Placenta mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a preparation method of placenta mesenchymal stem cells, which comprises the following steps: preserving a placenta in a preserving fluid for treatment, taking out the placenta, placing the placenta in a sterile tray, clipping bright red chorion tissues in the center of the placenta, and removing epithelial layers and calcified layers on two sides; shearing the obtained placenta tissue material containing the mesenchymal stem cells to obtain a sheared placenta tissue material; digesting the sheared placenta tissue material by using a combined digestive enzyme to obtain a digested tissue mixture; taking the digested tissue mixture supernatant suspension and centrifuging; adding a red blood cell lysis buffer into the precipitate to obtain a mixture subjected to red blood cell lysis treatment; performing centrifugal precipitation on the mixture subjected to erythrocyte splitting decomposition treatment to obtain mesenchymal stem cells; and performing serum-free amplification passage on the mesenchymal stem cells to obtain the placenta mesenchymal stem cells. According to the method, the digested cells do not need to be filtered, manual operation steps are few, and industrial and standardized operation is easy.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical technology field, in particular to a placental mesenchymal stem cell and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSC) are a kind of stem cells, named for being able to differentiate into mesenchymal tissues, and have the potential of sub- totipotency, and can be induced to differentiate into various tissue cells under specific in vivo and in vitro environments, such as nerve, heart, liver, bone, cartilage, tendon, fat, epithelial and other cells. Mesenchymal stem cells have the commonness of stem cells, that is, the ability of self-renewal, multi-directional differentiation and homing. Mesenchymal stem cells are widely distributed in bone marrow, periosteum, cancellous bone, adipose tissue, synovial membrane, skeletal muscle, fetal liver, deciduous teeth, umbilical cord, and umbilical cord blood. The content of mesenchymal stem cells in most tissues is very small, and it is difficult to obtain and widely used. It was first discovered in bone marrow, and then isolated from adipose tissue, bone, muscle, lung, liver, pancreas, and umbilical cord, umbilical cord blood and placental tissue. Due to its strong proliferation capacity, strong differentiation capacity, low immunogenicity and immune regulation function, its clinical application value is more and more concerned by people.

[0003] As a cell product to be applied to clinical cell therapy, it should have the characteristics of convenient tissue sampling, wide source, simple isolation process and no ethical restrictions. However, MSCs derived from bone marrow are difficult to obtain, and the cell proliferation and differentiation capacity is limited by the age of the donor, and there are strong immune rejection in allogeneic transplantation, so it has become a research hotspot to seek a source of MSCs that is rich in stem cells, easy to obtain, non-invasive and not subject to ethical restrictions. Mesenchymal stem cells have the potential of self-renewal and multi-directional differentiation, have low immunogenicity, and are not subject to immune rejection after allogeneic and xenogeneic transplantation, and are not tumorigenic, which provides a new choice for cell replacement therapy for various diseases.

[0004] At present, the existing stem cells isolated from placenta and umbilical cord have the disadvantages of insufficient purity and quantity, and cannot meet the use requirements. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a placental mesenchymal stem cell and a preparation method thereof to solve the above problems.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a preparation method of a placental mesenchymal stem cell, the specific steps are as follows: Step one, the placenta is preserved in a preservation solution, and is processed within 48-72 hours. The placenta is taken out and placed in a sterile tray. The fresh red chorionic membrane tissue in the center of the placenta is cut. The epithelial layer and calcified layer on both sides are removed. Step two, the placental tissue material containing mesenchymal stem cells obtained is subjected to a cutting treatment to obtain cut placental tissue material. Step three, the cut placental tissue material is subjected to a digestion treatment with a combined digestive enzyme to obtain a digested tissue mixture. Step four, the supernatant of the digested tissue mixture is taken and subjected to a centrifugal treatment to obtain a precipitate. Step five, a red blood cell lysis solution is added to the precipitate to obtain a mixture subjected to a red blood cell lysis treatment. Step six, the mixture subjected to the red blood cell lysis treatment is subjected to a centrifugal treatment to obtain a precipitate, i.e., a generation of mesenchymal stem cells. Step seven, the generation of mesenchymal stem cells is subjected to a serum-free amplification passage to obtain placental mesenchymal stem cells.

