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Culture method and application for stent-free three-dimensional gel of mesenchymal stem cells

A mesenchymal stem cell and three-dimensional gel technology, which is applied in the field of stem cells and regenerative medicine, can solve the problems of the lack of a three-dimensional culture system for mesenchymal stem cells, and achieve the effects of improving treatment efficiency, flexible application methods, and less pain in collection

Pending Publication Date: 2020-02-04
山东省齐鲁细胞治疗工程技术有限公司
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Problems solved by technology

[0005] In order to overcome the lack of a three-dimensional culture system suitable for culturing and directional differentiation of mesenchymal stem cells in the prior art, the present invention provides a scaffold-free three-dimensional gel culture method for mesenchymal stem cells

Method used

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  • Culture method and application for stent-free three-dimensional gel of mesenchymal stem cells
  • Culture method and application for stent-free three-dimensional gel of mesenchymal stem cells
  • Culture method and application for stent-free three-dimensional gel of mesenchymal stem cells

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Embodiment 1

[0024] The isolation and passage of adipose-derived mesenchymal stem cells and the induction of scaffold-free three-dimensional gel, the experimental operation is described in detail as follows:

[0025] 1. Separation and subculture of adipose-derived mesenchymal stem cells: Remove the fascia, skin, blood vessels and other tissues above the fat with solid fat, clean the fat block, and cut it into pieces with scissors. After that, transfer the shredded fat or liquid fat from liposuction to a 250mL centrifuge tube, add an appropriate amount of saline to it, blow it with a straw 10-20 times, then let it stand for about 1min, and use a straw to suck out the liquid in the lower layer of the tissue. Repeat 2-3 times until the sucked liquid is transparent without blood cells. Add 0.1% collagenase type I / PBS digestion solution at a volume ratio of 1:1 to the centrifuge tube containing adipose tissue; seal the centrifuge tube and place it in a constant temperature shaker at 37°C, shake...

Embodiment 2

[0028] Adipose-derived mesenchymal stem cell scaffold-free three-dimensional gel treatment of cartilage damage, the experimental operation is described in detail as follows

[0029] 1. Preparation of animal model: intramuscular injection of 30 mg of ketamine hydrochloride and 6 mg of xylazine mixed solution per kg body weight of anesthetized experimental pigs. Routine shaving, disinfection, and sterile towels. The cartilage at the medial femoral condyle was exposed, and a hole was drilled at the upper cartilage with a diameter of 5 mm and a depth of 2 mm.

[0030] 2. Experimental grouping: 9 experimental pigs were used as blank control in the left leg femur, and no treatment was given after the defect; stem cell gel transplantation was performed in the right leg femur defect.

[0031] 3. Treatment: transplant adipose-derived mesenchymal stem cell scaffold-free three-dimensional gel to the damaged cartilage. The gel has a certain adhesion ability, and its water-like properties...

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Abstract

The invention provides a culture method for a stent-free three-dimensional gel culture system of mesenchymal stem cells. The culture method comprises the following steps that the adipose mesenchymal stem cells are inoculated into a culture dish, inducing culture is carried out in an inducing medium containing ascorbic acid-2-phosphate (ASC2P) gel, collagen is secreted from the adipose mesenchymalstem cells under the action of the inducing medium to complete intercellular bonding, and the stent-free three-dimensional gel of the mesenchymal stem cells with a hydrogel state with a three-dimensional structure is formed. According to the culture method, autologous fat is used as a source of stem cells, the stent-free three-dimensional gel of the mesenchymal stem cells of an external organizational framework is not added, the hydrogel state is showed, cartilage damages of different shapes can be adopted, a rejection reaction is avoided, and cartilage is advantageously regenerated and repaired.

Description

technical field [0001] The invention relates to the field of stem cells and regenerative medicine, in particular to a preparation method and application of a scaffold-free three-dimensional gel culture system for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are a kind of pluripotent stem cells with self-renewal and multilineage differentiation potential existing in various tissues in the body. Due to the characteristics of easy separation and expansion, self-renewal, multi-directional differentiation and low immunogenicity, it has become an ideal seed cell in tissue engineering and regenerative medicine, bringing new hope to some diseases that are difficult to treat with traditional drugs. Mesenchymal stem cells have achieved good results in the treatment of arthritis, but the selection and application of different mesenchymal stem cells are limited by their source, in vitro proliferation and other factors. [0003] In 2001, Zuk et al. isolat...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/00
CPCC12N5/0662C12N5/0062
Inventor 王芝辉张剑慧马贺然张慧慧谭毅韩镇
Owner 山东省齐鲁细胞治疗工程技术有限公司
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