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Experimental method for enzymolysis, digestion and acquisition in vitro of rabbit knee cartilage unit

A technology of articular cartilage and experimental methods, applied in the direction of bone/connective tissue cells, vertebrate cells, animal cells, etc., can solve problems such as failure to form hyaline cartilage tissue, decrease in viscoelasticity, and degeneration of chondrocyte function, and achieve the acquisition method Simple, repeatable effect

Inactive Publication Date: 2010-09-15
THE SECOND HOSPITAL OF SHANXI MEDICAL UNIV +1
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AI Technical Summary

Problems solved by technology

However, in the process of in vitro culture and subculture of chondrocytes, the viscoelasticity gradually decreases, the fragility increases, and the function of chondrocytes degenerates relatively quickly. During the process of repairing cartilage defects in vivo, hyaline cartilage tissue that conforms to the characteristics of normal cartilage cannot be formed, which has restricted the development of tissue engineering technology. one of the biggest obstacles

Method used

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Experimental program
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Embodiment Construction

[0016] Concrete steps of the present invention are as follows:

[0017] (1) Rabbit knee joints were collected under aseptic conditions. The knee joint was opened in an ultra-clean workbench, and the full-thickness cartilage of the femur and tibial plateau of the knee joint was scraped with a sharp knife. The articular cartilage was repeatedly cut into pieces with small scissors, rinsed with sterile D-Hanks solution, the supernatant was discarded, weighed, and placed in a sterile Erlenmeyer flask for later use.

[0018] (2) Add 0.3% dispase (a neutral lyase, Sigma, USA) and 0.2% type II collagenase (Sigma, USA) to the Erlenmeyer flask at the rate of 12 mL DMEM-F12 culture solution / g cartilage. Both enzymes were dissolved in sterile DMEM-F12 culture solution (HyClone, USA), filtered through a 0.22 μm disposable filter, and sterilized. Place the Erlenmeyer flask containing the culture solution at 37°C, 5% CO 2 On the magnetic stirrer in the incubator, stir at a slow speed, and...

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Abstract

The invention belongs to the technical field of the construction in vitro of tissue engineered cartilage and in particular relates to an experimental method for anzymolysis, digestion and acquisition in vitro of a rabbit knee cartilage unit. The experimental method comprises the following steps of: taking a rabbit knee joint, repeatedly shearing joint cartilages into fragment tissues, washing the fragment tissues, removing supernate and putting the fragment tissues in an aseptic cone bottle for later use; adding dispase enzyme and II type collagenase into the cone bottle in the proportion of 12ml DMEM-F12 culture solution per gram of cartilages, stirring the mixture and digesting to form digesting suspension; and filtering and centrifugating the digesting suspension and adding primary chondrocyte culture solution into the obtained product to prepare aseptic cartilage unit suspension. The experimental method has the advantages that: the acquisition method is simple and the repetitiveness is high; the experimental method more accords with the growing environment and the biological characteristics of the chondrocyte in vitro; and compared with the pure cell, the cartilage unit has obviously improved biomechanics characteristics and the defect of poor biomechanics characteristics of the chondrocyte serving as a seeded cell is overcome.

Description

technical field [0001] The invention belongs to the technical field of in vitro construction of tissue engineered cartilage, and specifically relates to an experimental method for obtaining cartilage units by in vitro enzymolysis and digestion with rabbit knee articular cartilage as an experimental object. Background technique [0002] During the in vitro construction of tissue-engineered cartilage, the selection of ideal seed cells is the most critical and difficult point in current research. At present, in domestic and foreign literature reports, the most commonly used seed cells for articular cartilage tissue engineering are chondrocytes. The conventional enzymatic digestion method in vitro is: 0.4% Pronase enzyme is used to digest 12mL DMEM-F12 culture solution / g cartilage at 37°C for 90 minutes. After replacing the culture solution, 0.025% type II collagenase is digested overnight to obtain knee joint chondrocytes. However, in the process of in vitro culture and subcul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
Inventor 卫小春段王平孙振伟李琦
Owner THE SECOND HOSPITAL OF SHANXI MEDICAL UNIV
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