Amniotic mesenchymal stem cell culture medium and method for isolated culture of amniotic mesenchymal stem cells

Abstract

The invention discloses an amniotic mesenchymal stem cell culture medium and a method for isolated culture of amniotic mesenchymal stem cells. The amniotic mesenchymal stem cell culture medium consists of fibronectin, an epidermal growth factor (EGF), a basic fibroblast growth factor (bFGF), fetal calf serum and a basal culture medium. According to the method, fibronectin and growth factors are added into the culture medium, the climbing-out time of the amniotic mesenchymal stem cells can be greatly shortened, the cell yield is remarkably increased, cell damage is low, the motility rate can reach 95% or above, and the purity of the amniotic mesenchymal stem cells is effectively improved by conducting isolated culture through the improved tissue adhesion method.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, in particular to an amniotic mesenchymal stem cell culture medium and a method for isolating and culturing amniotic mesenchymal stem cells. Background Art

[0002] The amniotic membrane is primarily composed of mesenchymal and epithelial cell layers. Common isolation methods include conventional tissue attachment and enzymatic digestion, but each has its own advantages and disadvantages. Conventional tissue attachment involves attaching amniotic tissue directly to a culture dish surface, leveraging the cells' natural ability to adhere to the surface. This method has the advantage of avoiding the use of enzymes such as collagenase and DNase, thereby reducing potential damage to the cells. However, this method also has significant disadvantages: the yield of mesenchymal stem cells is low, and the isolation process is time-consuming, making it difficult to meet the needs of large-scale industrialization. In contrast, enzymatic digestion uses specific enzymes (such as collagenase and DNase) to break down the extracellular matrix of the amniotic tissue, thereby releasing the mesenchymal stem cells. This method offers higher isolation efficiency and yields a higher number of cells. However, currently used enzymatic digestion methods also have some drawbacks. First, the use of enzymes may affect cell surface markers and biological properties. Second, the enzymatic digestion process requires precise control of enzyme concentration and digestion time, otherwise it may cause cell damage or death. Furthermore, the enzymatic digestion method is relatively expensive and the operation is complex.

[0003] Therefore, each method for isolating amniotic mesenchymal stem cells has its own advantages and disadvantages. It is of great significance to develop a more efficient and low-cost isolation method to promote the widespread application of amniotic mesenchymal stem cells in the medical field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention provides an amniotic membrane mesenchymal stem cell culture medium and a method for isolating and culturing amniotic membrane mesenchymal stem cells. The present amniotic membrane mesenchymal stem cell culture medium contains fibronectin and growth factors, significantly shortening the time it takes for the cells to crawl out and significantly increasing cell yield. Cell damage is minimized, with a viability exceeding 95%. Furthermore, the present invention utilizes a modified tissue adherence method for isolation and culturing, effectively increasing the purity of the amniotic membrane mesenchymal stem cells.

[0005] The technical solutions of the present invention are as follows:

[0006] The invention discloses an amniotic membrane mesenchymal stem cell culture medium, which consists of fibronectin, epidermal growth factor EGF, basic fibroblast growth factor bFGF, fetal bovine serum and basal culture medium.

[0007] Furthermore, the concentration of the fibronectin is 0.04-0.1 mg / mL; the concentration of the epidermal growth factor EGF is 2.5 ng / mL; the concentration of the basic fibroblast growth factor bFGF is 2.5 mg / mL; and the volume percentage concentration of the fetal bovine serum is 10-15%.

[0008] Furthermore, the basal culture medium is α-MEM basal culture medium.

[0009] A method for preparing the amniotic membrane mesenchymal stem cell culture medium is characterized in that fibronectin, fetal bovine serum and basal culture medium are mixed to obtain the amniotic membrane mesenchymal stem cell culture medium.

[0010] A method for isolating and culturing amniotic mesenchymal stem cells, comprising the following steps:

[0011] (1) Collection and cleaning: The perinatal placenta tissue was collected, the amniotic membrane was peeled off and placed in a culture dish, and the tissue was washed with PBS buffer and blood was removed by scraping;

[0012] (2) Digestion: Cut the amniotic membrane into pieces of 1-2 cm 2 The fragments were digested with trypsin solution, filtered through a cell mesh, rinsed 2 to 3 times with PBS buffer, and filtered again to collect the amniotic tissue fragments;

[0013] (3) Cell culture: The amniotic tissue fragments are evenly distributed in a culture dish, air-dried, and then added to the amniotic mesenchymal stem cell culture medium for culture. Fresh amniotic mesenchymal stem cell culture medium is supplemented or replaced every 3-4 days. After 6-8 days of culture, the amniotic tissue fragments are removed and the culture is continued. When the cell clone fusion degree is ≥80%, the amniotic mesenchymal stem cells are picked and placed in a centrifuge tube. After centrifugation, the supernatant is discarded and the supernatant is added with α-MEM basal culture medium containing 10-15% fetal bovine serum by volume to resuspend. After re-inoculation and culture for 3-4 days, the amniotic mesenchymal stem cells are obtained.

