under the conditions of 37° C. and 5% CO ₂ , 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%.

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

Preferably, in step 6), the cultured product of step 5) is digested with trypsin, which specifically includes adding trypsin to the cultured product of step 5) and digesting it at room temperature for at least 2 minutes; then adding three times the volume of the trypsin stem cell culture medium , the digestion was terminated; the mixture was centrifuged, the supernatant was discarded, and the cell pellet was obtained; the cell pellet was resuspended in physiological saline, and then the supernatant was discarded by centrifugation; .

The beneficial effects of this application are:

The method for obtaining maternal-derived mesenchymal stem cells from the placenta of the present application solves the technical problem of separating maternal-derived mesenchymal stem cells from the placenta, and the prepared maternal-derived mesenchymal stem cells have a large quantity, high purity, and are not easily mixed with newborns. It can meet the needs of clinical application; the method of the present application solves the problems that the number of maternal-derived mesenchymal stem cells obtained from the decidua basalis is small and the decidua basalis is easy to lose, and provides a method for obtaining maternal-derived mesenchymal stem cells from the placenta. A new approach and approach.

Description of drawings

Fig. 1 is the cross-sectional schematic diagram of the placenta tissue in the embodiment of the present application;

2 is a partial result diagram of the identification of STR typing in the peripheral blood of the mother who gave birth to the placenta in the embodiment of the present application;

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

4 is a partial result diagram of STR typing and identification of mesenchymal stem cells harvested in Test 2 in the embodiment of the present application;

5 is a partial result diagram of STR typing and identification of mesenchymal stem cells harvested in Test 3 in the embodiment of the present application;

6 is a graph showing the results of flow cytometry detection of the surface marker CD73 in cells obtained in Test 1 of the embodiment of the present application;

7 is a graph showing the results of flow cytometry detection of the surface marker CD90 in cells obtained in Test 1 of the embodiment of the present application;

8 is a graph showing the results of flow cytometry detection of the surface marker CD105 in cells obtained in Test 1 of the embodiment of the present application;

9 is a graph showing the results of cell flow detection of negative indicators of surface markers in Test 1 of the embodiment of the present application;

10 is a graph showing the results of flow cytometry detection of the surface marker CD73 in cells obtained in Test 2 of the embodiment of the present application;

Figure 11 is a graph showing the results of flow cytometry detection of the surface marker CD90 in cells obtained in Test 2 of the embodiment of the present application;

Fig. 12 is a graph showing the results of flow cytometry detection of the surface marker CD105 in Test 2 of the embodiment of the present application;

FIG. 13 is a graph showing the results of cell flow detection of negative indicators of surface markers in Test 2 of the embodiment of the present application;

Figure 14 is a graph showing the results of flow cytometry detection of the surface marker CD73 in cells obtained in Test 3 of the embodiment of the present application;

Figure 15 is a graph showing the results of flow cytometry detection of the surface marker CD90 in cells obtained in Test 3 of the embodiment of the present application;

Figure 16 is a graph showing the results of cell flow detection of the surface marker CD105 in Test 3 of the embodiment of the present application;

FIG. 17 is a graph showing the results of cell flow detection of negative indicators of surface markers in Test 3 of Example 3 of the present application.

Detailed ways

At present, except for the isolation of high-purity maternal-derived mesenchymal stem cells from the decidua basalis of the placenta, there is no other technology that can isolate maternal-derived mesenchymal stem cells from the rest of the placenta. That is to say, the existing technical solutions are to separate maternal-derived mesenchymal stem cells from the decidua basalis of the placenta by enzymatic digestion, but not from other parts of the placenta.

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 technique; there is currently no effective protocol to obtain adequate maternal-derived mesenchymal stem cells from other parts of the placenta than the decidua basalis.

The study of this application found that in the placenta, in addition to the decidua basalis, which can obtain maternal-derived mesenchymal stem cells, 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-derived mesenchymal stem cells; especially It is combined with the improved enzyme digestion solution and enzyme digestion treatment of the present application, which can obtain higher-purity maternal-derived mesenchymal stem cells, which can meet the needs of clinical applications; and, compared with the same amount of decidua basalis, the present application can obtain a large number of , Maternal-derived mesenchymal stem cells with high purity, which solves the problems that the decidua basalis obtains the maternal-derived mesenchymal stem cells in small quantities and the decidua basalis is easily lost.

The present application will be further described in detail below through specific embodiments. The following examples are only to further illustrate the application, and should not be construed as a limitation to the application.

Example

1. Main reagents and materials

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, collagenase, hyaluronidase, neutral protease and papain were purchased from Gibco.

