Nested Microwell Device for Colony Assay Stability
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Solution Overview
Problem
Standard colony forming cell (CFC) assays in semisolid media face challenges such as colony disturbance during handling, overlapping colonies, and difficulty in distinguishing between individual colonies and multi-centric colonies, leading to subjective enumeration and contamination issues, while microwell devices struggle with colony spread and incorrect counting due to multiple progenitor cells per well.
Innovation Solution
A cell culture device with integrally formed microwells and sub-microwells, designed to compartmentalize cells effectively, featuring a frustoconical or frustopyramidal shape to prevent cell spread and overlapping, and a magnetic or magnetizable grid for precise cell immobilization, allowing for objective enumeration and live cell staining without fixation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cells are cultured in semisolid media to prevent movement, then colony formation is enabled, but colony disturbance during handling and contamination issues occur
Solution Approach 1:
The device divides the culture space into multiple microwells within a single well, creating discrete compartments for individual colonies. This segmentation allows colonies to be physically separated and stabilized in fixed positions, preventing disturbance during handling while maintaining the semisolid medium environment for reliable colony formation.
Solution Approach 2:
The device implements a nested structure with microwells containing sub-microwells, creating multiple levels of compartmentalization. This nested design provides robust entrapment of cells and colonies at different scales, ensuring stability during handling operations while maintaining the culture environment necessary for reliable colony formation.
2Ease of operation
If microwell devices are used to entrap individual cells, then cell manipulation is enabled, but colony spread and incorrect counting occur due to multiple progenitor cells per well
Solution Approach 1:
The device segments the microwell into multiple sub-microwells, creating finer compartments that reduce the probability of multiple progenitor cells occupying the same compartment. This segmentation maintains ease of cell manipulation while improving colony counting accuracy by ensuring more exclusive one-cell-per-compartment conditions.
Solution Approach 2:
The device creates local variations in compartment size and structure through the microwell-sub-microwell hierarchy, optimizing local conditions for single-cell entrapment. This local quality enhancement improves measurement precision for colony counting while preserving the overall ease of operation for cell manipulation.
3Reliability
If colonies are allowed to grow without compartmentalization, then natural colony formation occurs, but overlapping colonies and subjective enumeration result
Solution Approach 1:
The device provides physical compartmentalization through microwells and sub-micrawells that prevent colony overlap while allowing authentic colony formation within each compartment. This segmentation enables objective enumeration by creating discrete, non-overlapping colonies that can be clearly distinguished and counted, eliminating subjectivity in colony enumeration.
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
The device enables robust entrapment of colonies, reducing dispersal and contamination, facilitating objective enumeration and classification of colonies, and allowing for sensitive live cell staining, thereby improving the accuracy and reliability of colony forming assays.
Implementation Method 1
a magnetic or magnetizable member positioned below the sub-micrawells. The magnetizable member may be a wire grid.
Data Source
AI summary
A cell culture device comprises a well. A plurality of microwells are within the well, and a first common fluid volume is within the well above the microwells. A set of sub-microwells are within each microwell, and a second common fluid volume is within each microwell above the set of sub-microwells.