[0014] Furthermore, in step (2), the concentration of trypsin is 0.1 g / L, the volume mass ratio of the trypsin solution to the amniotic tissue fragments is 1-2 mL:1 g; the digestion temperature is 37° C., the shaking speed is 150-200 rpm, and the time is 45-60 min.

[0015] Furthermore, in step (2), the mesh size of the cell sieve is 100 mesh.

[0016] Furthermore, in step (3), the cell culture conditions are 37° C. and a volume concentration of 5% CO 2 ; and the number of the amniotic membrane tissue fragments in the culture dish is 40 to 60 per 150 mm culture dish.

[0017] Furthermore, in step (3), the air-drying temperature is 20-25° C., and the time is 30-45 min; the centrifugal speed is 1500 rpm, and the time is 5 min.

[0018] Furthermore, in step (3), the inoculation density is 4000-6000 cells / cm 2 .

[0019] The beneficial technical effects of the present invention are:

[0020] The present invention discloses an amniotic membrane mesenchymal stem cell culture medium, to which fibronectin and growth factors are added, and is used for isolating and culturing amniotic membrane mesenchymal stem cells, which can significantly shorten the crawling time of amniotic tissue cells and improve the yield and viability of primary cells. The present invention adopts a clonal cell picking method to select cells from primary amniotic membrane mesenchymal stem cells, which can significantly improve the cell purity. The method of isolating and culturing amniotic membrane mesenchymal stem cells of the present invention can significantly improve the yield and purity of primary cells, while causing little damage to the cells, and achieving a cell viability of more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the expression of surface markers of high-purity P1 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0023] The technology of the present invention uses trypsin digestion to remove most of the amniotic epithelial cell layer to a large extent. Then, the optimized tissue adhesion method is used for culture. The key technologies of the optimized tissue adhesion method include: 1. Adding fibronectin, epidermal growth factor EGF, and basic fibroblast growth factor bFGF to promote the crawling out of amniotic mesenchymal stem cells, thereby greatly increasing the primary yield. 2. When the primary cells are harvested, the stem cells with typical mesenchymal morphology (fibroblast-like cells, swirling adherent growth) are picked using the cloning method to continue to culture, and high-purity mesenchymal stem cells can be obtained.

[0024] Example 1

[0025] A method for preparing an amniotic membrane mesenchymal stem cell culture medium is provided, wherein the culture medium is prepared by adding the following components to an α-MEM basal culture medium and mixing the mixture. The culture medium comprises the following components:

[0026] Fibronectin: 0.04 mg / mL;

[0027] Epidermal growth factor (EGF): 2.5 ng / mL;

[0028] Basic fibroblast growth factor (bFGF): 2.5 mg / mL;

[0029] Fetal bovine serum FBS: 15% (volume percentage concentration):

[0030] Basal culture medium: α-MEM basal medium.

[0031] The prepared amniotic mesenchymal stem cell culture medium is used to culture amniotic mesenchymal stem cells, comprising the following steps:

[0032] (1) Collection and cleaning: Healthy perinatal placental tissue was collected, the amniotic membrane was peeled off and placed in a culture dish, and the tissue was washed with PBS buffer and blood was removed using a cell scraper;

[0033] (2) Digestion: Use surgical scissors to cut the amniotic membrane into pieces of 1-2 cm in size. 2 Weigh 5 g of amniotic membrane tissue fragments into a 50 mL centrifuge tube, add 7.5 mL of 0.1 g / L trypsin solution, and digest at 37 ° C and 150 rpm for 60 min. After digestion, filter through a 100-mesh cell sieve, rinse the amniotic membrane tissue fragments three times with PBS buffer, and filter again to collect the amniotic membrane tissue fragments;