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 compound enzyme digestion solution is 0.4% by mass, The concentration of hyaluronidase was 0.1% by mass, the concentration of neutral protease was 200 U/mL, and the concentration of papain was 0.05% by mass.

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.

2. Preparation of Mesenchymal Stem Cells

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

Test 1

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 a distance of more than 0.5 cm from the fetal surface; the sampling site is shown in Figure 1, and Figure 1 is a cross-section of the sampling site Schematic diagram, in which the upper surface is the maternal surface, the lower surface is the fetal surface, and the position of the black box is the sampling site.

2) Use tissue tweezers to clamp the tissue, try to remove the rich capillary tissue on the maternal surface, and leave the part containing the interstitial tissue. After the separation, put the tissue into a petri dish, and use normal saline. Sodium chloride injection, rinsed to remove residual blood, and repeated washing with sodium chloride injection until the cleaning solution was clear;

3) Take 3g of the cleaned villi and put it into a 50mL centrifuge tube, and use surgical scissors to further shred the tissue to a size of 1-3mm ³ ;

4) Add 10 mL of prepared compound enzyme digestion solution to the centrifuge tube of the chopped tissue pieces, namely collagenase, hyaluronidase, neutral protease and papain dissolved in PBS solution to make compound enzyme digestion solution, cover Put the cap on the centrifuge tube, invert it upside down and mix well, put it into a constant temperature shaker, set the temperature to 37°C, the rotation speed of 190RPM, and digest for 20 minutes;

5) Step 4) After the digestion is completed, pass the contents of the centrifuge tube through a cell sieve with a pore size of 50 μm, discard the filtrate, put the material on the cell sieve into a 50 mL centrifuge tube, add 10 mL of compound enzyme digestion solution, and continue. Digestion for 40 minutes;

6) Step 5) After the digestion 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 screen to remove debris, and collect the filtrate. Centrifuge at 800g for 5min and remove the supernatant;

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

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

9) When the cells grow to a confluence of 80%-90%, pour out the medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, shake gently to wash, then pour out, repeat Wash with 10 mL of sodium chloride injection for a total of 2 times;

10) Add 2 mL of trypsin to the culture flask to digest for 2 minutes, tap the bottom of the culture flask lightly to make the cells fall off, and shake left and right several times to mix evenly;

11) Add 6 mL of stem cell culture medium to terminate the digestion, suck out the cell suspension in all the culture flasks into a 50 mL centrifuge tube, centrifuge at 500 g for 5 minutes at 20°C, and after centrifugation, discard the supernatant to obtain a cell pellet;

12) Add 15 mL of sodium chloride injection to the cell pellet, resuspend the cell pellet, set the parameters to 500g and centrifuge at 20°C for 5 minutes, after centrifugation, pour the supernatant to obtain the cell pellet;

13) Add 10 mL of stem cell complete medium to the centrifuge tube of the cell precipitation, and gently pipette and mix with a pipette to obtain a medium cell suspension;

14) The cell suspension of step 13) was added to the T175 culture flask at a density of 8000 cells/cm ² , the stem cell medium was added to dilute the volume to 25 mL, and the culture flask was placed in a carbon dioxide incubator for cultivation. The parameters were set as The temperature is 37°C, the carbon dioxide concentration is 5%, and the relative humidity is 95%;

15) When the cells grow to a confluence of 80%-90%, harvest the cells according to steps 9-12;

16) The harvested cells were subjected to flow cytometry and STR typing according to the Forensic Science DNA Laboratory Testing Specification (GA/T383-2002) standard to determine the genotype of the harvested cells. At the same time, STR typing was performed on the blood of the mother who delivered the placenta using the same method to determine the maternal genotype of the placenta.

Test 2

In this experiment, the placental tissue obtained in experiment 1 was used for enzymatic digestion with different enzyme digestion solutions to prepare mesenchymal stem cells in this experiment. details as follows:

1) Take 3 g of the cleaned villi and put it into a 50mL centrifuge tube, and use surgical scissors to further cut the tissue to a size of ^1-3 mm; The back hair clumps" are the same;

2) Add 10 mL of the prepared control enzyme digestion solution to the centrifuge tube containing the tissue block, that is, the control enzyme digestion solution prepared by dissolving collagenase in PBS solution, cover the centrifuge tube, invert it upside down and mix well and put it in a constant temperature. In the shaker, set the temperature to 37°C, the rotation speed of 190RPM, and digest for 60 minutes;

3) Step 2) After the digestion 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 screen to remove debris, and collect the filtrate. Centrifuge at 800g for 5min and remove the supernatant;

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

5) After culturing for 24 hours, suck out the medium in the culture bottle with a pipette and discard it, then add 30 mL of stem cell culture medium, put it back into the carbon dioxide incubator, and continue to cultivate;

