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Method for inducing directed cartilage differentiation of stem cells

A technology of stem cells and cartilage, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as decomposition, high price, and influence on characteristics, achieve good cartilage differentiation ability, prevent protease decomposition, and help effect on secretion

Active Publication Date: 2020-01-14
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used methods for delivering growth factors to effectively induce chondrogenic differentiation of stem cells are: 1) Add protein growth factors exogenously, but protein growth factors are expensive and unstable in the body, easily decomposed by proteases, lack of long-term stability
2) Through the design and preparation of biological materials, growth factors are loaded and controlled release, but the loading process and conditions will have a certain impact on the characteristics of macromolecular growth factors
However, although non-viral vectors can be operated safely, the transfection efficiency has not been ideal; while viral vectors have high transfection efficiency, but the transfection process will cause some damage to the seed cells, and their safety is still controversial.

Method used

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  • Method for inducing directed cartilage differentiation of stem cells
  • Method for inducing directed cartilage differentiation of stem cells
  • Method for inducing directed cartilage differentiation of stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Transfection of adenovirus carrying TGF-β3 into chondrocytes

[0034] (1) Construct an adenovirus vector carrying transforming growth factor TGF-β3,

[0035] Purchase the Adeno-XTM ViraTrakTM ZsGreen1 Expression System 2 kit, add Xhol / NotI recognition sites at both ends of the transforming growth factor TGF-β3 sequence by PCR, and then use DNA recombination technology to insert the TGF-β3 sequence into the adenovirus vector ( Green fluorescent protein adenoviral vector), construct an adenoviral vector (Ad-T3) carrying TGF-β3, Ad-T3 amplified to >10 9IFU / mL and then frozen for later use. The specific construction method refers to the following literature: Hao, J. H., Yao, Y. C., Varshney, R. R., Wang, L. C., Prakash, C., Li, H., Wang, D. A., GeneTransfer and Living Release of Transforming Growth Factor-Beta 3 for Cartilage Tissue Engineering Applications, Tissue Engineering Part C Methods, 14 (2008), 4: 273-280.

[0036] (2) Isolation and culture of chondroc...

Embodiment 2

[0039] Example 2 Observation of the culture process of ATDC5 cells and transfected chondrocyte co-culture system (named 3A1C) and detection of directional cartilage differentiation

[0040] (1) Purchase ATDC5 cells from Abgent (San Diego) (this cell is recognized as a substitute for stem cells as model cells), using F12 medium [containing 0.5% FBS (fetal bovine serum) and 1% penicillin-streptomycin penicillin-streptomycin (Double Antibody)] Passage ATDC5 cells to 3 passages and freeze them for later use;

[0041] (2) ATDC5 cells and transfected chondrocytes were digested, collected, counted, mixed at a ratio of 3:1, and 1.2% sodium alginate solution was added to prepare a cell density of 6×10 6 cells / mL of cell suspension. Take 50uL of the above cell suspension with a special pipette tip, and quickly inject it into 102mM CaCl 2 solution to form a cell gel (results image 3 ), and after 10 min, remove the cell gel with a long spoon and place it on a 24-well plate, one capsul...

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Abstract

The invention belongs to the technical field of tissue engineering, and particularly relates to a method for inducing directed cartilage differentiation of stem cells. Based on a co-culture system ofcartilage cells and the stem cells, the cartilage cells and the stem cells are subjected to division and cooperation of labor through the co-culture system, an adenovirus vector is adopted to introduce a transforming growth factor (TGF-beta 3) into the stem cells, the continuous, local and over-expressed TGF-beta 3 are provided for the stem cells through transfected cartilage cells, directed cartilage cell differentiation of the stem cells is stimulated, exogenous addition of TGF-beta 3 is not needed, thus the growth factor is prevented from being decomposed by protease, damage possibly generated in the gene transfection process to the stem cells is avoided, and the higher survival rate and the better cartilage differentiation ability of the stem cells are maintained. In addition, throughthe mode of co-culture of the cartilage cells and the stem cells, directed cartilage differentiation of the stem cells in a cartilage cell conditional culture medium is facilitated, induction to the growth factor is acted more sensitively, and secretion of cartilage epimatrix is more facilitated.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering, and in particular relates to a method for inducing directional cartilage differentiation of stem cells. Background technique [0002] Articular cartilage has the functions of buffering mechanical impact, reducing friction between joints, and increasing joint adaptability. Its slight damage will lead to osteoarthritis (OA). Since articular cartilage is a tissue without nerves and blood vessels, its self-repair ability very bad. At present, there are many methods for repairing cartilage damage, but each has its own defects, and none of them can solve this problem well. Moreover, chondrocytes are surrounded by extracellular matrix, so it is difficult for exogenous drugs or various factors to act on it. Therefore, the repair of articular cartilage damage is still a medical problem. [0003] In recent years, with the rapid development of tissue engineering, some therapeutic strategies ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/10C12N11/10C12N11/04
CPCC12N5/0655C12N11/04C12N11/10C12N2501/15C12N2502/1317
Inventor 姚咏嫦张廷帅陈汉政陈艺张姝江
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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