[0034] (3) Cell culture: 50 amniotic membrane tissue fragments were evenly distributed in a 150 mm culture dish, air-dried at 20°C for 30 min, and then 10 mL of the prepared amniotic membrane mesenchymal stem cell culture medium was added. The culture was incubated at 37°C in a 5% CO2 incubator. On the 4th day of culture, 15 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was added. On the 8th day of culture, a large number of spindle-shaped adherent cells were observed crawling out of the tissue under microscopic examination. The amniotic membrane tissue fragments and the old culture medium were removed, and 30 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was replaced and culture was continued. On the 11th day of culture, 21 cell clones with a cell fusion degree of more than 80% were observed under microscopic examination in the culture dish. The amniotic membrane mesenchymal stem cells were picked and placed in a centrifuge tube. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and 5 mL of α-MEM basal culture medium containing 15% fetal bovine serum by volume was added for re-suspending to obtain P0 cells. The cells were then passed through Countstar Rigel The cell count using an S2 fluorescence cell counter showed that the P0 cell yield in the culture dish was 9.5×10 5 , the activity rate is 97.0%. Then according to 5000 / cm 2 The cells were re-seeded into culture flasks at a seeding density of 100 μg / mL and cultured for 3 to 4 days to obtain highly pure P1 generation amniotic mesenchymal stem cells.

[0035] The cell viability of the high-purity P1 generation amniotic mesenchymal stem cells obtained in this example was detected by Countstar Rigel S2 fluorescence cell counter, and was 98.5%. In addition, the expression of surface markers of the high-purity P1 generation amniotic mesenchymal stem cells obtained in this example was detected by BD FACSAria Fusion flow cytometer, including positive markers (CD73, CD90, CD105), and negative markers: CD11b, HLA-DR, CD19, CD34, CD45. Figure 1 As shown. Figure 1 It can be seen from the results that the purity of the P1 generation amniotic mesenchymal stem cells obtained in this example reached over 99.5%.

[0036] Example 2

[0037] A method for preparing an amniotic membrane mesenchymal stem cell culture medium is provided, wherein the culture medium is prepared by adding the following components to an α-MEM basal culture medium and mixing the mixture. The culture medium comprises the following components:

[0038] Fibronectin: 0.06 mg / mL;

[0039] Epidermal growth factor (EGF): 2.5 ng / mL;

[0040] Basic fibroblast growth factor (bFGF): 2.5 mg / mL;

[0041] Fetal bovine serum FBS: 12% (volume percentage concentration):

[0042] Basal culture medium: α-MEM basal medium.

[0043] The prepared amniotic mesenchymal stem cell culture medium is used to culture amniotic mesenchymal stem cells, comprising the following steps:

[0044] (1) Collection and cleaning: Healthy perinatal placental tissue was collected, the amniotic membrane was peeled off and placed in a culture dish, and the tissue was washed with PBS buffer and blood was removed using a cell scraper;

[0045] (2) Digestion: Use surgical scissors to cut the amniotic membrane into pieces of 1-2 cm in size. 2 Weigh 5 g of amniotic membrane tissue fragments into a 50 mL centrifuge tube, add 5 mL of 0.1 g / L trypsin solution, and digest at 37°C and 180 rpm for 50 min. After digestion, filter through a 100-mesh cell sieve, rinse the amniotic membrane tissue fragments twice with PBS buffer, and filter again to collect the amniotic membrane tissue fragments;

[0046] (3) Cell culture: 40 amniotic membrane tissue fragments were evenly distributed in a 150 mm culture dish, air-dried at 25°C for 40 min, and then 10 mL of the prepared amniotic membrane mesenchymal stem cell culture medium was added. The cells were cultured in a 37°C incubator containing 5% CO2. On the 4th day of culture, 15 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was added. On the 8th day of culture, a large number of spindle-shaped adherent cells were observed crawling out of the tissue under microscopic examination. The amniotic membrane tissue fragments and the old culture medium were removed, and 30 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was replaced and culture was continued. On the 11th day of culture, 18 cell clones with a cell fusion degree of more than 80% were observed under microscopic examination in the culture dish. The amniotic membrane mesenchymal stem cells were picked and placed in a centrifuge tube. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and 5 mL of α-MEM basal culture medium containing 12% fetal bovine serum by volume was added for re-suspending to obtain P0 cells. The cells were then passed through Countstar Rigel The cell count was performed using an S2 fluorescence cell counter, and the yield of P0 cells in each culture dish was 8.2×10 5 , the activity rate is 96.7%. Then according to 4000 / cm 2 The cells were re-seeded into culture flasks at a seeding density of 100 μg / mL and cultured for 3 to 4 days to obtain highly pure P1 generation amniotic mesenchymal stem cells.

