Extracting method of dog adipose-derived stem cells, and preparation and application of dog adipose-derived stem cells

A technique for adipose stem cells and an extraction method is applied in the field of preparation of allogeneic adipose stem cells for the treatment of chronic kidney disease in dogs, which can solve the problems of high cost, large body damage in dogs, and long preparation cycle, and achieves low cost, sufficient sources, and reduced The effect of null cells

Pending Publication Date: 2019-02-22
NANJING AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the current treatment of dogs with chronic kidney disease, the use of autologous stem cells for the treatment of dogs with large damage to the dog's body, long preparation cycle, and high cost problems, the present invention uses canine allogeneic adipose stem cells to prepare and treat canine chronic kidney disease The preparation specifically provides a method for extracting canine allogeneic adipose stem cells. In another aspect, the present invention provides a canine allogeneic adipose stem cell preparation. In another aspect, the present invention also provides a method for preparing allogeneic adipose stem cells to treat canine chronic kidney disease. Application in preparation

Method used

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  • Extracting method of dog adipose-derived stem cells, and preparation and application of dog adipose-derived stem cells
  • Extracting method of dog adipose-derived stem cells, and preparation and application of dog adipose-derived stem cells
  • Extracting method of dog adipose-derived stem cells, and preparation and application of dog adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Isolation of Canine Adipose Stem Cells

[0048] Step 1: Under aseptic conditions, the adipose tissue attached to the peritoneum and bilateral ovaries of adult bitches is surgically removed, the adipose tissue is washed with normal saline, and then disinfected with medical alcohol, and the adipose tissue is placed in a preservation solution at a constant temperature of 2-8°C save. The obtained adipose tissue was washed with 0.9% sodium chloride injection, and repeated 2 to 3 times to remove blood stains. The whole adipose tissue was sterilized by submerging in 75% ethanol. Sodium chloride injection was washed repeatedly to remove residual ethanol. Use sterile surgical scissors to cut the adipose tissue into several sections of about 2-5 cm, and remove the congestion and clots in the small blood vessels of the adipose tissue. Use ophthalmic tweezers to peel off the fascia and blood vessels surrounding the adipose tissue, put it into a sterile plate, add an ap...

Embodiment 2

[0052] Example 2 The cultivation of canine adipose-derived stem cells

[0053] Step 5: P0 generation cell culture: place the above culture bottle in a carbon dioxide incubator for culture, 37°C, 5% CO 2 and saturated humidity for 24 hours to make MSCs adhere to the wall, replace the culture medium to remove non-adherent cells, and then replace the culture medium every 2 to 3 days; when the cell confluency reaches 70 to 90%, use 0.25% trypsin for routine digestion and harvest to obtain For the P0 generation cells, use the self-set freezing procedure to freeze the cells and establish the P0 generation master cell bank;

[0054] Step 6: P1 generation cell culture: the P0 generation cells were divided into about 5×10 3 piece / cm 2 Density inoculation in culture flasks, placed in a carbon dioxide incubator at 37°C, 5% CO 2 Cultivate for no more than 4 days, and replace the medium every 2 to 3 days. During this period, the cell morphology and growth state were observed under a mi...

Embodiment 3

[0057] Example 3 Identification and Safety Inspection of Canine Adipose Stem Cells

[0058] Step 9: The P3 generation cells are further tested for specific surface antigens, differentiation potential identification, endotoxin test and mycoplasma inspection, and the P3 generation cells that can be used to prepare canine allogeneic adipose stem cell preparations for the treatment of canine chronic kidney disease are screened out.

[0059] 1. Identification of cell surface antigens

[0060] Transfer the P3 generation ADSCs sample prepared in Example 2 into the corresponding flow tube, add 1ml PBS to mix well, centrifuge at 500g for 5 minutes, discard the supernatant, and repeat the washing twice; add an appropriate amount of PBS to suspend the cells, filter, count, and The cell concentration was adjusted to (2.0~6.0)×10 6 pcs / ml for use; take out several tributary tubes, add each group of antibodies and isotype control reagents (operate in the dark, see Table 1 for specific reag...

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Abstract

The invention discloses an extracting method of dog adipose-derived stem cells, and a preparation and application of the dog adipose-derived stem cells. The preparation for treating dog chronic nephrosis is prepared from the dog allogeneic adipose-derived stem cells. The extracting method of the adipose-derived stem cells comprises the steps: the dog abdominal adipose tissue is obtained, digesting, filtering and centrifuging are conducted, red blood cell lysis buffer resuspending is conducted, and the cells of P0-P3 generations are cultured; and the obtained adipose-derived stem cells of the P3 generation are subjected to surface antigen testing, the adipose-derived stem cells which have more than 70% of CD29 and MHC-1 expressions, less than 2% of CD34 and CD45 expressions, and the high differentiative potential, achieve adipogenesis, osteogenesis and chondrogenesis, and have normal chromosomes, feminine endotoxin and nont-detected mycoplasma are screened out and resuspended with the dosage of 1*10<6> cells/kg-5*10<6> cells/kg through normal saline of 0.5-1.0 mL to prepare the application of the dog allogeneic adipose-derived stem cells, and the preparation is used for treating dogchronic nephrosis. The stem cell preparation is weak in immunogenicity and is not involved in argument in the aspects of society, ethic and law; a transplanted person does not need to provide autologous stem cells, and nearly no damage is caused; and a separating method is simple, and the cells are very high in activity and easy to increase massively.

Description

technical field [0001] The invention relates to the field of stem cell preparation for treating diseases. Specifically, the present invention provides a method for extracting canine adipose stem cells and its preparation, and its use in preparing allogeneic adipose stem cells for treating canine chronic kidney disease. Background technique [0002] The kidney is an important substantive organ in animals, responsible for excreting metabolic waste, regulating water and electrolyte acid-base balance, and important functions of endocrine. However, due to the complexity of its own structure, it often causes irreversible damage to the kidney after being subjected to acute or chronic factors, and then gradually develops into chronic renal failure. Chronic kidney disease (Chronickidney disease, CKD) is a chronic disease characterized by permanent kidney damage in animals, which belongs to a broad range of diseases. Animals with persistent (more than 3 months) structural and functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P13/12
CPCA61K35/28A61P13/12C12N5/0667C12N2509/00
Inventor 赖晓云张海彬吴文达文依信唐卓丰汪恒
Owner NANJING AGRICULTURAL UNIVERSITY
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