Accutase Enzyme Dissociation for Viable hESC Single Cell Suspension
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for generating single cell suspensions from embryonic stem cells, such as mechanical and enzymatic dissociation, result in high cell death and differentiation, making it difficult to maintain viable and pluripotent cells for research and therapeutic applications.
Innovation Solution
The use of ACCUTASE®, a mixture of proteolytic and collagenolytic enzymes, for enzymatic dissociation of embryonic stem cell colonies to produce a high percentage of viable and pluripotent single cells, which can be further flow-sorted and cultured, maintaining undifferentiated morphology and preventing genetic abnormalities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mechanical or enzymatic dissociation is used to generate single cell suspensions, then cell suspension is produced, but cell death and differentiation increase
Solution Approach 1:
The patent changes the chemical parameters of the dissociation process by using a specific enzyme combination (Accutase containing proteases and collagenases) with optimized concentrations and incubation conditions. This chemical parameter optimization allows gentle dissociation that maintains cell viability and pluripotency while effectively generating single cell suspensions, resolving the contradiction between productivity and reliability.
2Ease of operation
If traditional dissociation methods are used, then cell detachment is achieved, but cell damage and death occur
Solution Approach 1:
The patent introduces an intermediary substance - a specific enzyme mixture (Accutase) containing proteases and collagenases - that mediates the cell detachment process. This intermediary enzyme combination selectively degrades adhesion molecules and extracellular matrix components in a controlled manner, achieving easy cell detachment while minimizing cell damage and death through gentle enzymatic action rather than harsh mechanical forces.
3Duration of action of stationary object
If subculturing is performed to maintain cells in culture, then cell population is renewed, but cell viability decreases
Solution Approach 1:
The patent optimizes the parameters of the subculturing process by using a specific enzyme mixture (Accutase) with defined protease and collagenase concentrations, along with controlled incubation time and temperature conditions. These parameter changes enable gentle dissociation during passaging that preserves cell viability, allowing long-term culture maintenance without significant viability loss.
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
ACCUTASE® dissociation allows for efficient and reproducible production of viable pluripotent single embryonic stem cells with high plating efficiency and undifferentiated cell maintenance, overcoming the limitations of traditional methods by reducing cell death and differentiation, and enabling scalable and automated cell culture and differentiation.
Implementation Method 1
enzymatic dissociation of embryonic stem cell colonies to produce a high percentage of viable and pluripotent single cells
Data Source
AI summary
We used ACCUTASE®, a commercially available cell detachment solution, for single cell propagation of pluripotent hESCs. Unlike trypsin dissociation, ACCUTASE® treatment does not significantly affect the plating efficiency of hESC dissociation into single cells. Cultures dissociated with ACCUTASE® to single cells at each passage maintain a higher proportion of pluripotent cells as compared to collagenase-passaged hESCs. ACCUTASE®-treated hESCs can be grown to a high density as monolayers, and yet retain their pluripotency.