Culture medium and application thereof and method for inducing tendon stem cells to differentiate into bone cells

A technology of tendon stem cells and culture medium, applied in the field of stem cells, can solve the problem that the differentiation mechanism of tendon stem cells has not been clearly studied.

Inactive Publication Date: 2019-11-08
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, some scholars have proposed that the abnormal differentiation of tendon stem cells may be the basis of tendinopathy, but the differentiation mechanism of tendon stem cells has not yet been clearly studied

Method used

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  • Culture medium and application thereof and method for inducing tendon stem cells to differentiate into bone cells
  • Culture medium and application thereof and method for inducing tendon stem cells to differentiate into bone cells
  • Culture medium and application thereof and method for inducing tendon stem cells to differentiate into bone cells

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Experimental program
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Effect test

Embodiment 1

[0036] Human Achilles tendon tissue was collected under sterile conditions, rinsed with PBS three times, part of the connective tissue on the surface was cut off, and the Achilles tendon tissue was separated. The Achilles tendon was cut into pieces of 1mm×1mm×1mm, digested with 3mg / mL type Ⅰ collagenase at 37°C for 2h, and filtered through a 70μm filter to form a single cell suspension. Centrifuge the single cell suspension at 300×g for 5 min, discard the supernatant, and resuspend the cell pellet in complete medium (L-DMEM medium containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin) , at 500 pieces / cm 2 The cell density was seeded into a 10cm-diameter petri dish, cultured for 10 days, digested with 0.25% trypsin, mixed, and labeled as primary cells. Subculture after the cells cover 90% of the bottom of the bottle, discard the culture medium, wash twice with sterile PBS, digest with 0.25% trypsin for 3 minutes, add complete medium to stop digestion; centrifuge the ...

Embodiment 2

[0040] 1), prepare medium according to Table 1:

[0041] Table 1 medium formula

[0042]

[0043]

[0044] 2), take the 3rd passage cells, with 5×10 4 Cells / well were seeded in 6-well plates and divided into osteogenic induction group and control group. Among them, the osteogenic induction group used the medium of groups 1-6 in Table 1, and the control group used the complete medium. After the adherent cells in the osteogenic induction group grew and fused, 2 mL of the osteogenic induction medium was replaced, and the control group continued to be cultured with 2 mL of complete medium. The culture medium was changed every 3 days, and Alizarin red staining was performed at 4 weeks for identification.

[0045] 3) After 20 days of culture, the morphology of cells in groups 1 to 5 gradually changed, showing polygonal shape, and the intercellular substance contained calcified nodules of mineral salt deposition, and Alizarin red staining was positive; after 28 days of culture...

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Abstract

The invention relates to the technical field of stem cells, in particular to a culture medium and application thereof and a method for inducing tendon stem cells to differentiate into bone cells. According to the culture medium and application thereof and the method for inducing the tendon stem cells to differentiate into the bone cells, FBS, vitamin C, dexamethasone, beta- glycerophosphate and BMP-2 are added into the basic culture medium as an induction culture solution for tendon stem cell osteogenic differentiation. The number of tendon stem cells which are differentiated into bone cells is increased and the differentiation time is shortened. An experiment shows that the culture solution is used for inducing the tendon stem cells, and the characteristics of bone cells can appear within21days, the cell morphology is changed and the number of alizarin red staining cells is increased; and after 28 days of induction, the morphology of the cells is changed gradually, the cells are in apolygonal shape, calcified nodules with mineral deposits exist in the intercellular stroma, and alizarin red staining is positive.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a culture medium and its application and a method for inducing tendon stem cells to differentiate into bone cells. Background technique [0002] Tendinopathy is common in people who exercise heavily, and is characterized clinically by localized pain, swelling, and dysfunction. The common disease sites are the rotator cuff, supraspinatus muscle, patellar tendon, and Achilles tendon. At present, the pathogenesis of tendinopathy has not been fully studied, and the treatment methods for tendinopathy are limited and the curative effect is not clear, which is related to the lack of in-depth understanding of the pathological mechanism of the disease. [0003] In 2007, American scholars isolated a kind of cells with self-renewal ability from human and rabbit tendons, and named them tendon stem cells or progenitor cells (tendon-derived stem cells, TDSCs). In 2010, Hong Kong scholars a...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0654C12N2500/38C12N2500/42C12N2501/155C12N2501/39C12N2506/133
Inventor 陈东煌陈海佳葛啸虎王小燕黄幸李学家
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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