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Composition, differentiation inducing culturing liquid containing composition and inducing method

A technology of differentiation induction and composition, applied in the field of cells, can solve the problems of lack of mechanical properties and durability of hyaline cartilage, and unsatisfactory results, and achieve the effects of low immunogenicity, promotion of repair and regeneration, and easy expansion in vitro

Inactive Publication Date: 2019-04-16
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional treatment methods such as subchondral bone drilling, cartilage transplantation, periosteum or perichondrium transplantation, and chondrocyte transplantation cannot achieve satisfactory results because the repair tissue is mainly fibrocartilage, which lacks the mechanical properties and durability of normal hyaline cartilage.

Method used

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  • Composition, differentiation inducing culturing liquid containing composition and inducing method
  • Composition, differentiation inducing culturing liquid containing composition and inducing method
  • Composition, differentiation inducing culturing liquid containing composition and inducing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Primary isolation culture, identification and passage of DPSCs

[0035] Samples: Complete premolars or third molars extracted for orthodontics from 10-20-year-old healthy donors were quickly immersed in 4°C pre-cooled PBS containing 3 times double antibody.

[0036] Rinse: Rinse the teeth with PBS containing 3 times the double antibody to remove blood stains thoroughly. Repeat 3 times.

[0037] Pulp separation and extraction: Wrap the tooth in sterile gauze and crack the tooth with forceps to expose the pulp tissue; use sterile forceps to grasp the pulp tissue, and remove the pulp tissue at the root tip of 1 mm. Cut the pulp tissue to 1mm with ophthalmic curved scissors 3 Left and right size tissue blocks.

[0038] Digestion: Add 5-20 times the volume of collagenase-dispase enzyme 1:1 mixed digestion solution (collagenase 3g / L, dispase enzyme 4g / L), and place it in a constant temperature shaker at 37°C for 45-50min. After digestion, centrifuge at 1500r / min...

Embodiment 2

[0057] Take the 3rd generation DPSCs, press 5×10 5 The cell / tube was inoculated in a 15mL centrifuge tube, centrifuged at 1000rpm for 5min, so that the cells gathered at the bottom of the centrifuge tube, loosen half of the cap, and placed in 37°C, 5% CO 2 Culture in an incubator.

[0058] Discard the old solution after 24 hours, and replace with 1 mL of the experimental composition of chondrogenic induction solution: containing 10 μg / L TGF-β3, 50 μg / L proline, 5% PRP, 1% ITS + Premix high glucose DMEM medium. Gently flick the centrifuge tube to float the cell mass, loosen half of the tube cap, and place at 37°C, 5% CO 2 Culture in an incubator.

[0059] After 21 days of induced differentiation, the cells were subjected to paraffin section, safranin O-fast green staining and type II collagen immunohistochemistry experiments.

[0060] Safranin O-fast green staining experiment results are as follows image 3 As shown, the cell section is colored, and the colored area is obv...

Embodiment 3

[0062] Take the 3rd generation DPSCs, press 5×10 5 The cell / tube was inoculated in a 15mL centrifuge tube, centrifuged at 1000rpm for 5min, so that the cells gathered at the bottom of the centrifuge tube, loosen half of the cap, and placed in 37°C, 5% CO 2 Culture in an incubator.

[0063] Discard the old solution after 24 hours, and replace with 1 mL of the experimental composition of chondrogenic induction solution: containing 100 μg / L TGF-β3, 10 μg / L proline, 1% PRP, 5% ITS + Premix high glucose DMEM medium. Gently flick the centrifuge tube to float the cell mass, loosen half of the tube cap, and place at 37°C, 5% CO 2 Culture in an incubator.

[0064] After 21 days of induced differentiation, the cells were subjected to paraffin section, safranin O-fast green staining and type II collagen immunohistochemistry experiments.

[0065] Safranin O-fast green staining experiment results are as follows image 3 As shown, the cell section is colored, and the colored area is ob...

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Abstract

The invention relates to the field of cells, in particular to a composition, differentiation inducing culturing liquid containing the composition and an inducing method. Selected dental pulp stem cells (dental pulp stem cell, DPSCs) are same with MSCs of other sources, have self-renewing and multi-directional differentiation capacity and have the advantages that cell proliferation in vitro is easy, and the immunogenicity of the cells is low, and the selected dental pulp stem cells can be taken as seed cells to promote repairing and regeneration of injured tissue. Thedifferentiation inducing culturing liquid can effectively promote specificity differentiation, towards cartilage cells, of DPSCs. Based on the differentiation inducing culturing liquid, the provided method ofchondrogenesisdifferentiation for the DPSCs subjected to in-vitro inducing culturingcan successively achieve chondrogenesis differentiation inducing on the DPSCs.

Description

technical field [0001] The invention relates to the field of cells, in particular to a composition, a differentiation induction culture medium containing the composition and an induction method. Background technique [0002] With the development of social economy, the improvement of living standards and the phenomenon of population aging, the incidence of diseases such as osteoarthritis, mainly manifested by cartilage surface destruction, is increasing day by day. Therefore, how to minimally invasively repair damaged cartilage, alleviate the pain of patients, and improve the quality of life has long been one of the key research directions in the field of orthopedic medicine. Osteoarthritis is a common clinical disease in orthopedics. Articular cartilage defects caused by degenerative diseases and trauma have become an important cause of disability and affecting the quality of life. Articular cartilage is an avascular tissue, and chondrocytes mainly rely on synovial fluid an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0655C12N5/0664C12N2500/25C12N2500/32C12N2500/84C12N2501/15
Inventor 葛啸虎陈海佳王小燕戚康艺姜交华
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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