Methods of culturing retinal pigmented epithelium cells, including xeno-free production, RPE enrichment, and cryopreservation

A method of producing RPE cells from pluripotent cells under xeno-free culture conditions, comprising

• (a) culturing pluripotent cells on a first non-cellular xeno-free substrate in a xeno-free growth medium supplemented with one or more compositions which substantially maintains pluripotent cells in a pluripotent state, wherein the pluripotent cells are embryonic stem cells or induced pluripotent stem cells;
• (b) replacing the growth medium with a xeno-free growth medium lacking any composition which maintains cells in a pluripotent state, wherein the cultured cells have not reached confluence are not overgrown at the time of such replacement of growth medium;
• (c) allowing the cultured cells on the first non-cellular xeno-free substrate to undergo spontaneous differentiation, which results in some of the pluripotent cells differentiating into RPE cells;
• (d) physically separating the RPE cells from the first non-cellular xeno-free substrate;
• and
• (e) culturing the isolated RPE cells on a second non-cellular xeno-free substrate in xeno-free growth medium.