Method for purification of retinal pigment epithelial cells

Patent metadata

Field	Value
Publication number	US-11492593-B2
Application number	US-201415028158-A
Country	US
Kind code	B2
Filing date	Oct 9, 2014
Priority date	Oct 9, 2013
Publication date	Nov 8, 2022
Grant date	Nov 8, 2022

Abstract

The present invention provides a method of purifying highly pure retinal pigment epithelial cells from a cell population obtained by induction of differentiation of pluripotent stem cells into retinal pigment epithelial cells, by a simple and easy operation in a short period. The purification method of the present invention includes a step of introducing a cell population containing retinal pigment epithelial cells obtained by differentiation induction of pluripotent stem cells on laminin or a fragment thereof on a filter, and obtaining a cell population that passed the filter.

First claim

The invention claimed is:

1. A method of purifying a retinal pigment epithelial cell, comprising
(1) a step of inducing differentiation of pluripotent stem cells into a cell population on laminin-511E8 fragment, wherein the cell population contains (a) retinal pigment epithelial cells without forming a mixture with a jelly-like substance and (b) other visual cells contained in the jelly-like substance,
(2) a step of recovering the entire cell population containing (a) and (b) obtained in step (1) by treating it with a cell separating solution,
(3) a step of dissociating adhesion between retinal pigment epithelial cells contained in the entire cell population containing (a) and (b) obtained in step (2),
(4) a step of introducing the entire cell population containing (a) and (b) obtained in step (3) on a filter, and
(5) a step of obtaining retinal pigment epithelial cells that passed the filter, wherein the filter has a pore size of 15-100 μm.

2. The method according to claim 1 , wherein the step of dissociating adhesion between retinal pigment epithelial cells is reciprocating pipetting several times.

3. The method according to claim 1 , wherein the cell separating solution comprises trypsin.

4. The method according to claim 1 , wherein the filter has a pore size of 20-70 μm.

5. The method according to claim 1 , wherein step (3) further comprises, after dissociating adhesion between retinal pigment epithelial cells contained in the cell population, removing the cell separating solution and residual impurities thereof and matrix components in the cell population together with supernatant by centrifugation.

6. A method of producing a retinal pigment epithelial cell from a pluripotent stem cell, comprising
(1) a step of inducing differentiation of pluripotent stem cells into a cell population on laminin-511E8 fragment, wherein the cell population contains (a) retinal pigment epithelial cells without forming a mixture with a jelly-like substance and (b) other visual cells contained in the jelly-like substance,
(2) a step of recovering the entire cell population containing (a) and (b) obtained in step (1) by treating it with a cell separating solution,
(3) a step of dissociating adhesion between retinal pigment epithelial cells contained in the entire cell population containing (a) and (b) obtained in step (2), and
(4) a step of introducing the entire cell population containing (a) and (b) obtained in step (3) on a filter to obtain retinal pigment epithelial cells that passed the filter, wherein the filter has a pore size of 15-100 μm.

7. The method according to claim 6 , wherein the step of dissociating adhesion between retinal pigment epithelial cells is reciprocating pipetting several times.

8. The method according to claim 6 , wherein the cell separating solution comprises trypsin.

9. The method according to claim 6 , wherein the filter has a pore size of 20-70 μm.

10. The method according to claim 6 , wherein step (3) further comprises, after dissociating adhesion between retinal pigment epithelial cells contained in the cell population, removing the cell separating solution and residual impurities thereof and matrix components in the cell population together with supernatant by centrifugation.

Assignees

• Healios Kk
• Univ Osaka

Inventors

• Sawada Masanori
• Sekiguchi Kiyotoshi

Classifications

• C12N5/0621 Primary

  Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062) · CPC title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• A61K9/0048

  Eye, e.g. artificial tears · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title