6) When the cells grow to 80%-90% confluence, pour out the medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, shake gently to wash, then pour out, repeat Wash with 10 mL of sodium chloride injection for a total of 2 times;

7) Add 2 mL of trypsin to the culture flask to digest for 2 minutes, tap the bottom of the culture flask gently to make the cells fall off, and shake left and right several times to mix evenly;

8) Add 6 mL of culture medium to terminate the digestion, suck out the cell suspension in all the culture flasks into a 50 mL centrifuge tube, centrifuge at 500 g for 5 minutes at 20°C, and after centrifugation, discard the supernatant to obtain a cell pellet;

9) Add 15 mL of sodium chloride injection to the cell pellet, resuspend the cell pellet, set the parameters to 500g and centrifuge at 20°C for 5 minutes, after centrifugation, pour the supernatant to obtain the cell pellet;

10) Add 10 mL of stem cell complete medium to the centrifuge tube of the cell precipitation, and gently pipette and mix with a pipette to obtain a medium cell suspension;

11) Add the cell suspension of step 10) to the T175 culture flask at a density of 8000 cells/cm ² , add stem cell culture medium to dilute to 25 mL, and put the culture flask into a carbon dioxide incubator for cultivation. The parameters are set as The temperature is 37°C, the carbon dioxide concentration is 5%, and the relative humidity is 95%;

12) When the cells grow to a confluence of 80%-90%, harvest the cells according to steps 6-9; the harvested cells are detected by flow cytometry and tested according to the "Forensic Science DNA Laboratory Inspection Specifications" (GA/T383-2002) Standard assay STR typing to determine the genotype of harvested cells.

Trial 3

1) Using the same placenta sample of test 1, use scissors to cut the placenta tissue outside the circle marked by sample 1 with a radius of 5 cm from the umbilical cord and close to the fetal surface;

2) Use tissue tweezers to clamp the tissue, remove the rich capillary tissue as much as possible, and leave the part containing the interstitial tissue. After the separation, put the tissue into a petri dish, and use normal saline. In this case, chloride is used. Sodium injection, wash to remove residual blood, repeat washing with sodium chloride injection until the cleaning solution is clear;

3) Take 3g of the cleaned villi and put it into a 50mL centrifuge tube, and use surgical scissors to further shred the tissue to a size of 1-3mm ³ ;

4) Add 10 mL of prepared compound enzyme digestion solution to the centrifuge tube of the chopped tissue pieces, namely collagenase, hyaluronidase, neutral protease and papain dissolved in PBS solution to make compound enzyme digestion solution, cover Put the cap on the centrifuge tube, invert it upside down and mix well, put it into a constant temperature shaker, set the temperature to 37°C, the rotation speed of 190RPM, and digest for 20 minutes;

5) Step 4) After the digestion is completed, pass the contents of 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, add 10 mL of compound enzyme digestion solution, and continue. Digestion for 40 minutes;

6) Step 5) After the digestion 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 screen to remove debris, and collect the filtrate. Centrifuge at 800g for 5min and remove the supernatant;

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

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

9) When the cells grow to a confluence of 80%-90%, pour out the medium in the culture flask, add 10 mL of sodium chloride injection with a pipette, shake gently to wash, then pour out, repeat Wash with 10 mL of sodium chloride injection for a total of 2 times;

10) Add 2 mL of trypsin to the culture flask to digest for 2 minutes, tap the bottom of the culture flask lightly to make the cells fall off, and shake left and right several times to mix evenly;

11) Add 6 mL of stem cell medium to terminate the digestion, suck out the cell suspension in all the culture flasks into a 50 mL centrifuge tube, centrifuge at 500 g for 5 minutes at 20°C, and after centrifugation, discard the supernatant to obtain a cell pellet;

12) Add 15 mL of sodium chloride injection to the cell pellet, resuspend the cell pellet, set the parameters to 500g and centrifuge at 20°C for 5 minutes, after centrifugation, pour the supernatant to obtain the cell pellet;

13) Add 10 mL of stem cell complete medium to the centrifuge tube of the cell precipitation, and gently pipette and mix with a pipette to obtain a medium cell suspension;

14) The cell suspension of step 13) was added to the T175 culture flask at a density of 8000 cells/cm ² , the stem cell medium was added to dilute the volume to 25 mL, and the culture flask was placed in a carbon dioxide incubator for cultivation. The parameters were set as The temperature is 37°C, the carbon dioxide concentration is 5%, and the relative humidity is 95%;

15) When the cells grow to a confluence of 80%-90%, harvest the cells according to steps 9-12; the harvested cells are detected by flow cytometry and tested according to the "Forensic Science DNA Laboratory Test Specifications" (GA/T383-2002) Standard assay STR typing to determine the genotype of harvested cells.

