Preparation method of human primary cartilage cells with high yield rate

A high-yield technology for chondrocytes, applied in cell dissociation methods, bone/connective tissue cells, biochemical equipment and methods, etc., can solve the problem of low yield of chondrocytes and achieve the effect of improving the yield

Inactive Publication Date: 2016-10-12
GUANGDONG SECOND PROVINCIAL TRADITIONAL CHINESE MEDICINE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the traditional culture method of human primary chondrocytes has the problem of low chondrocyte yield, so it is necessary to develop a high-yield human primary chondrocyte preparation method

Method used

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  • Preparation method of human primary cartilage cells with high yield rate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] In this embodiment 1, based on the needs of the implementation process, the instruments and equipment used in the implementation are as follows:

[0024] HH-6 digital display constant temperature water bath (Jintan Fuhua Instrument Co., Ltd.), Varioskan Flash full-wavelength multi-functional microplate reader (Thermo, USA), 3K30 refrigerated high-speed centrifuge (Sigma, Germany), 5450 small High-speed centrifuge (Eppendorf, Germany), Class II Type B2 biological safety cabinet (Esco, Singapore), CO 2 Cell incubator (Thermo, USA), Cellometer Mini automatic cell counter (Nexcelom, USA), DMI8 inverted fluorescence microscope (Leica, Germany).

[0025] The implementation process proceeds in the following steps:

[0026] With the approval of the Ethics Committee and the patient’s informed consent, the knee articular cartilage tissue of the joint replacement patient was taken as the cultured chondrocytes; the obtained cartilage tissue was weighed, put into DPBS buffer (15mL ...

Embodiment 2

[0028] In this embodiment, based on the needs of the implementation process, the instruments and equipment used in the implementation are as follows:

[0029] HH-6 digital display constant temperature water bath (Jintan Fuhua Instrument Co., Ltd.), Varioskan Flash full-wavelength multi-functional microplate reader (Thermo, USA), 3K30 refrigerated high-speed centrifuge (Sigma, Germany), 5450 small High-speed centrifuge (Eppendorf, Germany), Class II Type B2 biological safety cabinet (Esco, Singapore), CO 2 Cell incubator (Thermo, USA), Cellometer Mini automatic cell counter (Nexcelom, USA), DMI8 inverted fluorescence microscope (Leica, Germany).

[0030] The implementation process proceeds in the following steps:

[0031] With the consent of the Ethics Committee and the patient’s informed consent, the cartilage tissue from the knee joint replacement patient was taken as cultured chondrocytes; the obtained cartilage tissue was shaken and washed 3 times in DPBS buffer (15mL cent...

Embodiment 3

[0033] In this embodiment, based on the needs of the implementation process, the instruments and equipment used in the implementation are as follows:

[0034] HH-6 digital display constant temperature water bath (Jintan Fuhua Instrument Co., Ltd.), Varioskan Flash full-wavelength multi-functional microplate reader (Thermo, USA), 3K30 refrigerated high-speed centrifuge (Sigma, Germany), 5450 small High-speed centrifuge (Eppendorf, Germany), Class II Type B2 biological safety cabinet (Esco, Singapore), CO 2 Cell incubator (Thermo, USA), Cellometer Mini automatic cell counter (Nexcelom, USA), DMI8 inverted fluorescence microscope (Leica, Germany).

[0035] The implementation process proceeds in the following steps:

[0036]With the consent of the Ethics Committee and the patient’s informed consent, the cartilage tissue from the knee joint replacement patient was taken as cultured chondrocytes; the obtained cartilage tissue was shaken and washed 3 times in DPBS buffer (15mL centr...

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Abstract

The invention belongs to the field of cell in-vitro culture, and particularly discloses a preparation method of human primary cartilage cells with high yield rate. The preparation method comprises the following steps of obtaining a cartilage tissue, and shearing the cartilage tissue by a pair of ophthalmology scissors; adding trypsinization, adsorbing supernatant, and discarding; adding II-type collagen digestion, staying overnight, filtering, adding into a culture medium, terminating digestion, centrifuging for 5min at the speed of 1000-1500rpm, obtaining precipitate of cartilage tissue, planting into a culture dish, and culturing, so as to obtain the human primary cartilage cells with high yield rate. The preparation method is mainly used for improving the shearing and centrifuging speeds of cartilage tissue blocks. The preparation method has the advantages that the centrifuging rotation speed is increased to 1000-1500rpm, the damage to the cartilage cells is avoided, and the yield rate of the cartilage cells is greatly improved; compared with the traditional cartilage cell preparation method, the yield rate of the cartilage cells is increased by about 3.55 times; after proofing by experiments, the cartilage cell has good cellular morphology.

Description

technical field [0001] The invention belongs to the field of in vitro culture of cells, and relates to a preparation method of human primary chondrocytes, in particular to a preparation method of high-yield human primary chondrocytes. Background technique [0002] In the body, chondrocytes are the only cell type in articular cartilage. When screening drugs for the prevention and treatment of osteoarthritis, primary human chondrocytes need to be cultured in vitro. However, usually after 3-4 passages of human primary chondrocytes in vitro, the cell morphology gradually becomes hypertrophic, and the expression of chondrocyte-specific genes such as type II collagen and proteoglycan is lost, so its phenotype is similar to that of cartilage in vivo. The cell phenotypes are quite different, so it is not suitable for scientific research needs. In order to ensure the smooth development of scientific research such as drug screening experiments, cells that have been passaged for 1-3 t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2509/00
Inventor 许学猛
Owner GUANGDONG SECOND PROVINCIAL TRADITIONAL CHINESE MEDICINE HOSPITAL
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