RPE Cell Selection via Autofluorescence and FACS

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Solution Overview

Problem

Current protocols for deriving retinal pigmented epithelial (RPE) cells from pluripotent stem cells yield mixed populations, making it difficult to obtain pure populations of pigmented cells for research and therapy due to operator-dependent and inconsistent mechanical dissection methods.

Innovation Solution

A method using fluorescence-activated cell sorting (FACS) to select RPE cells based on autofluorescence, CD81 expression, and light scattering properties, combined with specific markers like bestrophin and CRALBP, to achieve pure populations of RPE cells from mixed cell cultures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If mechanical dissection is used to isolate RPE cells, then operator-dependent isolation can be performed, but the method is work intensive, inconsistent, and cannot provide pure populations of pigmented cells

Engineering Contradiction:

Improveease of isolationVSAvoidpurity of RPE cell population

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The patent replaces mechanical dissection with fluorescence-activated cell sorting (FACS), substituting a manual mechanical process with an automated optical sorting system. This allows operator-independent isolation of RPE cells based on their autofluorescence properties, eliminating the inconsistency and subjectivity of manual methods while achieving pure populations of pigmented cells

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent utilizes the autofluorescence parameter of RPE cells (due to melanin content) to differentiate and sort them from non-pigmented cells. By detecting and sorting based on this optical parameter, the method achieves high purity without mechanical intervention

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If FACS sorting is attempted on differentiated RPE cells, then cell separation may be achieved, but Klimanskaya et al. teach that it is difficult to dissociate RPE cells following differentiation and accordingly sorting by FACS is not recommended

Engineering Contradiction:

Improvepurity of RPE cell populationVSAvoiddifficulty of cell dissociation and sorting

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent performs FACS sorting at an early stage during the differentiation process, before RPE cells fully differentiate and develop strong cell-cell adhesion. This preliminary sorting action avoids the dissociation difficulties that arise when attempting to sort mature, tightly adherent RPE cells

Inventive Principle:

Principle #10Preliminary action

3Productivity

If clusters of RPE cells are mechanically dissected and isolated based on pigmentation, then isolation can be performed, but the approach is operator dependent, work intensive, and inaccurate

Engineering Contradiction:

Improveisolation efficiencyVSAvoidaccuracy of pigmented cell identification

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent replaces operator-dependent visual identification and mechanical dissection with automated FACS sorting based on autofluorescence detection. This eliminates subjective judgment and provides consistent, accurate identification and isolation of pigmented RPE cells

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The RPE cells' inherent autofluorescence property (self-characteristic) is utilized for their own identification and sorting. The cells effectively sort themselves based on their natural pigmentation without requiring external staining or operator intervention

Inventive Principle:

Principle #25Self-service

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The method effectively purifies RPE cells, ensuring a mature phenotype and expression of specific markers, thereby providing a reliable source for research and therapeutic applications.

Implementation Method 1

cells which autofluorescence above a predetermined threshold

Methodology Applied

Scientific EffectAutofluorescence: Fluorescence

Implementation Method 2

cells which scatter light perpendicular to a laser beam above a predetermined threshold

Methodology Applied

Scientific EffectLight scattering: Scattering

Data Source

PatentUS11035857B2Methods of selecting retinal pigmented epithelial cells

Publication Date: 2021.06.15 HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD

AI summary

A method of selecting retinal pigmented epithelial (RPE) cells from a mixed population of cells is disclosed. The method comprises:(a) analyzing the cells of the mixed population of cells for at least one of the following parameters:(i) cells which autofluorescence above a predetermined threshold;(ii) cells which express CD81 above a predetermined threshold; and(iii) cells which scatter light perpendicular to a laser beam above a predetermined threshold; and(b) selecting cells which are positive for at least one of the parameters, thereby sorting RPE cells from a mixed population of cells.