A method of three-dimensional culture and differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells

A kind of stem cell, three-dimensional culture technology, applied in the direction of animal cells, nervous system cells, vertebrate cells, etc., to achieve the effect of maintaining self-renewal and multi-directional differentiation characteristics, promoting connection, and strong cell proliferation ability

Active Publication Date: 2016-03-09
冯文峰
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the use of three-dimensional culture to induce human adipose-derived mesenchymal stem cells to differentiate into neural-like stem cells

Method used

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  • A method of three-dimensional culture and differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells
  • A method of three-dimensional culture and differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells
  • A method of three-dimensional culture and differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Obtain human adipose-derived mesenchymal stem cells in vitro.

[0020] Take 5g of adipose tissue, cut it into pieces with sterile surgical scissors or a small knife, and the length should not exceed 3mm, then collect it in a 15ml centrifuge tube, add an equal volume of phosphate buffer, shake it vigorously, and let it stand at room temperature for 5 minutes. After being divided into two layers, carefully collect the upper layer (including mesenchymal stem cells, adipocytes, phosphate buffered saline and red blood cells), remove the oil / lipid in the lower layer, and wash the collected upper layer 3 times with phosphate buffered saline; Add an equal volume of 0.075% collagenase I, and bathe in water at 37°C for 30 minutes; add an equal volume of DMEM containing 10% fetal bovine serum to stop digestion, mix well, and leave at room temperature for 10 minutes to separate layers; remove the upper layer (lipid / debris), 20°C, centrifuge at 280g for 5min to collect the lowe...

Embodiment 2

[0027] (1) Obtaining isolated human adipose-derived mesenchymal stem cells: same as in Example 1.

[0028] (2) Inducing the differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells.

[0029] Polymerization of three-dimensional scaffolds: Add 2.5mg / mL polylactic acid solution to a 12-well culture plate, 25 microliters / well, and polymerize in a 37°C incubator for 30 minutes to obtain a porous polymerized three-dimensional scaffold.

[0030] Add the mixed human adipose-derived mesenchymal stem cell suspension to the three-dimensional scaffold that has been polymerized to make the cell density 5×10 5 cells / well. Add human adipose-derived mesenchymal stem cell culture medium (L-DMEM+10%FBS+100u / L penicillin+100u / L streptomycin), and place in an incubator at 37°C, 5%CO 2 Culture was carried out, and the culture medium was replaced every other day.

[0031] (3) Obtain neural-like stem cells.

Embodiment 3

[0033] (1) Obtaining isolated human adipose-derived mesenchymal stem cells: same as in Example 1.

[0034] (2) Inducing the differentiation of human adipose-derived mesenchymal stem cells into neural-like stem cells.

[0035] Polymerization of three-dimensional scaffolds: Add 5mg / mL silk fibroin solution to a 12-well culture plate, 25 microliters / well, and polymerize in a 37°C incubator for 30 minutes to obtain a porous polymerized three-dimensional scaffold.

[0036] Add the mixed human adipose-derived mesenchymal stem cell suspension to the three-dimensional scaffold that has been polymerized to make the cell density 5×10 5 cells / well. Add human adipose-derived mesenchymal stem cell culture medium (L-DMEM+10%FBS+100u / L penicillin+100u / L streptomycin), and place in an incubator at 37°C, 5%CO 2 Culture was carried out, and the culture medium was changed every other day.

[0037] (3) Obtain neural-like stem cells.

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Abstract

The invention discloses a method for differentiation of human adipose tissue-derived stromal cells into neuron-like stromal cells through three-dimensional culture. The method comprises the following steps of focusing a three-dimensional material at 37 DEG C for 30 minutes, next, adding the human adipose tissue-derived stromal cells, culturing with 5% of CO2 at 37 DEG C, and replacing a culture solution every other day, wherein the focused solution is a type I collagen solution, a polylactic acid solution or a silk fibroin solution. The method is characterized in that an in-vivo like three-dimensional growing environment is provided for neural differentiation through the three-dimensional culture technology, so that connection between cells is promoted, cells grow at a plurality of layers, and in the meantime, the self-renewal and multi-directional differentiation characteristics of the stromal cells can be maintained better; without the action of a chemical inductor factor, the differentiated neuron-like stromal cells can be directly applied to subsequent clinical tests, and are safer.

Description

technical field [0001] The invention relates to a method for inducing human adipose-derived mesenchymal stem cells to differentiate into neural-like stem cells, in particular to a method for human adipose-derived mesenchymal stem cells without chemical induction factors to differentiate into neural-like stem cells after three-dimensional culture. Background technique [0002] Neural stem cells (neural stem cells, NSCs) are a kind of mother cells with division potential and self-renewal ability, which can produce various types of cells of nerve tissue through asymmetric division. For a long time, it has been believed that the nerve cells in the adult mammalian brain do not have the ability to regenerate, and cannot regenerate once damaged or even dead. This point of view has greatly limited the treatment of central nervous system diseases. Although traditional medicine, surgery and rehabilitation have made some progress, they still cannot achieve satisfactory results. The d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797
Inventor 冯文峰佘志勇方海庆陈建兴
Owner 冯文峰
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