[0007] As a further scheme of the present application, the preservation solution is a mixture of trehalose solution, red blood cell storage solution, calcium heparin solution, human blood albumin injection and Taizishen cyclic peptide C.

[0008] As a further scheme of the present application, the preservation solution comprises the following components by weight: 25-30 parts of 0.2% trehalose solution, 20-25 parts of red blood cell storage solution, 3-5 parts of 0.2% calcium heparin solution, 35-40 parts of human blood albumin injection, and 1-2 parts of Taizishen cyclic peptide C. The 0.2% trehalose solution, red blood cell storage solution, 0.2% calcium heparin solution, human blood albumin injection and Taizishen cyclic peptide C are uniformly mixed at room temperature to form a mixed solution.

[0009] As a further scheme of the present application, the combined digestive enzyme in step three comprises collagenase IV with a final concentration of 50 U / ml-100 U / ml, 0.005 wt%-0.01 wt% trypsin, 0.2-0.35 mg / ml DNAase, and 0.03-0.08% trypsin-EDTA.

[0010] As a further scheme of the present application, the collagenase IV, trypsin, DNAase and trypsin-EDTA are simultaneously added. The digestion conditions include: digestion at 37±2°C; and digestion for 15-20 hours.

[0011] As a further scheme of the present application, the centrifugal treatment in step four is performed at a speed of 2000-3000 rpm for 10-15 min.

[0012] As a further scheme of the present application, the ratio of the precipitate to the red blood cell lysate in step five is 1:3-5; and the lysis is performed at 15-25 DEG C for 10-15 min.

[0013] As a further scheme of the present application, the centrifugal treatment in step six is performed at a speed of 2000-2500 rpm for 15-20 min.

[0014] A placental mesenchymal stem cell is prepared by using a preparation method of the placental mesenchymal stem cell.

[0015] The placental mesenchymal stem cell is used in the preparation of a cell preparation for treating diseases, cosmetics, health products and drugs.

[0016] Due to the above technical scheme, the present application has the following advantages and positive effects: 1. The cells after digestion do not need to be filtered, and the manual operation steps are less, so that the standardization operation is easy to industrialize. 2. The placental mesenchymal stem cell prepared by the method has good differentiation potential, and the cell shape and size are uniform, the volume is small, and the normal karyotype is maintained after multiple passage operations, which is beneficial to the later clinical application. DETAILED DESCRIPTION

[0017] Example 1 The preparation method of the placental mesenchymal stem cell comprises the following steps: Step one, the placenta is stored in a storage solution, and is processed within 48 hours; the placenta is taken out and placed in a sterile tray; the fresh red chorionic membrane tissue in the center of the placenta is cut; and the epithelial layer and calcified layer on both sides are removed. Step two, the placental tissue material containing mesenchymal stem cells is cut and processed to obtain the cut placental tissue material. Step three, the cut placental tissue material is digested by using a combination of digestive enzymes to obtain a digested tissue mixture. Step four, the supernatant suspension of the digested tissue mixture is taken and centrifuged to obtain a precipitate. Step five, red blood cell lysate is added to the precipitate to obtain a mixture treated by red blood cell lysis. Step six, the mixture treated by red blood cell lysis is centrifuged to obtain a precipitate, which is a generation of mesenchymal stem cells. Step seven, the generation of mesenchymal stem cells is subjected to serum-free amplification and passage to obtain placental mesenchymal stem cells. The preservation solution is a mixture of trehalose solution, red blood cell storage solution, calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclic peptide C; the preservation solution comprises the following components in parts by weight: 30 parts of 0.2% trehalose solution, 25 parts of red blood cell storage solution, 5 parts of 0.2% calcium heparin solution, 40 parts of human blood albumin injection, and 2 parts of radix pseudoginseng cyclic peptide C; the 0.2% trehalose solution, red blood cell storage solution, 0.2% calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclic peptide C are uniformly mixed at room temperature to form a mixed solution; the combined digestive enzyme in step three comprises collagenase IV with a final concentration of 100 U / ml, 0.01 wt% trypsin, 0.35 mg / ml DNase, and 0.08% trypsin-EDTA; the collagenase IV, trypsin, DNase and trypsin-EDTA are simultaneously added; and the digestion conditions comprise: digestion at 39 DEG C; digestion for 20 hours; the centrifugal treatment in step four is performed at a speed of 3000 rpm for 15 min; the ratio of the precipitate to the red blood cell lysis solution in step five is 1:5; lysis at 25 DEG C for 15 min; and the centrifugal treatment in step six is performed at a speed of 2500 rpm for 20 min.

