Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells

A technology of directional differentiation and stem cells, which is applied in the field of regenerative medicine, can solve the problems of unfavorable cell culture and tissue repair of degradation products, lack of special extracellular matrix components that promote differentiation, and cells do not have normal physiological functions in vivo, so as to achieve induction efficiency and Improved cell function, low cost, improved differentiation efficiency and functional effects

Inactive Publication Date: 2014-09-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Description
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Problems solved by technology

At present, the three-dimensional system that regulates the directional differentiation of stem cells in vitro uses decellularized scaffolds, collagen scaffolds, or polylactic acid scaffolds. Some of these scaffolds have high immunogenicity, and some lack special extracellular matrix components that promote differentiation. The degradation products are not conducive to cell culture and tissue repair, which leads to the low efficiency of stem cell directional differentiation in vitro, and the obtained cells do not have normal physiological functions in vivo.
[0004] Due to the above-mentioned defects in the existing induced differentiation system, the further development and clinical application of stem cell directed differentiation technology in vitro are severely limited. Therefore, there is an urgent need to develop a convenient and practical new method for inducing three-dimensional differentiation of stem cells, that is, by using three-dimensional culture and adding matrix components to construct It can simulate the microenvironment of tissues in vivo to realize the regulation of stem cell directional pre-differentiation in vitro, and further promote the application of stem cells in the field of regenerative medicine

Method used

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  • Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells
  • Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells
  • Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells

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Embodiment 1

[0026] Example 1: Three-dimensional induction system regulates the directional differentiation of human mesenchymal stem cells into chondrocytes in vitro

[0027] Sodium alginate (molecular weight 430kDa, ratio of guluronic acid to mannuronic acid 1.5) powder was dissolved in physiological saline to obtain a 3% (W / V, g / ml) solution. Chondroitin sulfate (average molecular weight 20kDa) powder was dissolved in saline to obtain a 5% (W / V, g / ml) solution. Mix 3% (W / V, g / ml) sodium alginate, 5% (W / V, g / ml) chondroitin sulfate solution and normal saline to obtain a final concentration of sodium alginate of 2% (W / V , g / ml), the mixed solutions of sodium alginate / chondroitin sulfate mass ratios of 1.5, 4 and 9, respectively. The fifth generation human umbilical cord mesenchymal stem cells were mixed with the mixed solution, and the cell density was adjusted to 6×10 6 cells mL -1 , the cell suspension was dripped into 100mmol·L via a syringe pump -1 CaCl 2 Calcification in the sol...

Embodiment 2

[0028] Example 2: Three-dimensional induction system regulates the directional differentiation of human mesenchymal stem cells into neural precursor cells in vitro

[0029] First, arginyl-glycyl-aspartic acid (RGD) modified sodium alginate was prepared. Sodium alginate (molecular weight 500kDa, ratio of guluronic acid to mannuronic acid 2) was dissolved in 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 0.5M NaCl (pH 6.5), to obtain 1% (W / V, g / ml) sodium alginate solution. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and RGD polypeptide, and stir at room temperature for 24 hours. The molar ratio of EDC to sodium alginate is 1:20, the molar ratio of EDC to sulfo-NHS is 2:1, and the mass ratio of RGD polypeptide to sodium alginate is 1:1000. Then dialyzed and freeze-dried to obtain RGD-modified sodium alginate.

[0030] RGD modified sodium alginate powder was dissolved in saline to obtain a 3% (W / V, g / ml) solu...

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Abstract

The invention relates to the technical field of regenerative medicine and discloses a method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells. The method includes following steps: embedding Stem cells into calcium alginate micro gel beads containing chondroitin sulfate, adding an inductive differentiation culture liquid, adjusting and controlling the in vitro directed differentiation of the stem cells by changing a mass ratio between the sodium alginate and the chondroitin sulfate during the preparation of the micro gel beads, dissolving the calcium alginate micro gel beads in a sodium citrate solution after the differentiation process being finished, and then obtaining pure differentiation cells. The method is simple in operation and low in cost, the calcium alginate micro gel beads containing chondroitin sulfate can simulate an in vivo three-dimensional extracellular matrix so that a three-dimensional directed differentiating microenvironment which approximates an in vivo microenvironment is supplied for the stem cells, thereby improving directed differentiation efficiency and a biological function of the stem cells. The method plays an important role in applications of regenerative medicine.

Description

technical field [0001] The invention relates to the field of regenerative medicine, and is a method for regulating three-dimensional directional differentiation of stem cells in vitro. Background technique [0002] Stem cells are ideal seed cells for repairing damaged tissues due to their ability to self-renew and differentiate. However, when stem cells are implanted in the damaged part of the body, in addition to differentiating into the required tissue cells and participating in tissue repair, the stem cells also differentiate into other types of cells, and even have high tumorigenicity. Pre-differentiation of stem cells in vitro, so that they can be pre-differentiated into mature tissue cells or precursor cells before transplantation, can improve the efficiency of directed differentiation, reduce the ability of stem cells to differentiate into other types of cells in vivo, thereby improving the effect of tissue repair and reducing Tumor risk. [0003] In vivo cells grow...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/077C12N5/079C12N11/10
Inventor 马小军刘洋王淑君孙广炜
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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