Method for preparing mesenchymal stem cell from umbilical cord of dog
A technology of stem cells and umbilical cord, applied in the field of mesenchymal stem cells in the treatment of canine diseases, can solve problems such as being unsuitable for canine animals
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Embodiment 1
[0042] Example 1. Preparation of Mesenchymal Stem Cells from Dog's Umbilical Cord
[0043] (1) Disinfection and cleaning: in the biosafety cabinet, disinfect the surface of the dog's umbilical cord tissue (fresh isolated umbilical cord tissue) with disinfectant solution (75% ethanol), cut the umbilical cord, and lay it flat (on a surface with a diameter of 10cm culture dish), the umbilical cord tissue was washed by buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to reduce the red blood cells on the umbilical cord tissue;
[0044] (2) Digestion treatment: cut the umbilical cord tissue obtained in step (1) into tissue blocks (cubic blocks of about 0.1 cubic centimeters) in another cell culture plate, and put the tissue blocks into a digestive enzyme solution (preparation method: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, and then filter it with a 0.45 μm filter to obtain the digestion solution), digest it for 0.5-3 hours (in this example, digest it at 3...
Embodiment 2
[0048] Example 2, preparation of mesenchymal stem cells from the umbilical cord of dogs
[0049] (1) Disinfection and cleaning: in the biosafety cabinet, disinfect the surface of the dog's umbilical cord tissue (fresh isolated umbilical cord tissue) with disinfectant solution (75% ethanol), cut the umbilical cord, and lay it flat (on a surface with a diameter of 10cm culture dish), the umbilical cord tissue was washed by buffer (0.025M sodium dihydrogen phosphate buffer at pH 6.5) to reduce the red blood cells on the umbilical cord tissue;
[0050] (2) Digestion treatment: cut the umbilical cord tissue obtained in step (1) into tissue blocks (cubic blocks of about 0.1 cubic centimeters) in another cell culture plate, and put the tissue blocks into a digestive enzyme solution (preparation method: Add 0.1 g of type I collagenase to 100 ml of DMEM-F12, and then filter it with a 0.45 μm filter to obtain the digestion solution), digest it for 0.5-3 hours (in this example, digest ...
Embodiment 3
[0055] Embodiment 3, biological characteristic identification of umbilical cord MSC
[0056] The following tests were carried out on the canine umbilical cord 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.
[0057] 1. Cell growth and morphological characteristics
[0058] Through the isolation and culture of Examples 1 and 2, the obtained canine umbilical cord mesenchymal stem cells can be clearly seen under the microscope after cultured for 48 hours. Spindle-shaped adherent cells will be formed in about 8 days. Turbine-shaped cell clones will be formed after digestion and passage. An adherent layer with about 70% fusion was 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 characteris...
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