[0047] The cell viability of the highly pure P1 amniotic mesenchymal stem cells obtained in this example was determined to be 98.2% using a Countstar Rigel S2 fluorescence cell counter. Furthermore, the expression of surface markers for the highly pure P1 amniotic mesenchymal stem cells obtained in this example, including positive markers (CD73, CD90, and CD105) and negative markers (CD11b, HLA-DR, CD19, CD34, and CD45), was determined using a BD FACSAria Fusion flow cytometer. The results demonstrated that the purity of the P1 amniotic mesenchymal stem cells obtained in this example reached greater than 99.5%.

[0048] Example 3

[0049] A method for preparing an amniotic membrane mesenchymal stem cell culture medium is provided, wherein the culture medium is prepared by adding the following components to an α-MEM basal culture medium and mixing the mixture. The culture medium comprises the following components:

[0050] Fibronectin: 0.1 mg / mL;

[0051] Epidermal growth factor (EGF): 2.5 ng / mL;

[0052] Basic fibroblast growth factor (bFGF): 2.5 mg / mL;

[0053] Fetal bovine serum FBS: 10% (volume percentage concentration):

[0054] Basal culture medium: α-MEM basal medium.

[0055] The prepared amniotic mesenchymal stem cell culture medium is used to culture amniotic mesenchymal stem cells, comprising the following steps:

[0056] (1) Collection and cleaning: Healthy perinatal placental tissue was collected, the amniotic membrane was peeled off and placed in a culture dish, and the tissue was washed with PBS buffer and blood was removed using a cell scraper;

[0057] (2) Digestion: Use surgical scissors to cut the amniotic membrane into pieces of 1-2 cm in size. 2 Weigh 5 g of amniotic membrane tissue fragments into a 50 mL centrifuge tube, add 10 mL of 0.1 g / L trypsin solution, and digest at 37 ° C and 200 rpm for 45 minutes. After digestion, filter through a 100-mesh cell sieve, rinse the amniotic membrane tissue fragments three times with PBS buffer, and filter again to collect the amniotic membrane tissue fragments;

[0058] (3) Cell culture: 60 amniotic membrane tissue fragments were evenly distributed in a 150 mm culture dish, air-dried at 20°C for 45 min, and then 10 mL of the prepared amniotic membrane mesenchymal stem cell culture medium was added. The cells were cultured in a 37°C incubator containing 5% CO2. On the 4th day of culture, 15 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was added. On the 8th day of culture, a large number of spindle-shaped adherent cells were observed crawling out of the tissue under microscopic examination. The amniotic membrane tissue fragments and the old culture medium were removed, and 30 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was replaced and culture was continued. On the 11th day of culture, 26 cell clones with a cell fusion degree of more than 80% were observed under microscopic examination in the culture dish. The amniotic membrane mesenchymal stem cells were picked and placed in a centrifuge tube. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and 5 mL of α-MEM basal culture medium containing 10% fetal bovine serum by volume was added for re-suspending to obtain P0 cells. The cells were then passed through Countstar Rigel The cell count using an S2 fluorescence cell counter showed that the P0 cell yield in each culture dish was 9.8×10 5 , the activity rate is 95.8%. Then according to 6000 / cm 2 The cells were re-seeded into culture flasks at a seeding density of 100 μg / mL and cultured for 3 to 4 days to obtain highly pure P1 generation amniotic mesenchymal stem cells.

[0059] The cell viability of the highly pure P1 amniotic mesenchymal stem cells obtained in this example was determined to be 98.7% using a Countstar Rigel S2 fluorescence cell counter. Furthermore, the expression of surface markers for the highly pure P1 amniotic mesenchymal stem cells obtained in this example, including positive markers (CD73, CD90, and CD105) and negative markers (CD11b, HLA-DR, CD19, CD34, and CD45), was determined using a BD FACSAria Fusion flow cytometer. The results demonstrated that the purity of the P1 amniotic mesenchymal stem cells obtained in this example reached greater than 99.5%.

[0060] Comparative Example 1

[0061] A method for preparing an amniotic membrane mesenchymal stem cell culture medium is provided, wherein the culture medium is prepared by adding the following components to an α-MEM basal culture medium and mixing the mixture. The culture medium comprises the following components:

[0062] Fibronectin: 0.04 mg / mL;

[0063] Fetal bovine serum FBS: 15% (volume percentage concentration):

[0064] Basal culture medium: α-MEM basal medium.

