Method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells

Abstract

The invention relates to a method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells. It specifically relates to a cell cryopreservation solution for canine fetal membrane mesenchymal stem cells. The method for preparing canine fetal membrane mesenchymal stem cells includes the following steps: disinfection and cleaning, digestion treatment, cell culture, cell passage, and cell cryopreservation. The method for preparing mesenchymal stem cells from canine fetal membranes of the present invention has excellent technical effects, and the culture medium used can greatly improve the efficiency of preparing stem cells. The prepared mesenchymal stem cells can beneficially treat arthritis, fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated disease, dry eye, recurrent Uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease, skin disease.

Description

technical field

[0001] The invention belongs to the technical field of stem cell treatment of canine diseases, and relates to a method for preparing mesenchymal stem cells from canine fetal membranes and the use of the mesenchymal stem cells in treating canine diseases. The method for preparing mesenchymal stem cells from canine fetal membranes of the present invention has excellent technical effects. The prepared mesenchymal stem cells can beneficially treat arthritis, fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated disease, dry eye, recurrent Uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease, skin disease. Further, the present invention also relates to a method for cryopreserving the mesenchymal stem cells prepared from canine fetal membranes. The present invention also relates to the culture medium used in the process of preparing canine fetal membrane...

Examples

Embodiment 1

[0046] Example 1. Preparation of mesenchymal stem cells from canine fetal membranes

[0047] (1) Disinfection and cleaning: take out the placenta from the placenta sample collection box, put it in a stainless steel cup, use alcohol (75% alcohol) and saline to rinse the surface repeatedly (alcohol soaking disinfection time 30s)), and disinfect the placenta Then use surgical forceps to slowly tear off the fetal membrane, and place it in a glass dish. After removing the blood vessels, use phosphate buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to rinse again to remove surface dirty blood, impurities;

[0048] (2) Digestion treatment: in another cell culture plate, the fetal membrane tissue obtained in step (1) is cut into tissue blocks (cubic blocks of about 0.1 cubic centimeters), and the tissue blocks are put into digestive enzyme solution (preparation method : Add 0.1g of type I collagenase to 100ml of DMEM-F12, then filter it with a 0.45μm filter to obtain the ...

Embodiment 2

[0052] Example 2. Preparation of mesenchymal stem cells from canine fetal membranes

[0053] (1) Disinfection and cleaning: In the biological safety cabinet, take out the placenta from the placenta sample collection box, place it in a stainless steel cup, use alcohol (75% alcohol) and normal saline to rinse the surface repeatedly (alcohol soaking disinfection time 30s)) , disinfect the placenta; then use surgical forceps to slowly tear off the fetal membranes and place them in a glass dish. After removing the blood vessels, use phosphate buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to rinse again , remove surface dirt and impurities;

[0054] (2) Digestion treatment: in another cell culture plate, the fetal membrane tissue obtained in step (1) is cut into tissue blocks (cubic blocks of about 0.1 cubic centimeters), and the tissue blocks are put into digestive enzyme solution (preparation method : Add 0.1g of type I collagenase to 100ml of DMEM-F12, then filt...

Embodiment 3

[0061] Embodiment 3, biological characteristic identification of fetal membrane MSC

[0062] The following tests were carried out on the canine fetal membrane mesenchymal stem cells obtained in Example 1 and Example 2, and there was no difference in the test results of the two kinds of stem cells.

[0063] 1. Cell growth and morphological characteristics

[0064] Through the isolation and culture of Example 1 and Example 2, the obtained canine fetal mesenchymal stem cells can be clearly seen under the microscope after 48 hours of culture. Spindle-shaped adherent cells will form turbine-shaped cell clones in about 8 days. After digestion and passage An adherent layer with about 75% fusion will be formed. During the culture process, it was found that the cell shape was relatively uniform, the proliferation speed was fast, the adhesion speed was fast, and it was easily digested by trypsin. After passage to 3-15 generations, its shape and growth characteristics did not change s...

technical field

[0001] The invention belongs to the technical field of stem cell treatment of canine diseases, and relates to a method for preparing mesenchymal stem cells from canine fetal membranes and the use of the mesenchymal stem cells in treating canine diseases. The method for preparing mesenchymal stem cells from canine fetal membranes of the present invention has excellent technical effects. The prepared mesenchymal stem cells can beneficially treat arthritis, fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated disease, dry eye, recurrent Uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease, skin disease. Further, the present invention also relates to a method for cryopreserving the mesenchymal stem cells prepared from canine fetal membranes. The present invention also relates to the culture medium used in the process of preparing canine fetal membrane...

Examples

Embodiment 1

[0046] Example 1. Preparation of mesenchymal stem cells from canine fetal membranes

[0047] (1) Disinfection and cleaning: take out the placenta from the placenta sample collection box, put it in a stainless steel cup, use alcohol (75% alcohol) and saline to rinse the surface repeatedly (alcohol soaking disinfection time 30s)), and disinfect the placenta Then use surgical forceps to slowly tear off the fetal membrane, and place it in a glass dish. After removing the blood vessels, use phosphate buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to rinse again to remove surface dirty blood, impurities;

[0048] (2) Digestion treatment: in another cell culture plate, the fetal membrane tissue obtained in step (1) is cut into tissue blocks (cubic blocks of about 0.1 cubic centimeters), and the tissue blocks are put into digestive enzyme solution (preparation method : Add 0.1g of type I collagenase to 100ml of DMEM-F12, then filter it with a 0.45μm filter to obtain the ...

Embodiment 2

[0052] Example 2. Preparation of mesenchymal stem cells from canine fetal membranes

[0053] (1) Disinfection and cleaning: In the biological safety cabinet, take out the placenta from the placenta sample collection box, place it in a stainless steel cup, use alcohol (75% alcohol) and normal saline to rinse the surface repeatedly (alcohol soaking disinfection time 30s)) , disinfect the placenta; then use surgical forceps to slowly tear off the fetal membranes and place them in a glass dish. After removing the blood vessels, use phosphate buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to rinse again , remove surface dirt and impurities;

[0054] (2) Digestion treatment: in another cell culture plate, the fetal membrane tissue obtained in step (1) is cut into tissue blocks (cubic blocks of about 0.1 cubic centimeters), and the tissue blocks are put into digestive enzyme solution (preparation method : Add 0.1g of type I collagenase to 100ml of DMEM-F12, then filt...

Embodiment 3

[0061] Embodiment 3, biological characteristic identification of fetal membrane MSC

[0062] The following tests were carried out on the canine fetal membrane mesenchymal stem cells obtained in Example 1 and Example 2, and there was no difference in the test results of the two kinds of stem cells.

[0063] 1. Cell growth and morphological characteristics

[0064] Through the isolation and culture of Example 1 and Example 2, the obtained canine fetal mesenchymal stem cells can be clearly seen under the microscope after 48 hours of culture. Spindle-shaped adherent cells will form turbine-shaped cell clones in about 8 days. After digestion and passage An adherent layer with about 75% fusion will be formed. During the culture process, it was found that the cell shape was relatively uniform, the proliferation speed was fast, the adhesion speed was fast, and it was easily digested by trypsin. After passage to 3-15 generations, its shape and growth characteristics did not change s...