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 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.
2. The method of claim 1 , wherein
the dissociation agent is selected from the group consisting of trypsin, a trypsin-like enzyme, collagenase, dipsase, and EDTA.
3. The method of claim 1 , wherein
the dissociation agent solution is a xeno-free composition.
4. The method of claim 1 , wherein
the first time interval during which the cells are exposed to a solution comprising a dissociation agent does not exceed six minutes.
5. The method of claim 1 , wherein
the buffer or medium is Dulbecco's Phosphate Buffered Saline.
6. The method of claim 1 , wherein
the first xeno-free substrate and the second xeno-free substrate comprise a non-cellular substrate.
7. The method of claim 6 , wherein
the non-cellular substrate is human or recombinant vitronectin.
8. The method of claim 6 , wherein
the non-cellular substrate is parylene membrane coated with vitronectin, laminin, fibronectin, or peptides derived therefrom.
9. The method of claim 6 , wherein
the non-cellular substrate is a vitronectin-derived peptide coated with laminin, fibronectin, or peptides derived therefrom.
10. The method of claim 1 ,
further comprising the step of adding antibiotics subsequent to washing the substrate with buffer or medium.
11. The method of claim 1 ,
further comprising the step of filtering a suspension of the isolated RPE cells subsequent to physically separating the mature RPE cells from the first xeno-free substrate.
12. The method of claim 1 ,
further comprising the step of quantifying a concentration of the mature RPE cells.
13. The method of claim 12 , wherein
the quantification of the concentration of the mature RPE cells is accomplished using a cell counter with a gating diameter set between 6 μm and 20 μm.
14. The method of claim 1 , wherein
the pluripotent cells are human cells.
15. The method of claim 1 , wherein
the xeno-free growth media are MX-302 media or E8 media.
16. 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.
17. The method of claim 16 , wherein
the basic fibroblast growth factor is present in the xeno-free growth medium at a concentration between 50 μg/ml and 100 μg/ml.
18. 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.
19. The method of claim 18 , wherein
the transforming growth factor Beta-1 is present in the xeno-free growth medium at a concentration between 0.1 ng/ml and 1 ng/ml.