Cell Aggregate Particle Counting for Accurate Viability and Biomass
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Solution Overview
Problem
Existing automated cell counting instruments struggle to accurately determine cell viability and concentration in cell aggregate compositions, particularly for cell therapy applications, as their algorithms are limited when dealing with clustered or 3D aggregates, leading to inconsistent dose formulation.
Innovation Solution
A method involving the use of dissociation enzymes and particle counting instruments to quantify live cells and total cells without mechanical shearing, using a small fraction of the aggregate composition, and calculating viability based on particle size and biomass measurements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If automated cell counting instruments use labeling dyes and image analysis algorithms to distinguish live and dead cells, then cell viability can be determined for individualized cells, but the method becomes ineffective for cell clusters or 3D aggregates
Solution Approach 1:
The patent replaces mechanical dissociation methods with enzymatic dissociation using TRYPLETMTM or ACCUTASE® to separate cell aggregates into individual cells for counting. This enzymatic approach is more gentle and specific than mechanical methods, preserving cell viability while achieving effective dissociation for accurate measurement.
Solution Approach 2:
The patent introduces dissociation enzymes as intermediary substances that facilitate the separation of cell aggregates without directly affecting cell viability. These enzymes act as mediators between the aggregated cell state and the individualized cell state required for accurate counting, enabling the transition without mechanical damage.
2Ease of operation
If mechanical shearing methods are used to dissociate cell aggregates, then cells can be individualized for counting, but cell damage and death may occur affecting viability measurements
Solution Approach 1:
The patent substitutes mechanical shearing with enzymatic digestion using TRYPLETMTM or ACCUTASE®, which chemically break down cell adhesion molecules without applying physical force. This replacement eliminates mechanical damage to cells while achieving complete dissociation, thereby preserving cell viability and ensuring reliable viability measurements.
Solution Approach 2:
The patent changes the dissociation mechanism from mechanical force to biochemical reaction by using specific enzymes. This parameter change from physical to chemical dissociation allows for gentler, more controlled separation that maintains cell integrity and viability while achieving effective individualization for counting.
3Productivity
If a small fraction of the cell aggregate composition is used for counting, then the method is faster and requires less sample, but representative sampling must be ensured
Solution Approach 1:
The patent performs preliminary complete dissociation of the entire cell aggregate population using enzymatic treatment before taking a small fraction for counting. This preliminary action ensures that the fraction taken is representative of the whole population, as all cells have been uniformly individualized. The dissociation is performed on the bulk sample first, then a aliquot is taken for measurement, guaranteeing representativeness while maintaining efficiency.
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
Provides accurate and consistent determination of live cell concentration and viability in cell aggregates, eliminating the need for mechanical dissociation and dye labeling, thereby improving dose formulation accuracy.
Implementation Method 1
contacting the fraction of the cell aggregate composition with a dissociation enzyme to obtain a dissociated sample
Implementation Method 2
quantifying the number of live cells in the dissociated sample by counting the number of particles based on size
Data Source
AI summary
Provided herein are methods for using particle counting to determine live cell concentration, total cell concentration, cell viability and biomass of cell aggregate compositions, such as for accurate cell dosing for clinical applications.