[0018] A placental mesenchymal stem cell is prepared by using a preparation method of the placental mesenchymal stem cell.

[0019] The placental mesenchymal stem cell is used in the preparation of a cell preparation for treating diseases, cosmetics, health products and drugs.

[0020] Example 2 The preparation method of the placental mesenchymal stem cell comprises the following steps: In step one, the placenta is preserved in a preservation solution, and the placenta is taken out from the sterile tray within 55 hours, the fresh red chorionic membrane tissue in the center of the placenta is cut, and the epithelial layer and calcified layer on both sides are removed; In step two, the placental tissue material containing mesenchymal stem cells is cut and processed to obtain the cut placental tissue material; In step three, the cut placental tissue material is digested by using a combined digestive enzyme to obtain a digested tissue mixture; In step four, the supernatant of the digested tissue mixture is taken and centrifuged to obtain a precipitate; In step five, the red blood cell lysis solution is added to the precipitate to obtain a mixture treated by red blood cell lysis; In step six, the mixture treated by red blood cell lysis is centrifuged to obtain a precipitate, which is the mesenchymal stem cell; In step seven, the mesenchymal stem cell is subjected to serum-free amplification and passage to obtain the placental mesenchymal stem cell; The preservation solution is a mixture of trehalose solution, erythrocyte storage solution, heparin calcium solution, human serum albumin injection solution, and Codonopsis pilosula cyclic peptide C. The preservation solution comprises the following components by weight: 28 parts of 0.2% trehalose solution, 24 parts of erythrocyte storage solution, 4 parts of 0.2% heparin calcium solution, 38 parts of human serum albumin injection solution, and 2 parts of Codonopsis pilosula cyclic peptide C. The 0.2% trehalose solution, erythrocyte storage solution, 0.2% heparin calcium solution, human serum albumin injection solution, and Codonopsis pilosula cyclic peptide C are mixed evenly at room temperature to form a mixture. The combined digestive enzyme mentioned in step three has a final concentration of 80 U / m³. The following ingredients were added simultaneously: collagenase IV, 0.008 wt% trypsin, 0.31 mg / ml DNase, and 0.07% trypsin-EDTA; digestion conditions included: digestion at 38°C for 18 hours; centrifugation conditions in step four: 2850 rpm for 14 minutes; the ratio of precipitate to erythrocyte lysis buffer in step five was 1:4.5; lysis was performed at 21°C for 14 minutes; and centrifugation conditions in step six: 2400 rpm for 18 minutes.

[0021] A type of placental mesenchymal stem cell is prepared using a method for preparing placental mesenchymal stem cells.

[0022] Applications of placental mesenchymal stem cells in cell preparations for treating diseases, cosmetics, health products, and pharmaceuticals.

