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 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 xeno-free 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 at the time of such replacement of growth medium;
• (c) allowing the cultured cells on the first xeno-free substrate to undergo spontaneous differentiation, which results in some of the pluripotent cells differentiating into RPE cells;
• (d) treating the cultured cells on the first xeno-free substrate with a solution comprising a dissociation agent for a first interval of time, removing the solution comprising the dissociation agent, and immediately thereafter washing the first xeno-free substrate with buffer or medium;
• (e) physically separating cells other than mature RPE cells from the first xeno-free substrate;
• (f) treating the cultured cells remaining on the first xeno-free substrate with a solution comprising a dissociation agent for a second interval of time;
• (g) physically separating the mature RPE cells from the first xeno-free substrate to yield isolated RPE cells;
• and
• (h) culturing the isolated RPE cells on a second xeno-free substrate in xeno-free growth medium.