Microwell Cytotoxicity Assay for Single-Cell Serial Killing Analysis
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Solution Overview
Problem
Current methods for characterizing cell therapies fail to provide single-cell-specific information on the functional heterogeneity and serial killing capability of immune cells, and do not maintain optimal conditions for target cell viability during cytotoxicity assays.
Innovation Solution
A method using an open microwell microfluidic device with automated liquid exchange and periodic perfusion of media in microwells to maintain cell viability and assess cytotoxic activity of individual effector cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulk assays are used to characterize cell therapy products, then the analysis can be performed on the entire cell population, but single-cell-specific information on functional heterogeneity and serial killing capability cannot be obtained
Solution Approach 1:
The cell population is segmented into individual cells by distributing them into separate microwells, allowing single-cell analysis. Each microwell contains one effector cell and multiple target cells, enabling measurement of cytotoxicity at the single-cell level while maintaining functional heterogeneity information.
Solution Approach 2:
The microwell array serves as an intermediary structure that isolates individual effector cells while maintaining controlled interactions with target cells. This intermediary system enables single-cell resolution without requiring complex single-cell manipulation techniques.
2Duration of action of moving object
If target cells are maintained for 4-72 hours in cytotoxicity assays, then effector cell cytotoxic activity can be assessed, but target cell viability deteriorates due to unwanted death unrelated to effector-target interactions
Solution Approach 1:
Target cells are segmented into individual microwells with isolated effector cells, allowing assessment of each effector cell's cytotoxic activity over time. This segmentation enables longer assay durations while maintaining target cell viability through controlled local environments.
Solution Approach 2:
Multiple target cells are placed in each microwell with a single effector cell, creating an excess of target cells that allows assessment of serial killing capability. This partial action approach enables duration assessment without requiring all target cells to remain viable throughout the entire assay period.
3Measurement precision
If flow cytometry assays are used to define subpopulations, then phenotypic markers can be identified, but functional heterogeneity and cytotoxic activity cannot be traced at the single-cell level
Solution Approach 1:
The assay segments the cell population into individual microwell compartments, enabling functional characterization of each effector cell. This segmentation allows tracing of cytotoxic activity and serial killing capability at the single-cell level, providing functional heterogeneity information that flow cytometry cannot provide.
Solution Approach 2:
Each microwell creates a localized environment with specific effector-target cell interactions. This local quality approach allows functional assessment of individual effector cells while maintaining controlled conditions, enabling detection of functional heterogeneity without requiring complex flow cytometry protocols.
4Reliability
If automated liquid exchange is implemented in microwell devices, then periodic perfusion of media can be achieved to maintain cell viability, but device complexity increases
Solution Approach 1:
The system implements periodic perfusion of media through the microwell array, replacing spent media with fresh media at defined time intervals. This periodic action maintains target cell viability and nutrient supply throughout the extended assay duration of 4-72 hours, enabling reliable cytotoxicity assessment.
Solution Approach 2:
The microfluidic device integrates multiple functions including cell loading, media perfusion, and waste removal through a unified microwell array structure. This multi-functionality reduces the need for separate manual operations while maintaining target cell viability through automated periodic media exchange.
Data Source
AI summary
The present invention relates to a method for determining the efficacy of a cell therapy or immunotherapy, where said method comprises; a. Providing a first set of microwells each containing effector cells and target cells; b. optionally, providing a second set of microwells each containing target cells; c. Selecting those microwells comprising a single effector cell and n target cells, where n is between 1 and 50, preferably between 1 and 20; d. Keeping the cells in culture for a time t; e. Measuring the number of dead target cells in each of the microwells selected to contain a single effector cell; f. Calculating a potency score consisting of the weighted average of the number of target cells killed by each effector cell.