Extraction method of amniotic mesenchymal stem cells

Abstract

The invention discloses an extraction method of amniotic mesenchymal stem cells, which comprises the following steps: separating an amniotic membrane from a placenta, and then cleaning and cutting the amniotic membrane to obtain an amniotic membrane sample; the amnion sample is placed in a mixed solution of type I collagenase, trypsin and papain to be digested and cleaned, and residual amnion tissue is obtained; placing the residual amniotic tissues in a mixed solution of type IV collagenase and DNA enzyme for digestion treatment, sieving to remove tissues and cell clusters, and centrifuging filtrate to obtain the amniotic mesenchymal stem cells. According to the extraction method of the amniotic mesenchymal stem cells, through limitation of a specific process, impurity components can be sufficiently removed, the mesenchymal stem cells can be sufficiently separated and extracted from amniotic membranes, and the separated mesenchymal stem cells have high purity and yield.

Description

Technical Field

[0001] The invention relates to the technical field of stem cell extraction, and in particular to a method for extracting amniotic mesenchymal stem cells. Background Art

[0002] The amnion is a thin membrane composed of a single layer of epithelial cells connected to each other. The single layer of epithelial cells has the function of secreting amniotic fluid. As the embryo grows and develops, the amnion and the chorion are closely attached to form the fetal membrane. As the amniotic cavity gradually expands, the fetal membrane becomes a translucent film with no blood vessels and is tough (the fetal membrane is also the cyst wall of the amniotic cavity). The liquid filled in the amniotic cavity is amniotic fluid. The amnion only covers the fetal surface of the placenta and does not penetrate into the placental tissue. As the pregnancy progresses, the amniotic cavity gradually expands and occupies the entire uterine cavity. The amnion is the innermost layer of the fetal membrane. It is a translucent film that is connected to the amniotic layer covering the placenta and umbilical cord.

[0003] Amniotic mesenchymal stem cells are a multifunctional stem cell with the ability to self-renew and regenerate. They can differentiate into various types of cells under certain conditions and have a wide range of medical application potential. The main effects of amniotic mesenchymal stem cells include: Tissue repair and regeneration: Amniotic mesenchymal stem cells can differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes, thereby promoting tissue repair and regeneration. This plays an important role in fracture healing, cartilage repair, and skin regeneration. Immunomodulation: Amniotic mesenchymal stem cells have immunomodulatory functions, which can inhibit inflammatory responses and regulate the activity of immune cells. This helps to reduce the occurrence and development of autoimmune diseases and reduce rejection reactions in situations such as organ transplantation. Angiogenesis: These stem cells have the ability to promote the formation of new blood vessels, which can improve blood circulation and promote tissue repair. This is very important in the treatment of cardiovascular diseases, ischemic diseases, and vascular damage. Anti-tumor effects: Recent studies have shown that amniotic mesenchymal stem cells may have anti-tumor effects, which can inhibit tumor growth and spread and improve the efficacy of chemotherapy and radiotherapy.

[0004] At present, the isolation of amniotic mesenchymal stem cells can generally be obtained through tissue culture and enzymatic hydrolysis. However, the amniotic mesenchymal stem cells isolated by existing separation methods contain impurities and have low purity. In addition, due to incomplete digestion, there is a problem of low yield.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for extracting amniotic mesenchymal stem cells, which can fully separate and extract mesenchymal stem cells from amniotic membrane, and the separated mesenchymal stem cells have high purity and yield.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The first aspect of the present invention provides a method for extracting amniotic mesenchymal stem cells, the extraction method comprising the following steps:

[0009] (a) separating the amniotic membrane from the placenta, washing it, and cutting it into pieces to obtain an amniotic membrane sample;

[0010] (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue;

[0011] (c) placing the remaining amniotic membrane tissue in a mixture of type IV collagenase and DNase for digestion, sieving to remove the tissue and cell clusters, and centrifuging the filtrate to obtain the amniotic membrane mesenchymal stem cells.

[0012] Preferably, in step (a), the solvent used for washing is physiological saline or PBS solution containing 1% dual antibody.

[0013] Preferably, in step (b), in the mixed solution of type I collagenase, trypsin and papain, the concentration of type I collagenase is 0.5-1 mg / mL, the concentration of trypsin is 2-4 mg / mL, and the concentration of papain is 1-3 mg / mL.

[0014] Preferably, in step (b), the digestion temperature is 37° C. and the digestion time is 40 to 60 minutes.

[0015] Preferably, in step (c), the concentration of type IV collagenase in the mixed solution of type IV collagenase and DNase is 3-6 mg / mL, and the concentration of DNase is 1-2 mg / mL.

[0016] Preferably, in step (c), the digestion treatment temperature is 37° C. and the time is 60 to 80 minutes.

[0017] Preferably, in step (c), the mesh size of the sieve used for sieving is 140 mesh.

[0018] Preferably, in step (c), the centrifugal speed is 1000-1200 rpm and the time is 4-8 min.

[0019] Preferably, the step (c) further comprises inoculating the amniotic mesenchymal stem cells into a culture medium for culturing.

