Method for differentiating MSC into Schwann cells, culture medium and system
A cell culture and culture medium technology, applied in the field of cell biology, can solve the problems of consumption of embryonic tissue, limited access to SC, and immature preparation process steps.
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[0061] (g) Preparation of peripheral neuronal cells (iPN)
[0062] Peripheral neural stem cells (NP) were treated with 0.5~3.0×10 4 piece / cm 2 Inoculate the cell culture vessel treated with recombinant laminin (Lm, animal-free, instead of mouse-derived Matrigel, etc.), using animal-free peripheral nerve induction medium-02 (pNiM-02AF) After culturing, the culture medium is replaced every 24-36 hours, and the peripheral neuron cells (iPN) are analyzed after 10-12 days of culture to detect their purity and yield.
[0063] S3: Differentiation of mesenchymal stem cell-derived neural precursor cells into Schwann cells
[0064] Neural precursor cells (NPC) derived from mesenchymal stem cells were mixed with 25 NPC clumps / cm 2 Inoculated at the density of recombinant laminin (Lm)-treated cell culture dishes, cultured with animal-derived Schwann cell induction medium-01 (SCiM-01AF) for 8 to 10 days, and then treated with trypsin substitutes And collect the cells, with 0.5~1.5×10 ...
Embodiment 1
[0067] Example 1 Culture of mesenchymal stem cells and preparation of neural precursor cells
[0068] (1) Isolation of mesenchymal stem cells (MSCs) and primary culture;
[0069] (2) subculture and seed the mesenchymal stem cells after primary culture on the surface of the cell culture substrate, add cell culture medium, and perform the first subculture, and the cell culture substrate is treated with "promoting cell adhesion";
[0070] (3) Subculture and seed the mesenchymal stem cells after the first subculture on the surface of the cell culture substrate, add serum-free cell culture medium, and carry out the second subculture. The cell culture substrate has not undergone "promoting cell "Adherence" treatment; the formulation of the serum-free cell culture medium is shown in Table 1.
[0071] Among them, cell culture substrates, such as cell culture dishes and cell culture plates, are made of polystyrene after the treatment of promoting cell adhesion (Tc-Treated, referred to...
Embodiment 2
[0075] Example 2 Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Peripheral Neuronal Cells
[0076] (1) Culture of iPSCs
[0077] Divide iPSCs at 1.5×10 4 piece / cm 2 The density is inoculated on the cell culture dish (the example culture dish: 6-well plate) that is processed through vitronectin (Vc), uses E8 to cultivate (the amount of the example: 2mL per well of the 6-well plate), and replaces the culture every 24 hours Base, after 3 to 4 days, iPSCs were treated with EDTA (0.5mM) when the fusion degree of iPSCs reached 60 to 80% (EDTA treatment time range: 3 to 8 minutes, example treatment time: 5 minutes), and the iPSCs were collected. For subculture, the same 1.5×10 4 piece / cm 2 The density was inoculated on the Vc-treated cell culture dish.
[0078] (2) NP differentiation of iPSCs
[0079] Convert iPSCs to 2.0 × 10 4 piece / cm 2 The density was inoculated on cell culture dishes treated with recombinant laminin (Lm), using animal-derived peripheral n...
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