[0023] Example 3 This invention discloses a method for preparing placental mesenchymal stem cells, the specific steps of which are as follows: Step 1: The placenta is preserved in a preservation solution and processed within 65 hours. The placenta is removed and placed in a sterile tray. The bright red chorionic membrane tissue in the center of the placenta is cut off, and the epithelial layer and calcified layer on both sides are removed. Step 2: The obtained placental tissue material containing mesenchymal stem cells is minced to obtain minced placental tissue material. Step 3: The shredded placental tissue material is digested with a combination of digestive enzymes to obtain a digested tissue mixture; Step 4: Take the supernatant suspension of the digested tissue mixture and centrifuge it to obtain a precipitate; Step 5: Add red blood cell lysis buffer to the precipitate to obtain a mixture treated with red blood cell lysis. Step six: The mixture treated with erythrocyte lysis is centrifuged to obtain a precipitate, which is the mesenchymal stem cell. Step 7: The mesenchymal stem cells are expanded and passaged in serum-free conditions to obtain placental mesenchymal stem cells. The preservation solution is a mixture of trehalose solution, red blood cell storage solution, calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclic peptide C; the preservation solution comprises the following components in parts by weight: 27 parts of 0.2% trehalose solution, 23 parts of red blood cell storage solution, 3 parts of 0.2% calcium heparin solution, 37 parts of human blood albumin injection and 1 part of radix pseudoginseng cyclic peptide C; the 0.2% trehalose solution, red blood cell storage solution, 0.2% calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclic peptide C are uniformly mixed at room temperature to form a mixed solution; the combined digestive enzyme in step three comprises collagenase IV with a final concentration of 65 U / ml, 0.007 wt% trypsin, 0.25 mg / ml DNase and 0.05% trypsin-EDTA; the collagenase IV, trypsin, DNase and trypsin-EDTA are simultaneously added; and the digestion conditions comprise: digestion at 37°C; digestion for 17 hours; the centrifugal treatment in step four is performed at a speed of 2400 rpm for 12 min; the ratio of the precipitate to the red blood cell lysis solution in step five is 1:3; lysis at 18°C for 13 min; and the centrifugal treatment in step six is performed at a speed of 2200 rpm for 16 min.

[0024] A placental mesenchymal stem cell is prepared by using a preparation method of the placental mesenchymal stem cell.

[0025] The placental mesenchymal stem cell is used in the preparation of a cell preparation for treating diseases, cosmetics, health products and drugs.

[0026] Example 4 The preparation method of the placental mesenchymal stem cell comprises the following steps: In step one, the placenta is preserved in a preservation solution, and the processing is completed within 72 hours; the placenta is taken out and placed in a sterile tray; the fresh red chorionic membrane tissue in the center of the placenta is cut; and the epithelial layer and calcified layer on both sides are removed; In step two, the placental tissue material containing mesenchymal stem cells is cut and processed to obtain cut and processed placental tissue material; In step three, the cut and processed placental tissue material is digested with a combined digestive enzyme to obtain a digested tissue mixture; In step four, the supernatant of the digested tissue mixture is taken and centrifuged to obtain a precipitate; In step five, red blood cell lysis solution is added to the precipitate to obtain a mixture treated by red blood cell lysis; In step six, the mixture treated by red blood cell lysis is centrifuged to obtain a precipitate, which is a generation of mesenchymal stem cells; In step seven, the generation of mesenchymal stem cells is subjected to serum-free amplification and passage to obtain placental mesenchymal stem cells; The storage solution is a mixture of trehalose solution, red blood cell storage solution, calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclopeptide C; the storage solution comprises the following components in parts by weight: 25 parts of 0.2% trehalose solution, 20 parts of red blood cell storage solution, 3 parts of 0.2% calcium heparin solution, 35 parts of human blood albumin injection, and 1 part of radix pseudoginseng cyclopeptide C; the 0.2% trehalose solution, red blood cell storage solution, 0.2% calcium heparin solution, human blood albumin injection and radix pseudoginseng cyclopeptide C are mixed uniformly at room temperature to form a mixed solution; the combined digestive enzyme in step three comprises collagenase IV with a final concentration of 50 U / ml, 0.005 wt% trypsin, 0.2 mg / ml DNase, and 0.03% trypsin-EDTA; the collagenase IV, trypsin, DNase and trypsin-EDTA are added simultaneously; and the digestion conditions comprise: digestion at 35°C; digestion for 15 hours; the centrifugal treatment in step four is performed at a speed of 2000 rpm for 10 min; the ratio of the precipitate to the red blood cell lysis solution in step five is 1:3; lysis at 15°C for 10 min; the centrifugal treatment in step six is performed at a speed of 2000 rpm for 15 min.

