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High-activity primary cartilage cell preparing method

A technology of chondrocytes and high vitality, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of primary chondrocytes falling off and low yield, shorten the time of digestion and improve the yield rate, damage avoidance

Active Publication Date: 2015-09-16
SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the current primary culture process, primary chondrocytes are not easy to fall off from the cartilage matrix, resulting in a problem of low yield. Therefore, it is urgent to develop a culture method to increase the yield of primary chondrocytes

Method used

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  • High-activity primary cartilage cell preparing method
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. At 24 hours after birth, 6 male or female mice are not limited to 6 young mice. Take the cartilage from the joints of the limbs and try to remove the clean soft tissue. The remaining transparent cartilage tissue is placed in a petri dish filled with PBS buffer and washed several times until there is residual The blood is rinsed clean. Preparation of PBS buffer: PBS tablets were purchased from Biowest, and the PBS tablets were directly dissolved in water to obtain PBS buffer.

[0030] 2. Add a small amount (about 1ml) of 0.1% mass fraction of type II collagenase (C6885, Sigma-Aldrich) to keep the tissue moist during the cutting process. Since the cartilage tissue is elastic and comes from young mice, the tissue mass is relatively small. Ordinary ophthalmic scissors are easy to slip off when cut, and they are not easy to cut, which directly affects the degree of collagenase digestion and ultimately affects the yield of cartilage cells. In the present invention, a surgica...

Embodiment 2

[0034] 1. At 24 hours after birth, 6 male or female mice are not limited to 6 young mice. Take the cartilage from the joints of the limbs and try to remove the clean soft tissue. The remaining transparent cartilage tissue is placed in a petri dish filled with PBS buffer and washed several times until there is residual The blood is rinsed clean. Preparation of PBS buffer: PBS tablets were purchased from Biowest, and the PBS tablets were directly dissolved in water to obtain PBS buffer.

[0035] 2. Add a small amount (about 1ml) of 0.1% mass fraction of type II collagenase (C6885, Sigma-Aldrich) to keep the tissue moist during the cutting process. Since the cartilage tissue is elastic and comes from young mice, the tissue mass is relatively small. Ordinary ophthalmic scissors are easy to slip off when cut, and they are not easy to cut, which directly affects the degree of collagenase digestion and ultimately affects the yield of cartilage cells. In the present invention, a surgica...

Embodiment 3

[0039] 1. At 24 hours after birth, 6 male or female mice are not limited to 6 young mice. Take the cartilage from the joints of the limbs and try to remove the clean soft tissue. The remaining transparent cartilage tissue is placed in a petri dish filled with PBS buffer and washed several times until there is residual The blood is rinsed clean. Preparation of PBS buffer: PBS tablets were purchased from Biowest, and the PBS tablets were directly dissolved in water to obtain PBS buffer.

[0040] 2. Add a small amount (about 1ml) of 0.1% mass fraction of type II collagenase (C6885, Sigma-Aldrich) to keep the tissue moist during the cutting process. Since the cartilage tissue is elastic and comes from young mice, the tissue mass is relatively small. Ordinary ophthalmic scissors are easy to slip off when cut, and they are not easy to cut, which directly affects the degree of collagenase digestion and ultimately affects the yield of cartilage cells. In the present invention, a surgica...

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Abstract

The invention discloses a high-activity primary cartilage cell preparing method. The high-activity primary cartilage cell preparing method includes the following steps that 1, cartilage tissues of joints in limbs of an immature rat are extracted; 2, a blade special for operations is adopted to cut up the cartilage tissues; 3, the cut-up cartilage tissues are placed in a constant temperature shaking table to be shaken and placed in a horizontal centrifuge for centrifugation by 1500-2000 rpm, cell sediments are left, the cartilage tissues continue to be placed in the shaking table to be digested, are separated from cartilage matrixes after being repeatedly blown and beaten by a gun head of 1 ml, and are placed in the horizontal centrifuge for centrifugation by 1500-2000 rpm, and cartilage cell sediments are left; and 4, the cartilage cell sediments are planted in a culture dish and cultured in a DMEM high-glucose culture medium containing 10% of FBS to obtain high-activity primary cartilage cells. Compared with a traditional cartilage cell extracting method, the high-activity primary cartilage cell preparing method shortens the time by a half and improves the yield of cartilage cells by at least ten times; experiments verify that the obtained cartilage cells have a good rate of increase and good cellular morphology.

Description

Technical field [0001] The invention relates to a method for preparing primary chondrocytes, in particular to a method for preparing high-viability primary chondrocytes, which can significantly improve the efficiency of primary chondrocyte culture. Background technique [0002] After primary chondrocytes are cultured in vitro generally after 3-4 passages and expansion, the cell morphology gradually becomes hypertrophy, and the expression of chondrocyte-specific genes such as type II collagen and proteoglycan is lost. These are not conducive to the needs of scientific research. . In order to ensure the reliability of the experimental results, cells that are passaged 1-2 times are usually taken, which requires a large number of chondrocytes. [0003] Traditional chondrocyte culture methods generally include the following steps: [0004] For pups within 24 hours of birth, take the cartilage from the joints of the limbs, wash them with PBS, cut them with ophthalmic scissors, add 5ml of...

Claims

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Application Information

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IPC IPC(8): C12N5/077
Inventor 丁道芳胡鸿扬丁立何杰杜国庆郑昱新詹红生
Owner SHUGUANG HOSPITAL AFFILIATED WITH SHANGHAI UNIV OF T C M
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