Stem Cell Culture Using Botulinum Hemagglutinin

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Solution Overview

Problem

Maintaining the undifferentiated state of pluripotent stem cells, such as human iPS cells, is challenging due to the emergence of deviated cells during subculture, which requires complex and expert-operated procedures to remove.

Innovation Solution

Culturing stem cells in the presence of hemagglutinin (HA) from the neurotoxin complex of Clostridium botulinum, which inhibits cell-cell adhesion, allowing for the removal of deviated cells through flotation and apoptosis, thereby maintaining the undifferentiated state.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If manual pipetting operations under microscope are used to remove deviated cells, then undifferentiated state can be maintained, but operation complexity increases and requires expert operators

Engineering Contradiction:

Improvemaintenance of undifferentiated stateVSAvoidoperation complexity

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The patent replaces manual mechanical pipetting operations with a chemical/biological mechanism. Hemagglutinin (HA) from Clostridium botulinum neurotoxin complex is added to the culture medium to selectively inhibit cell-cell adhesion of deviated cells, causing their automatic removal through flotation and apoptosis, eliminating the need for expert manual operations under microscope

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

Hemagglutinin (HA) acts as an intermediary substance between the culture medium and deviated cells. It specifically binds to deviated cells and inhibits their E-cadherin function, causing selective detachment and removal while leaving undifferentiated cells unaffected, thus maintaining colony integrity

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If subculture at low confluence is performed, then frequency of deviated cells decreases, but culture efficiency and productivity are reduced

Engineering Contradiction:

Improvefrequency of deviated cellsVSAvoidculture efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent changes the chemical parameter of the culture medium by adding hemagglutinin (HA) at specific concentrations (e.g., 1-100 μg/mL). This parameter change selectively affects deviated cells by inhibiting their adhesion molecules, causing their removal while allowing undifferentiated cells to maintain normal proliferation rates, thus resolving the trade-off between reliability and productivity

Inventive Principle:

Principle #35Parameter changes

3Reliability

If frequent colony sorting is performed to remove deviated cells, then undifferentiated state is maintained, but time consumption and operational burden increase

Engineering Contradiction:

Improveundifferentiated state maintenanceVSAvoidtime consumption

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent applies preliminary action by adding hemagglutinin (HA) to the culture medium before deviated cells can proliferate and contaminate the culture. The HA continuously acts on the culture, preventing deviated cell emergence and automatically removing them as they appear, eliminating the need for frequent manual sorting operations and saving significant time

Inventive Principle:

Principle #10Preliminary action

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 method enables the efficient removal of deviated cells from stem cell colonies, simplifying the process and allowing for the maintenance of undifferentiated stem cell colonies, even by non-experts, by inhibiting cell-cell adhesion and promoting apoptosis of deviated cells.

Implementation Method 1

hemagglutinin (HA) of a neurotoxin complex of Clostridium botulinum having an E-cadherin function inhibitory activity

Methodology Applied

Scientific EffectE-cadherin function inhibition:

Implementation Method 2

wherein said cell deviated from an undifferentiated state is removed during said culture by flotation and/or apoptosis

Methodology Applied

Scientific EffectApoptosis:

Data Source

PatentEP2940125B1Method for culturing pluripotent stem cells

Publication Date: 2019.05.01 OSAKA UNIVERSITY

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Figure 3A~3D

AI summary

Provided is a method by which cells deviated from the undifferentiated state, which emerge in a colony during culture of stem cells having pluripotency, can be removed. In one aspect, provided is a method for culturing stem cells having pluripotency, the method including performing cell culture in the presence of a substance that can inhibit cell-cell adhesion. In another aspect, provided is a method for removing cells deviated from the undifferentiated state, the cells being cells that have emerged or may possibly emerge during culture of stem cells having pluripotency, the method including performing cell culture in the presence of a substance that can inhibit cell-cell adhesion. In still another aspect, provided is a method for maintaining the undifferentiated state of stem cells having pluripotency, the method including performing cell culture in the presence of a substance that can inhibit cell-cell adhesion.