[0065] The prepared amniotic mesenchymal stem cell culture medium is used to culture amniotic mesenchymal stem cells, comprising the following steps:

[0066] (1) Collection and cleaning: Same as step (1) in Example 1.

[0067] (2) Digestion treatment: Same as step (2) in Example 1.

[0068] (3) Cell culture: 50 amniotic membrane tissue fragments were evenly distributed in a 150 mm culture dish, air-dried at 20°C for 30 min, and then 10 mL of the prepared amniotic membrane mesenchymal stem cell culture medium was added. The cells were cultured in a 37°C incubator containing 5% CO2. On the 4th day of culture, 15 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was added. On the 8th day of culture, a large number of spindle-shaped adherent cells were observed crawling out of the tissue under microscopic examination. The amniotic membrane tissue fragments and the old culture medium were removed, and 30 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was replaced and culture was continued. On the 11th day of culture, 16 cell clones with a cell fusion degree of more than 80% were observed under microscopic examination in the culture dish. The amniotic membrane mesenchymal stem cells were picked and placed in a centrifuge tube. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and 5 mL of α-MEM basal culture medium containing 15% fetal bovine serum by volume was added for re-suspending to obtain P0 cells. The cells were then passed through Countstar Rigel The cell count using an S2 fluorescence cell counter showed that the P0 cell yield in the culture dish was 5.5×10 5 , the activity rate is 95.2%. Then according to 5000 / cm 2 The cells were re-seeded into culture flasks at a seeding density of 100 μg / mL and cultured for 3 to 4 days to obtain highly pure P1 generation amniotic mesenchymal stem cells.

[0069] The cell viability of the highly pure P1 amniotic mesenchymal stem cells obtained in this example was determined to be 98.4% using a Countstar Rigel S2 fluorescence cell counter. Furthermore, the expression of surface markers for the highly pure P1 amniotic mesenchymal stem cells obtained in this example, including positive markers (CD73, CD90, and CD105) and negative markers (CD11b, HLA-DR, CD19, CD34, and CD45), was determined using a BD FACSAria Fusion flow cytometer. The results showed that the purity of the P1 amniotic mesenchymal stem cells obtained in this comparative example reached over 99%.

[0070] Comparative Example 2

[0071] A method for preparing an amniotic membrane mesenchymal stem cell culture medium is provided, wherein the culture medium is prepared by adding the following components to an α-MEM basal culture medium and mixing the mixture. The culture medium comprises the following components:

[0072] Fetal bovine serum FBS: 15% (volume percentage concentration):

[0073] Basal culture medium: α-MEM basal medium.

[0074] The prepared amniotic mesenchymal stem cell culture medium is used to culture amniotic mesenchymal stem cells, comprising the following steps:

[0075] (1) Collection and cleaning: Same as step (1) in Example 1.

[0076] (2) Digestion treatment: Same as step (2) in Example 1.

[0077] (3) Cell culture: 50 amniotic membrane tissue fragments were evenly distributed in a 150 mm culture dish, air-dried at 20°C for 30 min, and then 10 mL of the prepared amniotic membrane mesenchymal stem cell culture medium was added. The cells were cultured in a 37°C incubator containing 5% CO2. On the 4th day of culture, 15 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was added. On the 8th day of culture, a few spindle-shaped adherent cells were observed crawling out of the tissue under microscopic examination. The amniotic membrane tissue fragments and the old culture medium were removed, and 30 mL of fresh prepared amniotic membrane mesenchymal stem cell culture medium was replaced and culture was continued. On the 11th day of culture, 11 cell clones with a cell fusion degree of more than 80% were observed under microscopic examination in the culture dish. The amniotic membrane mesenchymal stem cells were picked and placed in a centrifuge tube. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and 5 mL of α-MEM basal culture medium containing 15% fetal bovine serum by volume was added for re-suspending to obtain P0 cells. The cells were then passed through Countstar Rigel The cell count using an S2 fluorescence cell counter showed that the P0 cell yield in the culture dish was 2.0×10 5 , the activity rate is 93.4%. Then according to 5000 / cm 2 The cells were re-seeded into culture flasks at a seeding density of 100 μg / mL and cultured for 3 to 4 days to obtain highly pure P1 generation amniotic mesenchymal stem cells.

