In Situ Cell Analysis Using Analyte Probes
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Solution Overview
Problem
Current methods for analyzing molecules in cell culture systems require isolation of cells, which interferes with cell growth and lacks suitable in-situ controls for monitoring intermediate products, necessitating a method that minimally intervenes in the culture while maintaining high sensitivity and specificity.
Innovation Solution
An in-situ method for determining cell surface or extracellular matrix molecules in two- or three-dimensional cell cultures using analyte probes with detection and identification elements that bind to molecules without significantly impairing cell growth, allowing for continued cell cultivation and sequential molecule detection over time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are isolated and analyzed by flow cytometry, then measurement precision and sensitivity are improved, but cell growth is impaired and cells must be discarded after analysis
Solution Approach 1:
The analysis system is segmented into two independent parts: the cell culture system where cells grow undisturbed, and the analysis system where probes are detected. This allows cells to remain in their natural growth environment while still enabling sensitive molecular analysis through probes that bind to cell surface molecules without requiring cell isolation
Solution Approach 2:
Analyte probes serve as intermediaries that bind to target molecules on cell surfaces. These probes carry detection elements (such as fluorescent labels or enzymatic markers) that enable sensitive detection without requiring physical isolation of cells. The probes mediate between the cell culture system and the detection system, allowing analysis while cells remain in situ
2Measurement precision
If fluorescent dyes are used for labeling living cells, then detection sensitivity is improved, but cell growth conditions are changed and culture is interfered with
Solution Approach 1:
Instead of directly labeling cells with fluorescent dyes that may affect cell physiology, the invention uses analyte probes that bind to cell surface molecules. The detection elements on these probes create a detectable signal that copies or represents the presence of target molecules without directly modifying cell properties or culture conditions
Solution Approach 2:
The analyte probes act as intermediaries between the target cell surface molecules and the detection system. Rather than applying fluorescent dyes directly to cells, the probes bind specifically to target molecules and carry the detection elements, thereby enabling sensitive detection while maintaining cell culture stability
3Measurement precision
If cells are removed from culture for analysis, then measurement specificity is improved, but continuous monitoring capability is lost
Solution Approach 1:
The method enables continuous monitoring by allowing cells to remain in the culture system throughout the analysis process. Analyte probes are applied to cells in situ, bound probes are detected without cell removal, and cells can be reused for subsequent analyses. This creates a continuous workflow where the same cell population can be monitored over time without repeated isolation and discarding
Solution Approach 2:
The invention performs preliminary binding of analyte probes to cell surface molecules while cells remain in culture. This preliminary action establishes the detection complex in situ before any measurement occurs, allowing subsequent detection and repeated analyses without requiring cell removal or re-isolation
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
Enables the detection of specific molecules in their qualitative environment within cell culture systems without substantial impairment of cell growth, allowing for continuous monitoring and maintaining the chemical integrity of molecules, thus improving the quality control of cell therapeutic products.
Implementation Method 1
binding of the analyte probe to the molecule in the cell culture system
Data Source
AI summary
The present invention includes an in-situ method, comprisinga) determining a molecule selected from the group consisting of cell surface molecules and extracellular matrix molecules in a two- or three-dimensional cell culture system comprising living cells and cell culture medium, comprising the steps ofi) providing an analyte probe consisting of a detection element, which binds the molecule, and one or more identification elements;ii) binding of the analyte probe to the molecule in the cell culture system, wherein the growth ability of the contained living cells is not substantially impaired by this step;iii) optionally removing unbound analyte probes;iv) releasing the analyte probe;v) transferring the analyte probe into a container which differs from the cell culture system;vi) detecting the identification element(s); andb) continuing the cell cultivation in the cell culture system.