Methods of culturing retinal pigmented epithelium cells, including xeno-free production, RPE enrichment, and cryopreservation
The production of high quality retinal pigmented epithelium (RPE) cells is necessary for research and potential therapeutic uses. Especially desirable are methods for the production of RPE cells using xeno-free culture conditions. Disclosed herein are novel methods for the production of RPE cells from pluripotent cells with high yields, including xeno-free production methods. Also provided are methods of efficiently isolating RPE cells from cultures containing heterogeneous cell types, allowing for substantially pure RPE cell cultures to be established. Additionally, novel methods for the cryopreservation of RPE cells are provided.
1. 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.
2. The method of claim 1 , wherein
the pluripotent cells are human cells.
3. The method of claim 1 wherein,
the first non-cellular xeno-free substrate is selected from the group consisting of human or recombinant vitronectin, laminin, poly-lysine-D, acrylate functionalized with peptides, parylene membrane coated with human or recombinant vitronectin, parylene membrane coated with laminin, and parylene membrane coated with human or recombinant fibronectin.
4. The method of claim 1 wherein
the xeno-free growth medium is MX-302 medium or E8 medium.
5. The method of claim 1 wherein
the one or more compositions which substantially maintains pluripotent cells in a pluripotent state is basic fibroblast growth factor.
6. The method of claim 5 , wherein
basic fibroblast growth factor is present in the growth medium at a concentration between 50 and 100 ug/ml.
7. The method of claim 1 wherein the one or more compositions which substantially maintains pluripotent cells in a pluripotent state is transforming growth factor Beta-1.
8. The method of claim 7 wherein the transforming growth factor Beta-1 is present in the growth medium at a concentration of 0.1 ng/ml to 1 ng/ml.
9. The method of claim 3 , wherein
the selected xeno-free substrate is human or recombinant vitronectin.
10. The method of claim 3 , wherein
the selected xeno-free substrate is parylene membrane coated with human or recombinant vitronectin.