Mixed Cell Population iPS Generation from Limited Blood

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Solution Overview

Problem

Current methods for generating induced pluripotent stem cells (iPS cells) from donor blood face challenges due to limited blood volume, low abundance of specific cell types, and low iPS cell establishment efficiency, leading to difficulties in obtaining sufficient cells and differentiating them into target cells, which can result in the need for repeated blood collection from donors.

Innovation Solution

A method involving the sequential extraction and reprogramming of monocytes, CD34-positive cells, and T cells from peripheral blood mononuclear cells, using density-gradient centrifugation and flow cytometry for separation, and electroporation or lipofection for reprogramming, with specific culture media to enhance cell proliferation and iPS cell generation efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If a single cell type (e.g., CD34-positive cells or monocytes) is used for iPS cell generation, then the DNA information of iPS cells matches the donor, but the iPS cell establishment efficiency is low and sufficient cells cannot be obtained

Engineering Contradiction:

ImproveDNA information match between iPS cells and donorVSAvoidiPS cell establishment efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent combines multiple cell types (CD34-positive cells, monocytes, and T cells) from the same blood sample into a mixed cell population for reprogramming. This merging approach increases the total number of cells available for iPS cell generation while maintaining DNA match with the donor, thereby resolving the contradiction between reliability and productivity.

Inventive Principle:

Principle #5Merging (Combining)

2Ease of manufacture

If T cells are used for iPS cell generation, then the process is simple, but recombination in T cell receptor genes causes DNA information to differ from the donor

Engineering Contradiction:

ImproveiPS cell generation process simplicityVSAvoidDNA information match between iPS cells and donor

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent merges T cells with other blood cell types (CD34-positive cells and/or monocytes) to create a mixed cell population for reprogramming. This approach maintains the ease of using T cells while compensating for their DNA recombination issue by including other cell types that preserve donor DNA information, thus resolving the contradiction between ease of manufacture and reliability.

Inventive Principle:

Principle #5Merging (Combining)

3Quantity of substance

If blood volume from donor is limited, then repeated blood collection is necessary, but this increases burden on donor and time for establishing iPS cells

Engineering Contradiction:

Improveavailable blood volumeVSAvoidtime for establishing iPS cells

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The patent merges multiple cell types from a single limited blood sample to maximize the utility of available blood volume. By utilizing CD34-positive cells, monocytes, and T cells together, the method increases the number of usable cells without requiring additional blood collections, thereby reducing both donor burden and overall establishment time.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent performs preliminary separation and preparation of multiple cell types from the blood sample before reprogramming. This preliminary action ensures that all available cell types are utilized efficiently, maximizing the output from limited blood volume and avoiding the need for repeated collections.

Inventive Principle:

Principle #10Preliminary action

4Reliability

If monocytes are used for iPS cell generation, then DNA information matches the donor, but iPS cell establishment efficiency is low

Engineering Contradiction:

ImproveDNA information match between iPS cells and donorVSAvoidiPS cell establishment efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent combines monocytes with other blood cell types (CD34-positive cells and/or T cells) to create a mixed cell population. This merging increases the total cell number available for reprogramming while maintaining the DNA match advantage of monocytes, thereby resolving the contradiction between reliability and productivity.

Inventive Principle:

Principle #5Merging (Combining)

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

This approach increases the probability of establishing iPS cells, reduces the risk of failure, and improves differentiation efficiency into target cells, thereby reducing the burden on donors by effectively utilizing limited blood samples and expanding the range of cell applications.

Implementation Method 1

extracting monocytes from mononuclear cells separated from whole blood

Methodology Applied

Scientific EffectDensity-gradient centrifugation: Centrifugal Separation

Implementation Method 2

using density-gradient centrifugation and flow cytometry for separation

Methodology Applied

Scientific EffectFlow cytometry:

Data Source

PatentUS20240344036A1METHOD OF GENERATING iPS CELL AND iPS CELL GENERATION SYSTEM

Publication Date: 2024.10.17 CANON KK

AI summary

According to one embodiment, a method of generating an iPS cell includes extracting monocytes from mononuclear cells separated from whole blood to separate the mononuclear cells into the monocytes and non-monocytes, reprogramming the separated monocytes to generate a monocyte-derived iPS cell, and culturing the separated non-monocytes in a first culture medium to prepare first raw material cells for iPS cell generation.