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Canine umbilical cord mesenchymal stem cells, preparation method and cryopreservation method thereof

A technology of mesenchymal stem cells and umbilical cords, applied in the field of mesenchymal stem cells in the treatment of canine diseases, can solve problems such as not suitable for canines

Active Publication Date: 2020-08-18
天津瑞博斯生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many methods have been reported for isolating stem cells from related human tissues and organs, for example, the Chinese Invention Patent Publication No. CN102660501 A (Chinese Patent Application No. 201210159918.1) of the applicant's research and development team discloses a method for isolating and expanding umbilical cord fresh tissue mesenchyme. The method of mesenchymal stem cells, however, may be due to species differences, the inventors of the present invention have found that the method of the above-mentioned patent documents is not suitable for direct application to canines

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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 37...

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

[0057] Embodiment 3, biological characteristic identification of umbilical cord MSC

[0058] 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.

[0059]1. Cell growth and morphological characteristics

[0060] 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 characterist...

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Abstract

The invention relates to canine umbilical cord mesenchymal stem cells as well as a preparation method and a cryopreservation method thereof. Specifically, the method comprises the following steps: disinfection and washing, digestion treatment, cell culture, cell passage, and cell cryopreservation. The method for preparing mesenchymal stem cells from a canine umbilical cord exhibits an excellent technical effect. The mesenchymal stem cells is beneficial for treatment of canine arthritis, fractures, muscle damage, ligament injury, cartilage damage, joint damage, cognitive dysfunction, immune-mediated diseases, dry eye, recurrence uveitis, liver disease, heart disease, kidney disease, diabetes, gastrointestinal disease, thyroid disease and skin disease.

Description

technical field [0001] The invention belongs to the technical field of stem cell therapy for canine diseases, and relates to a method for preparing mesenchymal stem cells from canine umbilical cords and the use of the mesenchymal stem cells in treating canine diseases. The method for preparing mesenchymal stem cells from canine umbilical cord 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 freezing the mesenchymal stem cells prepared from the canine umbilical cord. Background technique [0002] Mesenchymal stem cells (MSCs or MSCs) are adult stem cells that exist in the interst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A01N1/02A61K35/28A61P19/02A61P19/08A61P21/00A61P19/04A61P25/00A61P37/02A61P27/02A61P1/16A61P9/00A61P13/12A61P3/10A61P1/00A61P5/14A61P17/00
CPCA01N1/0221A61K35/28A61P1/00A61P1/16A61P3/10A61P5/14A61P9/00A61P13/12A61P17/00A61P19/02A61P19/04A61P19/08A61P21/00A61P25/00A61P27/02A61P37/02C12N5/0665C12N2509/00
Inventor 王小武迟大明朱春颖郭春明许晓椿
Owner 天津瑞博斯生物技术有限公司
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