[0027] A placental mesenchymal stem cell is prepared by using a preparation method of the placental mesenchymal stem cell.

[0028] Application of the placental mesenchymal stem cell in cell preparations for treating diseases, cosmetics, health products and drugs.

[0029] Although the specific embodiments of the present application are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to the present embodiments without departing from the principles and essence of the present application, and the protection scope of the present application is only defined by the appended claims.

Claims

1. A method for preparing placental mesenchymal stem cells, characterized by: Specific steps As follows: Step one, the placenta is preserved in a preservation solution, and processed within 48-72 hours. The placenta is taken out and placed in a sterile tray. The fresh red chorionic membrane tissue in the center of the placenta is cut and the epithelial layer and calcified layer on both sides are removed. Step two, the placental tissue material containing mesenchymal stem cells obtained is cut and processed to obtain cut placental tissue material. Step three, the cut placental tissue material is digested with a combination of digestive enzymes to obtain a digested tissue mixture. Step four, the supernatant of the digested tissue mixture is taken and centrifuged to obtain a precipitate. Step five, red blood cell lysis solution is added to the precipitate to obtain a mixture treated by red blood cell lysis. Step six, the mixture treated by red blood cell lysis is centrifuged to obtain a precipitate, which is the generation of mesenchymal stem cells. Step seven, the generation of mesenchymal stem cells is subjected to serum-free amplification and passage to obtain placental mesenchymal stem cells.

2. The method of claim 1, wherein the placental mesenchymal stem cells are prepared by the following steps of: The preservation solution is a mixture of trehalose solution, red blood cell storage solution, calcium heparin solution, human blood albumin injection and radix notoginseng cyclic peptide C. ​ 3. The method for preparing placental mesenchymal stem cells according to claim 2, characterized in that: The preservation solution comprises the following components by weight: 25-30 parts of 0.2% trehalose solution, 20-25 parts of red blood cell storage solution, 3-5 parts of 0.2% calcium heparin solution, 35-40 parts of human blood albumin injection, and 1-2 parts of radix notoginseng cyclic peptide C. The 0.2% trehalose solution, red blood cell storage solution, 0.2% calcium heparin solution, human blood albumin injection, and radix notoginseng cyclic peptide C are mixed uniformly at room temperature to form a mixed solution.

4. The method for preparing placental mesenchymal stem cells according to claim 1, characterized in that: The combination of digestive enzymes in step three includes collagenase IV with a final concentration of 50U / ml-100U / ml, 0.005wt%-0.01wt% trypsin, 0.2-0.35mg / ml DNAase, and 0.03-0.08% trypsin-EDTA.

5. The method for preparing placental mesenchymal stem cells according to claim 4, characterized in that: The collagenase IV, trypsin, DNAase and trypsin-EDTA are added simultaneously, and the digestion conditions include: digestion at 37±2℃; digestion for 15-20 hours.

6. The method for preparing placental mesenchymal stem cells according to claim 1, characterized in that: The centrifugation conditions in step four are: 2000-3000rpm for 10-15min.

7. The method for preparing placental mesenchymal stem cells according to claim 1, characterized in that: The ratio of precipitate to red blood cell lysis solution in step five is 1:3-5; lysis at 15-25℃ for 10-15min.

8. The method of claim 1, wherein the placental mesenchymal stem cells are prepared by the following steps of: The centrifugation conditions in step six are: 2000-2500rpm for 15-20min. ​ 9. A placental mesenchymal stem cell, characterized by being A method for preparing placental mesenchymal stem cells according to any one of claims 1-8 is used.

10. The placental mesenchymal stem cells of claim 9 for use in the preparation of cells for treating diseases, cosmetics, health products, and drugs.