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Digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation and separation culture method of tibial growth plate cartilage primary cells

A technology for separating, culturing and digesting enzyme liquid, applied in the field of cell culture

Inactive Publication Date: 2017-07-25
TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cause of the decline in viability and death in the in vitro culture process is still unclear. Therefore, a complete set of isolation and culture methods have not been successfully formed in the prior art.

Method used

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  • Digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation and separation culture method of tibial growth plate cartilage primary cells
  • Digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation and separation culture method of tibial growth plate cartilage primary cells
  • Digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation and separation culture method of tibial growth plate cartilage primary cells

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preparation example Construction

[0033] In the present invention, the preparation method of the digestive enzyme liquid comprises the following steps:

[0034] 1) Mix high-glucose DMEM-F12 medium, fetal bovine serum, vitamin C and penicillin to obtain complete medium;

[0035] 2) Mix the complete culture medium obtained in step 1) with collagenase IV and hyaluronidase, and then membrane filter to obtain a digestive enzyme solution.

[0036] In the present invention, the filter membrane for membrane filtration preferably has a pore size of 0.22 μm.

[0037] The present invention also provides a method for isolating and culturing primary poultry tibial growth plate cartilage cells, comprising the following steps:

[0038] 1) Rinse the leg bones stripped from poultry within 30 minutes of execution, soak them in 75% alcohol and rinse again to obtain pretreated leg bones;

[0039] 2) In a sterile environment, the leg bone in step 1) is stripped from the muscles at the tibial joint, the exposed tibial dry end is ...

Embodiment 1

[0070] Complete medium preparation: high-glucose DMEM-F12 medium is used as the basic medium, including the following components: fetal bovine serum with a volume concentration of 8%, vitamin C with a mass concentration of 5.5 mg / mL and 1 mg / mL of vitamin C penicillin streptomycin.

[0071] The configuration of the digestive enzyme solution: based on the complete medium, it contains collagenase IV with a mass concentration of 100 mg / mL and hyaluronidase with a mass concentration of 80 mg / mL.

[0072] The above-mentioned complete medium and digestive enzyme solution were used to separate and culture the primary cells of poultry tibial growth plate cartilage, and the specific steps were as follows:

[0073] (1) Prepare the required equipment and reagents, and place them in a sterile ultra-clean bench for at least 30 minutes of ultraviolet radiation.

[0074] (2) Three healthy 2-week-old broilers were killed by bloodletting from the jugular vein, the leg bones were peeled off fr...

Embodiment 2

[0089]Complete medium preparation: high-glucose DMEM-F12 medium is used as the basic medium, including the following components: fetal bovine serum with a volume concentration of 12%, vitamin C with a mass concentration of 4.5 mg / mL and 10 mg / mL of penicillin streptomycin.

[0090] The configuration of the digestive enzyme solution: based on the complete medium, it contains collagenase IV with a mass concentration of 80 mg / mL and hyaluronidase with a mass concentration of 100 mg / mL.

[0091] The above-mentioned complete medium and digestive enzyme solution were used to separate and culture the primary cells of poultry tibial growth plate cartilage, and the specific steps were as follows:

[0092] (1) Prepare the required equipment and reagents, and place them in a sterile ultra-clean bench for at least 30 minutes of ultraviolet radiation.

[0093] (2) Three healthy 2-week-old broilers were killed by bloodletting from the jugular vein, the leg bones were peeled off from the ou...

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Abstract

The invention provides digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation. A complete medium is used as basic liquid, and is prepared from the following ingredients including collagenase IV with the mass concentration being 80 to 100mg / mL and hyaluronidase with the mass concentration being 80 to 100mg / mL; the complete medium takes a high-glucose DMEM-F12 culture medium as a basic culture medium, and contains the following ingredients including fetal calf serum with the volume concentration being 8 to 12 percent, vitamin C with the mass concentration being 4.5 to 5.5mg / mL and mycillin with the mass concentration being 1 to 10mg / mL. Through the selection on digestive enzyme and the concentration optimization, the cell yield reaches 85 to 95 percent or higher; during the further culture on the digested cells, the cell upward trend is good; meanwhile, the cell viability of the digested primary cell reaches 85 to 95 percent or higher; the cell viability is relatively stable.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a digestive enzyme solution for isolating poultry tibia growth plate cartilage primary cells and a method for separating and culturing the tibial growth plate cartilage primary cells. Background technique [0002] Tibial chondrodysplasia (TD) is a cartilage metabolic disease that occurs in poultry. The disease often occurs in poultry such as broiler hens, layers and turkeys. The clinical manifestations are depression, loss of appetite, often crawling on the bottom, dyskinesia, difficulty standing, and sometimes walking with the support of wings. TD can reduce the disease resistance and production performance of chickens, induce breast cysts and osteomyelitis, reduce the quality and grade of chicken meat, endanger animal welfare, and cause important losses to the field of poultry farming. It occurs in all countries in the world, and causes huge economic losses to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2500/38C12N2500/84C12N2501/73C12N2501/734C12N2509/00
Inventor 李家奎张辉
Owner TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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