Method of producing retinal pigment epithelial cell

a retinal pigment and epithelial cell technology, applied in cell dissociation methods, cell culture active agents, drug compositions, etc., can solve the problems of easy cell loss, high workload of purification steps, easy cell loss, etc., to improve differentiation induction efficiency, simple and easy purification operation, and high yield

Pending Publication Date: 2016-08-18
HEALIOS KK +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]According to the present invention, retinal pigment epithelial cells can be produced in a high yield from pluripotent stem cells. In addition, differentiation induction efficiency is improved, and a cell population containing retinal pigment epithelial cells at a high concentration can be obtained, and highly pure retinal pigment epithelial cells can be produced by a simple and easy purification operation. According to the present invention, moreover, since retinal pigment epithelial cells can be stably adhered, the cells are not easily lost during medium exchange, and can be stably passaged.

Problems solved by technology

However, due to the low differentiation induction efficiency, these methods require plural steps combining adhesion culture and floating culture to obtain a highly concentrated cell population of retinal pigment epithelial cells, and have problems such as the required presence of a purification step with a high workload and a long time, which includes selectively picking up a colony of pigment cells under an optical microscope.
Moreover, these methods have a problem of easy cell loss during passage culture.
However, no report has documented utilization of such E8 fragment of laminin for other than culture of pluripotent stem cells, for example, differentiation induction of pluripotent stem cells and the like.
However, no report has documented use of the E8 fragment of laminin for the induction of differentiation of pluripotent stem cell into retinal pigment epithelial cell.

Method used

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  • Method of producing retinal pigment epithelial cell
  • Method of producing retinal pigment epithelial cell
  • Method of producing retinal pigment epithelial cell

Examples

Experimental program
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Effect test

example 1

Production of RPE Cell Derived from iPS Cell

Reagents

[0069]differentiation induction basic medium (GMEM medium (Invitrogen), KSR (Invitrogen), 0.1 mM MEM non-essential amino acid solution (Invitrogen), 1 mM pyruvic acid sodium (SIGMA), 0.1 M 2-mercaptoethanol (Wako Pure Chemical Industries, Ltd.), 100 U / ml penicillin-100 μg / ml streptomycin (Invitrogen))[0070]primary differentiation induction medium (differentiation induction basic medium containing 20% KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))[0071]secondary differentiation induction medium (differentiation induction basic medium containing 15% KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))[0072]tertiary differentiation induction medium (differentiation induction basic medium containing 10% KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))[0073]quaternary differentiation i...

example 2

Production of RPE Cell (Other iPS Cell)

[0079]By a method similar to that of Example 1 except that iPS cells (201B7, provide by Kyoto University) derived from human skin (fibroblast) were used instead of iPS cells (112007, provide by Kyoto University) derived from human peripheral blood (mononuclear cell) were used, pigment cells were obtained.

[0080]As a result, like Example 1, the rate of generation of pigment cells relative to the seeded iPS cells was drastically improved and the differentiation induction efficiency was markedly improved.

example 3

Amplification of RPE Cell Derived from iPS Cell

[0081]The cell population containing pigment cells on Day 47, which underwent adhesion culture in the culture dish in Example 1 and Example 2, was treated with 0.01% Trypsin-0.53 mM EDTA and cell aggregates were detached from the culture dish. Then, adhesion between the cells was detached by mild pipetting. Protease liquid and residual impurities thereof in the cell mixture were removed together with the supernatant by centrifugation, then, unnecessary cells were separated by filtration separation through a cell strainer (DB Falcon Cell Strainer 40 μm Nylon), and a cell population containing RPE cells was recovered (Day 48).

[0082]The obtained cells were seeded in RPE maintenance medium described in Example 1 in the same 5 culture dishes coated with laminin-511 E8 as in Example 1 at 9×106 cells / 9 cm dish, and standing culture was performed until around Day 50 when adhesion of the RPE cell colony was confirmed.

[0083]From Day 51 to Day 71,...

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Abstract

Provided are a production method of retinal pigment epithelial (RPE) cells that improves differentiation induction efficiency of pluripotent stem cells into RPE cells, and can provide highly pure RPE cells by a simple and easy operation in a short period, a culture method of RPE cells that can stably grow and culture a cell, a toxicity / efficacy evaluation method using RPE cells useful for transplantation therapy, and a therapeutic drug for a retinal disease. The invention relates to a production method of RPE cells, comprising adhesion culture of human pluripotent stem cells using a culture substrate coated with a laminin-E8 fragment, a culture method of RPE cells, comprising adhesion culture of RPE cells using a culture substrate coated with a laminin-E8 fragment, a toxicity or efficacy evaluation method using RPE cells obtained by producing or culturing by the method, and a therapeutic drug for a retinal disease, containing the RPE cells.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of efficiently producing retinal pigment epithelial (RPE) cells from human pluripotent stem cells, a method of stably culturing retinal pigment epithelial cells, and the like.Background Art[0002]As a method for producing retinal pigment epithelial cells from pluripotent stem cells, a method called SFEB method including culturing ES cells as a floating aggregate in a serum-free medium (patent document 1 etc.), a method including inducing differentiation of pluripotent stem cells in the presence of a differentiation-inducing factor on a culture substrate coated with a weakly cell adhesive coating agent and the like (non-patent document 1 etc.) are known. However, due to the low differentiation induction efficiency, these methods require plural steps combining adhesion culture and floating culture to obtain a highly concentrated cell population of retinal pigment epithelial cells, and have problems such as the required pres...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/079A61K9/00A61K35/30
CPCA61K35/30C12N5/0621C12N2506/45C12N2509/00A61K9/0048C12N2501/727C12N2533/52A61P27/02
Inventor SAWADA, MASANORITAKAHASHI, MASAYOSEKIGUCHI, KIYOTOSHI
Owner HEALIOS KK
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