3. Results and Analysis

1. Cell flow assay results

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 and Figure 6 to Figure 17 .

Table 1 Flow assay results of cells obtained by different methods in three experiments

surface markers CD73 CD90 CD105 Negative indicator Test 1 99.80% 99.68% 99.73% 1.21% Test 2 99.78% 99.60% 99.72% 1.15% Trial 3 99.76% 99.60% 99.46% 1.54%

Figure 6 is a graph showing the results of flow cytometric detection of the surface marker CD73 in cells obtained in Experiment 1, Figure 7 is a graph showing the results of flow cytometry detection of the surface marker CD90 in cells obtained in Experiment 1, and Figure 8 is a graph showing the results of flow detection of the surface marker CD105 in cells obtained in Experiment 1 Figure 9 is the result of the negative index of the cell flow detection surface marker obtained in test 1, Figure 10 is the result of the cell flow detection surface marker CD73 obtained in test 2, and Figure 11 is the cell flow obtained in test 2. The results of detecting the surface marker CD90, Figure 12 is the result of the cell flow detection of the surface marker CD105 obtained in the experiment 2, Figure 13 is the result of the cell flow detection of the negative index of the surface marker obtained in the experiment 2, and Figure 14 is the experiment 3. The results of the flow detection of the surface marker CD73 in the obtained cells, FIG. 15 is the result of the flow detection of the surface marker CD90 of the cells obtained in the experiment 3, and FIG. 16 is the result of the flow detection of the surface marker CD105 of the cells obtained in the experiment 3. FIG. 17 is a graph showing the results of cell flow detection of negative indicators of surface markers obtained in experiment 3. FIG.

The results shown in Table 1 and Figure 6 to Figure 17 show that the surface markers of mesenchymal stem cells obtained in Test 1, Test 2 and Test 3 are all qualified; indicating that all three methods can effectively obtain mesenchymal stem cells.

2. STR typing test results

In this example, STR typing was performed on the mesenchymal stem cells harvested in Experiment 1, Experiment 2 and Experiment 3. At the same time, STR typing was also performed on the blood of the mother who gave birth to the placenta. The test results are shown in Figures 2 to 5. Show.

Figure 2 shows the results of STR typing and identification of the peripheral blood of the mother who gave birth to the placenta, Figure 3 shows the STR typing and identification results of the mesenchymal stem cells harvested in Experiment 1, and Figure 4 shows the STR typing and identification results of the mesenchymal stem cells harvested in Experiment 2 The results of typing and identification, Figure 5 shows the results of STR typing and identification of the mesenchymal stem cells harvested in experiment 3.

Comparing the results in Figure 2 and Figure 3, it can be seen that the phenotypes of the two result maps are the same, indicating that the two are the same person, indicating that the mesenchymal stem cells obtained in Experiment 1 were derived from the placenta, that is, a high-purity maternal source was obtained. mesenchymal stem cells.

The results in Figure 4 show that there are three peaks at some sites, indicating that the second human DNA is mixed in this sample, which is caused by the mixing of fetal cells with maternal cells, indicating that the mesenchymal stem cells obtained in test 2 include cells derived from Maternal cells and cells from the fetus.

The results in Figure 5 show that there are three peaks at some sites, indicating that the second human DNA is mixed in this sample, which is caused by the mixing of fetal cells with maternal cells, indicating that the mesenchymal stem cells obtained in test 3 also include cells from the mother and cells from the fetus.

By comparing and analyzing the above results, it can be seen that using the sampling site in this example, that is, the placenta tissue within 5 cm of the radius of the placenta from the umbilical cord and more than 0.5 cm from the fetal surface, combined with the special compound enzyme digestion solution and enzyme digestion treatment of this example, it is possible to obtain the tissue samples. High-purity maternal-derived mesenchymal stem cells, namely test 1. Experiment 2 and Experiment 1 had the same sampling sites, but due to the different enzymatic digestion solution and specific conditions of enzyme digestion, certain neonatal cells would be mixed, which would affect 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 position of Experiment 3 was incorrect, and it was impossible to separate and obtain maternal-derived mesenchymal stem cells, and the obtained mesenchymal stem cells also contained maternal-derived mesenchymal stem cells. cells and neonatal cells.

The above content is a further detailed description of the present application in conjunction with specific embodiments, and it cannot be considered that the specific implementation of the present application is limited to these descriptions. For those of ordinary skill in the technical field to which the present application pertains, some simple deductions or substitutions can also be made without departing from the concept of the present application.

Claims (10)

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;

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.

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;

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.

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

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.

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.

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