[0020] Compared with the prior art, the beneficial effects of the present invention include at least:

[0021] The method for extracting amniotic mesenchymal stem cells of the present invention can fully remove impurities through the limitation of specific processes, and can fully separate and extract mesenchymal stem cells from the amniotic membrane, so that the separated mesenchymal stem cells have higher purity and yield. DETAILED DESCRIPTION

[0022] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0023] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0024] The embodiment of the present invention provides a method for extracting amniotic mesenchymal stem cells, the extraction method comprising the following steps:

[0025] (a) separating the amniotic membrane from the placenta, washing it, and cutting it into pieces to obtain an amniotic membrane sample;

[0026] (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue;

[0027] (c) placing the remaining amniotic membrane tissue in a mixture of type IV collagenase and DNase for digestion, sieving to remove the tissue and cell clusters, and centrifuging the filtrate to obtain the amniotic membrane mesenchymal stem cells.

[0028] The method for extracting amniotic mesenchymal stem cells of the present invention can fully remove impurities through the limitation of specific processes, and can fully separate and extract mesenchymal stem cells from the amniotic membrane, so that the separated mesenchymal stem cells have higher purity and yield.

[0029] In some embodiments, in step (a), the solvent used for washing is physiological saline or PBS solution containing 1% dual antibody.

[0030] In some embodiments, in step (b), in the mixed solution of type I collagenase, trypsin and papain, the concentration of type I collagenase is 0.5-1 mg / mL, the concentration of trypsin is 2-4 mg / mL, and the concentration of papain is 1-3 mg / mL.

[0031] In some embodiments, in step (b), the digestion temperature is 37° C. and the digestion time is 40 to 60 minutes.

[0032] In some embodiments, in step (c), the concentration of type IV collagenase in the mixed solution of type IV collagenase and DNase is 3-6 mg / mL, and the concentration of DNase is 1-2 mg / mL.

[0033] In some embodiments, in step (c), the digestion treatment temperature is 37° C. and the time is 60 to 80 minutes.

[0034] In some embodiments, in step (c), the mesh size of the sieve used for sieving is 140 mesh.

[0035] In some embodiments, in step (c), the centrifugal speed is 1000-1200 rpm and the time is 4-8 min.

[0036] In some embodiments, the step (c) further comprises inoculating the amniotic mesenchymal stem cells into a culture medium for culturing.

[0037] The technical solution of the present invention is further described in detail below through specific embodiments.

[0038] Example 1

[0039] This embodiment is a method for extracting amniotic mesenchymal stem cells, which comprises the following steps:

[0040] (a) separating the amniotic membrane from the placenta, washing it three times, and cutting it into pieces to obtain an amniotic membrane sample, wherein the washing solvent is physiological saline;

[0041] (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue, wherein the concentration of type I collagenase in the mixture of type I collagenase, trypsin and papain is 0.5 mg / mL, the concentration of trypsin is 4 mg / mL, and the concentration of papain is 1 mg / mL; the digestion temperature is 37° C., and the time is 40 min;

[0042] (c) placing the remaining amniotic tissue in a mixture of type IV collagenase and DNase for digestion, removing tissue and cell clusters through a 140-mesh sieve, and centrifuging the filtrate to obtain the amniotic mesenchymal stem cells, wherein the concentration of type IV collagenase in the mixture of type IV collagenase and DNase is 3 mg / mL, and the concentration of DNase is 2 mg / mL; the digestion temperature is 37° C., the time is 60 min; the centrifugal speed is 11200 rpm, and the time is 4 min.

[0043] Example 2

[0044] This embodiment is a method for extracting amniotic mesenchymal stem cells, which comprises the following steps:

[0045] (a) separating the amniotic membrane from the placenta, washing it three times, and cutting it into pieces to obtain an amniotic membrane sample, wherein the washing solvent is physiological saline;

[0046] (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue, wherein the concentration of type I collagenase in the mixture of type I collagenase, trypsin and papain is 1 mg / mL, the concentration of trypsin is 2 mg / mL, and the concentration of papain is 3 mg / mL; the digestion temperature is 37° C., and the time is 60 min;

[0047] (c) placing the remaining amniotic tissue in a mixture of type IV collagenase and DNase for digestion, removing tissue and cell clusters through a 140-mesh sieve, and centrifuging the filtrate to obtain the amniotic mesenchymal stem cells, wherein the concentration of type IV collagenase in the mixture of type IV collagenase and DNase is 6 mg / mL, and the concentration of DNase is 1 mg / mL; the digestion treatment temperature is 37° C., the time is 60 to 80 min; the centrifugal speed is 1000 rpm, and the time is 8 min.

[0048] Example 3

[0049] This embodiment is a method for extracting amniotic mesenchymal stem cells, which comprises the following steps:

[0050] (a) separating the amniotic membrane from the placenta, washing it three times, and cutting it into pieces to obtain an amniotic membrane sample, wherein the washing solvent is physiological saline;

[0051] (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue, wherein the concentration of type I collagenase in the mixture of type I collagenase, trypsin and papain is 0.8 mg / mL, the concentration of trypsin is 3 mg / mL, and the concentration of papain is 2 mg / mL; the digestion temperature is 37° C., and the time is 50 min;

[0052] (c) placing the remaining amniotic tissue in a mixture of type IV collagenase and DNase for digestion, removing tissue and cell clusters through a 140-mesh sieve, and centrifuging the filtrate to obtain the amniotic mesenchymal stem cells, wherein the concentration of type IV collagenase in the mixture of type IV collagenase and DNase is 4 mg / mL, and the concentration of DNase is 1.5 mg / mL; the digestion temperature is 37° C., the time is 70 min; the centrifugal speed is 1000 rpm, and the time is 6 min.

