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A method for constructing epiphyseal plate cartilage in vitro

A technology of cartilage and chondrocytes, applied in the direction of prosthesis, bone/connective tissue cells, tissue regeneration, etc., can solve the problems of restricting adult stem cells, high price, etc., and achieve the effect of easy acquisition and cost reduction

Inactive Publication Date: 2019-05-03
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as with articular chondrocytes, how to ensure the stability of adult stem cell traits during expansion remains to be resolved
More importantly, in the process of inducing the differentiation of adult stem cells into chondrocytes and osteoblasts in vitro, it is often necessary to add different cytokines such as tumor growth factor β. The price of these growth factors is usually very expensive, which seriously restricts the large-scale development of adult stem cells. clinical application
[0006] In addition, at present, tissue engineering methods are used to construct articular cartilage and bone tissue in vitro, while the construction of epiphyseal growth platecartilage in vitro has not been reported yet.

Method used

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  • A method for constructing epiphyseal plate cartilage in vitro
  • A method for constructing epiphyseal plate cartilage in vitro
  • A method for constructing epiphyseal plate cartilage in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] A method for constructing epiphyseal cartilage in vitro, comprising:

[0054] S11. After culturing the human bone marrow stem cells to form a cell monolayer, digest with trypsin, wash, and resuspend to obtain a cell suspension, and count for later use;

[0055] S12. Cut the pre-treated bone matrix gelatin material into a cylinder smaller than the diameter of the culture hole, and drill a hole on the cylinder that runs through the two bottom surfaces of the cylinder, and sterilize it; the cut bone matrix gelatin material Suspended and fixed in the culture well;

[0056] S13. Implant the cell suspension obtained in step S11 under the bone matrix gelatin material through the channel, and make the liquid surface infiltrate the bone matrix gelatin material, and cultivate epiphyseal plate cartilage;

[0057] Wherein, step S11 and step S12 have no sequence.

Embodiment 2

[0059] A method for constructing epiphyseal cartilage in vitro, comprising:

[0060] S21. Human cartilage C28 / I2 cell line cells frozen in liquid nitrogen were resuscitated and inoculated in 75mL culture flasks with an inoculation amount of 350,000 for cultivation; the culture conditions were: add 3.5mL of 28% fetal bovine serum to each culture flask , 110U / mL penicillin and streptomycin in DMEM / F12 culture medium, 4.5% CO 2 , Cultivate at 38°C; change the medium at intervals of 68 hours for the first time, and change the medium at intervals of 44 hours thereafter.

[0061] After the cells are cultured to form a monolayer of cells, discard the culture medium, wash the cells once with 3mL D-Hanks solution for each bottle of cells, add 3mL of digestive enzyme solution of 0.25% trypsin and 0.02% EDTA, and digest at 36°C for 12min;

[0062] Collect the cells in the 7 bottles of culture flasks into a centrifuge tube, centrifuge to discard the supernatant, and resuspend the cells i...

Embodiment 3

[0067] A method for constructing epiphyseal cartilage in vitro, comprising:

[0068] S31. Cut the pretreated bone matrix gelatin material into a cylinder slightly smaller than the diameter of the culture hole, and drill a channel on the cylinder that runs through the two bottom surfaces of the cylinder. The diameter of the channel is 1.2-1.4 mm; cobalt- 60 radioactive sterilization treatment; suspending and fixing the cut bone matrix gelatin material in the culture hole; sealing the gap between the bone matrix gelatin material and the edge of the culture hole with sterile agarose gel.

[0069] S32. The human cartilage C28 / I2 cell line cells frozen in liquid nitrogen were resuscitated and inoculated in a 75mL culture bottle with an inoculation amount of 450,000 for culture; the culture conditions were: add 4.5mL of 32% fetal bovine serum to each culture bottle , 90U / mL penicillin and streptomycin in DMEM / F12 culture medium, 5.5% CO 2 , Cultivate at 36°C; change the medium at i...

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Abstract

The invention relates to the technical field of tissue engineering, and in particular relates to an in-vitro construction method of epiphyseal plate cartilage. The in-vitro construction method of the epiphyseal plate cartilage comprises the following steps: (1) culturing human cartilage cell line cells or human bone marrow stem cells till cell monolayers are formed, carrying out trypsinization, washing and resuspension to obtain a cell suspension, and carrying out counting for later use; (2) cutting a pretreated bone matrix gelatin material into a cylinder with the diameter smaller than the diameter of each culture hole, drilling a pore passage running through the two bottom surfaces of the cylinder in the cylinder, carrying out sterilization treatment, and fixing the cut bone matrix gelatin material in the culture holes in a suspension manner; and (3) implanting the cell suspension obtained in the step (1) to be below the bone matrix gelatin material from the pore passage, enabling the liquid level to wet the bone matrix gelatin material, and carrying out culturing to obtain the epiphyseal plate cartilage. With the adoption of the technical scheme, a culture system of C28 / I2 cells and an in-vitro construction method of the epiphyseal plate cartilage by adopting the culture system are established, and therefore, the problem that the seed cells are difficultly available is solved; the cells are stable in character and low in culturing cost.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a method for constructing epiphyseal plate cartilage in vitro. Background technique [0002] Tissue engineering technology is a new strategy gradually used in the treatment of joint diseases in recent years. Its core is to use the method of tissue engineering to cultivate the cells or tissues of the lesion in vitro, and implant them into the lesion in the joint, such as the defect of articular cartilage, through injection or surgery, so as to promote the reconstruction and healing of the lesion. Eventually restore joint function. However, there are two basic prerequisites for in vitro culture using tissue engineering technology, namely, cell scaffolds and seed cells. In addition, the integration between seed cells and cell scaffolds is also an important factor for the success of tissue engineering. [0003] Bone Matrix Gelatin (BMG) is a scaffold material obtained b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077A61L27/36A61L27/50A61L27/22
CPCA61L27/222A61L27/3612A61L27/3654A61L27/3687A61L27/50A61L2430/06C08L89/00
Inventor 李思远曹峻岭罗明秀陆敏灵王加丽李锦陈静宏付强
Owner XI AN JIAOTONG UNIV
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