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
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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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