[0078] The cell viability of the highly pure P1 amniotic mesenchymal stem cells obtained in this example was determined to be 95.6% using a Countstar Rigel S2 fluorescence cell counter. Furthermore, the expression of surface markers for the highly pure P1 amniotic mesenchymal stem cells obtained in this example, including positive markers (CD73, CD90, and CD105) and negative markers (CD11b, HLA-DR, CD19, CD34, and CD45), was determined using a BD FACSAria Fusion flow cytometer. The results showed that the purity of the P1 amniotic mesenchymal stem cells obtained in this comparative example reached over 99%.

[0079] Table 1 Yield and viability of amniotic mesenchymal stem cells prepared in each embodiment / comparative example

[0080]

[0081]

[0082] As shown in Table 1, the present invention can significantly shorten the crawling time of amniotic tissue cells and improve the yield and viability of primary cells by adding fibronectin and growth factors to the culture medium for isolating and culturing amniotic mesenchymal stem cells.

[0083] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment. It is understood that other improvements and variations directly derived or imagined by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the scope of protection of the present invention.

Claims

1. An amniotic membrane mesenchymal stem cell culture medium, characterized in that The amniotic membrane mesenchymal stem cell culture medium consists of fibronectin, epidermal growth factor EGF, basic fibroblast growth factor bFGF, fetal bovine serum and basal culture medium.

2. The amniotic membrane mesenchymal stem cell culture medium according to claim 1, characterized in that The concentration of the fibronectin is 0.04-0.1 mg / mL; the concentration of the epidermal growth factor EGF is 2.5 ng / mL; the concentration of the basic fibroblast growth factor bFGF is 2.5 mg / mL; and the volume percentage concentration of the fetal bovine serum is 10-15%.

3. The amniotic membrane mesenchymal stem cell culture medium according to claim 1, characterized in that The basic culture medium is α-MEM basic culture medium.

4. A method for preparing the amniotic membrane mesenchymal stem cell culture medium according to any one of claims 1 to 4, characterized in that: Fibronectin, fetal bovine serum and basal culture medium are mixed to obtain the amniotic membrane mesenchymal stem cell culture medium.

5. A method for isolating and culturing amniotic mesenchymal stem cells, characterized in that: The method comprises the following steps: (1) Collection and cleaning: The perinatal placenta tissue was collected, the amniotic membrane was peeled off and placed in a culture dish, and the tissue was washed with PBS buffer and blood was removed by scraping; (2) Digestion: Cut the amniotic membrane into pieces of 1-2 cm 2 The fragments were digested with trypsin solution, filtered through a cell mesh, rinsed 2 to 3 times with PBS buffer, and filtered again to collect the amniotic tissue fragments; (3) Cell culture: The amniotic tissue fragments are evenly distributed in a culture dish, air-dried, and then added to the amniotic mesenchymal stem cell culture medium described in any one of claims 1 to 3 for culture. Fresh amniotic mesenchymal stem cell culture medium is supplemented or replaced every 3-4 days. After 6-8 days of culture, the amniotic tissue fragments are removed and the culture is continued. When the cell clone fusion degree is ≥80%, the amniotic mesenchymal stem cells are picked and placed in a centrifuge tube. After centrifugation, the supernatant is discarded and the supernatant is added with α-MEM basal culture medium containing 10-15% fetal bovine serum by volume to resuspend. After re-inoculation and culture for 3-4 days, the amniotic mesenchymal stem cells are obtained.

6. The method according to claim 5, characterized in that In step (2), the concentration of trypsin is 0.1 g / L, and the volume mass ratio of the trypsin solution to the amniotic tissue fragments is 1-2 mL:1 g; the digestion temperature is 37° C., the shaking speed is 150-200 rpm, and the time is 45-60 min.

7. The method according to claim 5, characterized in that In step (2), the mesh size of the cell sieve is 100 mesh.

8. The method according to claim 5, characterized in that In step (3), the cell culture conditions are 37° C. and 5% CO 2 by volume; the number of the amniotic membrane tissue fragments in the culture dish is 40 to 60 per 150 mm culture dish.

9. The method according to claim 5, characterized in that In step (3), the air-drying temperature is 20-25° C., and the time is 30-45 min; the centrifugal speed is 1500 rpm, and the time is 5 min.

10. The method according to claim 5, characterized in that In step (3), the inoculation density is 4000-6000 cells / cm 2 .

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