Method for purification of retinal pigment epithelial cells
Inventors
SAWADA, Masanori • Sekiguchi, Kiyotoshi
Assignees
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Publication Number
US-11492593-B2
Publication Date
2022-11-08
Expiration Date
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.
Core Innovation
The invention relates to purifying and producing retinal pigment epithelial cells from pluripotent stem cells by inducing differentiation on a laminin-511E8 fragment. The cell population contains retinal pigment epithelial cells without forming a mixture with a jelly-like substance, and also contains other visual cells contained in the jelly-like substance.
The method recovers the entire differentiated cell population by treating it with a cell separating solution. The method dissociates adhesion between retinal pigment epithelial cells contained in the entire cell population, and then introduces the entire cell population onto a filter to obtain retinal pigment epithelial cells that pass the filter.
The filter pore size is 15-100 μm. The document states that purity and yield are improved, and that downstream production of retinal pigment epithelial cells and a retinal pigment epithelial cell sheet is enabled for therapeutic and drug-evaluation uses.
Claims Coverage
The document contains two independent claims. Both independent claims share a common multi-step inventive concept: inducing differentiation on a laminin-511E8 fragment, obtaining a recovered cell population that avoids mixture with a jelly-like substance, treating with a cell separating solution, dissociating adhesion, and filtering with a defined filter pore size range of 15-100 μm.
Differentiation on laminin-511E8 fragment to obtain a jelly-like mixture-free RPE cell population
Inducing differentiation of pluripotent stem cells into a cell population on laminin-511E8 fragment, wherein the cell population contains retinal pigment epithelial cells without forming a mixture with a jelly-like substance and other visual cells contained in the jelly-like substance.
Recovery by treating with a cell separating solution
Recovering the entire cell population obtained in the differentiation step by treating it with a cell separating solution.
Dissociating adhesion between retinal pigment epithelial cells
Dissociating adhesion between retinal pigment epithelial cells contained in the entire cell population obtained after treating with a cell separating solution.
Filtering with a defined pore size range to obtain cells that pass
Introducing the entire cell population obtained after dissociating adhesion on a filter to obtain retinal pigment epithelial cells that passed the filter, wherein the filter has a pore size of 15-100 μm.
Producing retinal pigment epithelial cells using the same purification workflow
Inducing differentiation of pluripotent stem cells into a cell population on laminin-511E8 fragment; recovering the entire cell population by treating with a cell separating solution; dissociating adhesion between retinal pigment epithelial cells; and introducing the entire cell population on a filter to obtain retinal pigment epithelial cells that passed the filter, wherein the filter has a pore size of 15-100 μm.
Across both independent claims, the inventive coverage centers on differentiating pluripotent stem cells on a laminin-511E8 fragment to obtain a cell population defined by the absence of an RPE/jelly-like mixture, followed by recovering with a cell separating solution, dissociating adhesion, and filtering with a 15-100 μm pore size to obtain retinal pigment epithelial cells that pass the filter.
Stated Advantages
Improvement of differentiation/purity yield of retinal pigment epithelial cells.
Purification purity of about 80-95% and higher purity after re-filtration/amplification.
Documented Applications
Downstream production of retinal pigment epithelial cells/sheets for retinal disease transplantation therapy.
Downstream production of retinal pigment epithelial cells/sheets for drug-evaluation uses.
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