# US 10,246,682 B2 · Methods of culturing retinal pigmented epithelium cells, including Xeno-free production, RPE enrichment, and cryopreservation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,682 B2 - Document ID: US10246682B2 - Status: Granted - Kind: B2 - Application number: 15/816,912 - Filed: 2017-11-17 - Published: 2018-03-29 - Granted: 2019-04-02 - Earliest claimed: 2012-02-01 - Assignees: The Regents of the University of California; University of Southern California - Applicants: The Regents of the University of California - Inventors: Dennis Clegg; Sherry T. Hikita; Lincoln V. Johnson; Liane Miller; Marlene Tsie; Chelsea D. Presbrey; Lisa R. Conti; Michelle A. Maloney; David R. Hinton; Qirui Hu - Web page: https://patentgrep.com/patent/US10246682B2 ## CPC - C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)} - C12N 2500/98: Xeno-free medium and culture conditions - C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2) - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2506/02: from embryonic cells - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The method of claim 1, wherein the dissociation agent solution is a xeno-free composition. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) The method of claim 1, wherein the buffer or medium is Dulbecco's Phosphate Buffered Saline. ### Claim 6 (depends on 1) The method of claim 1, wherein the first xeno-free substrate and the second xeno-free substrate comprise a non-cellular substrate. ### Claim 7 (depends on 6) The method of claim 6, wherein the non-cellular substrate is human or recombinant vitronectin. ### Claim 8 (depends on 6) The method of claim 6, wherein the non-cellular substrate is parylene membrane coated with vitronectin, laminin, fibronectin, or peptides derived therefrom. ### Claim 9 (depends on 6) The method of claim 6, wherein the non-cellular substrate is a vitronectin-derived peptide coated with laminin, fibronectin, or peptides derived therefrom. ### Claim 10 (depends on 1) The method of claim 1, further comprising the step of adding antibiotics subsequent to washing the substrate with buffer or medium. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 1) The method of claim 1, further comprising the step of quantifying a concentration of the mature RPE cells. ### Claim 13 (depends on 12) 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. ### Claim 14 (depends on 1) The method of claim 1, wherein the pluripotent cells are human cells. ### Claim 15 (depends on 1) The method of claim 1, wherein the xeno-free growth media are MX-302 media or E8 media. ### Claim 16 (depends on 1) 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. ### Claim 17 (depends on 16) 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. ### Claim 18 (depends on 1) 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. ### Claim 19 (depends on 18) 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.