[0053] Comparative Example 1

[0054] This comparative example is a method for extracting amniotic mesenchymal stem cells, which comprises the following steps:

[0055] (a) separating the amniotic membrane from the placenta, washing it three times, and cutting it into pieces to obtain an amniotic membrane sample, wherein the washing solvent is physiological saline;

[0056] (b) placing the amniotic membrane sample in a mixture of type I collagenase and trypsin for digestion and washing to obtain the remaining amniotic membrane tissue, wherein the concentration of type I collagenase in the mixture of type I collagenase and trypsin is 0.8 mg / mL, and the concentration of trypsin is 3 mg / mL; the digestion temperature is 37° C., and the time is 50 min;

[0057] (c) placing the remaining amniotic tissue in a mixture of type IV collagenase and DNase for digestion, removing tissue and cell clusters through a 140-mesh sieve, and centrifuging the filtrate to obtain the amniotic mesenchymal stem cells, wherein the concentration of type IV collagenase in the mixture of type IV collagenase and DNase is 4 mg / mL, and the concentration of DNase is 1.5 mg / mL; the digestion temperature is 37° C., the time is 70 min; the centrifugal speed is 1000 rpm, and the time is 6 min.

[0058] Comparative Example 2

[0059] This comparative example is a method for extracting amniotic mesenchymal stem cells, which comprises the following steps:

[0060] (a) separating the amniotic membrane from the placenta, washing it three times, and cutting it into pieces to obtain an amniotic membrane sample, wherein the washing solvent is physiological saline;

[0061] (b) placing the amniotic membrane sample in a mixture of trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue, wherein the trypsin concentration in the mixture of trypsin and papain is 3 mg / mL and the papain concentration is 2 mg / mL; the digestion temperature is 37° C. and the time is 50 min;

[0062] (c) placing the remaining amniotic tissue in a mixture of type IV collagenase and DNase for digestion, removing tissue and cell clusters through a 140-mesh sieve, and centrifuging the filtrate to obtain the amniotic mesenchymal stem cells, wherein the concentration of type IV collagenase in the mixture of type IV collagenase and DNase is 4 mg / mL, and the concentration of DNase is 1.5 mg / mL; the digestion temperature is 37° C., the time is 70 min; the centrifugal speed is 1000 rpm, and the time is 6 min.

[0063] Experimental example

[0064] Obtain the amniotic mesenchymal stem cells prepared in Example 3 and Comparative Examples 1 to 3 respectively;

[0065] The content of cells expressing CD49d in human amniotic mesenchymal stem cells was detected, and the detection results are shown in Table 1;

[0066] Table 1

[0067] Group CD49d Example 3 98.3％ Comparative Example 1 73.9％ Comparative Example 2 82.6％

[0068] From Table 1, we can see that:

[0069] Compared with the comparative example, the technical solution of the present invention can better improve the purity of prepared amniotic mesenchymal stem cells by limiting the specific process in the embodiment.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for extracting amniotic mesenchymal stem cells, characterized in that: The extraction method comprises the following steps: (a) separating the amniotic membrane from the placenta, washing it, and cutting it into pieces to obtain an amniotic membrane sample; (b) placing the amniotic membrane sample in a mixture of type I collagenase, trypsin and papain for digestion and washing to obtain the remaining amniotic membrane tissue; (c) placing the remaining amniotic membrane tissue in a mixture of type IV collagenase and DNase for digestion, sieving to remove the tissue and cell clusters, and centrifuging the filtrate to obtain the amniotic membrane mesenchymal stem cells.

2. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (a), the solvent used for washing is physiological saline or PBS solution containing 1% double antibody.

3. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (b), in the mixed solution of type I collagenase, trypsin and papain, the concentration of type I collagenase is 0.5-1 mg / mL, the concentration of trypsin is 2-4 mg / mL, and the concentration of papain is 1-3 mg / mL.

4. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (b), the digestion temperature is 37° C. and the digestion time is 40 to 60 minutes.

5. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (c), the concentration of type IV collagenase in the mixed solution of type IV collagenase and DNase is 3-6 mg / mL, and the concentration of DNase is 1-2 mg / mL.

6. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (c), the digestion treatment temperature is 37° C. and the time is 60 to 80 minutes.

7. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (c), the mesh size of the sieve used for sieving is 140 mesh.

8. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: In the step (c), the centrifugal speed is 1000-1200 rpm and the time is 4-8 min.

9. The method for extracting amniotic mesenchymal stem cells according to claim 1, characterized in that: The step (c) also includes the process of inoculating the amniotic membrane mesenchymal stem cells into a culture medium for culturing.