3d suspension method for generating autologous melanocyte by inducing ips cells and application thereof

A method of inducing iPS cells to generate autologous melanocytes by using 3D suspension system, characterized in that, said method comprises the following steps:

• a) embryoid body formation by using single cell method
• iPS single cell dissociation enzyme is added into iPS clones for dissociation;
• mTeSR medium is added and cells are pipetted gently to form iPS single cell suspension;
• after centrifugation, the supernatant is discarded, then mTeSR medium is added to cell pellet for resuspension;
• iPS single cells are counted and inoculated into three dimensional culture plate;
• ROCK inhibitor is added;
• after culture, embryo bodies having uniform morphology and size are obtained;
• the embryo bodies are aspirated by gentle pipette and transferred to a low-attachment plate for continued culture, and the medium is changed every day;
• and
• b) 3D suspension differentiation:
• (1) 3D early-stage differentiation:
• the embryoid bodies obtained in step a) are transferred into differentiation medium for early-stage differentiation,
• (2) mid-stage attached differentiation:
• after the early-stage differentiation in the above step b)(1), the embryoid bodies are transferred to a fibronectin-coated culture plate for mid-stage attached culture, the differentiation medium components remain unchanged, and the embryoid bodies attach to the plate and grow, and
• (3) late-stage differentiation:
• after attached culture in the above step b)(2), the embryoid bodies are dissociated into single cells, inoculated into a fibronectin-coated culture plate, and subjected to late maturation induction in differentiation medium, when the cell density reaches 90%, passage is performed with dissociation enzymes, and mature melanocytes are obtained after 35 to 42 days of differentiation.