Photochemical Crosslinkers for Cell Selection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for selecting cells from heterogeneous populations based on complex dynamic or morphological features are limited, as they often require robust fluorescence signals, are not compatible with dynamic cellular responses, or affect cell viability, especially when cells need to be suspended or cultured on specialized substrates.
Innovation Solution
The development of photostick and photolift techniques, which use photochemical crosslinkers and photocleavable crosslinkers to selectively attach or release cells from a surface based on static or dynamic properties, allowing for high-throughput microscopy and automated feature detection, while preserving cell viability and compatibility with various cell types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence-activated cell sorting (FACS) is used to select cells, then cell selection based on fluorescence signal is achieved, but the method requires robust fluorescence signals and cannot detect small changes or weak signals due to high noise levels
Solution Approach 1:
The patent transitions from static fluorescence measurement in FACS to dynamic imaging of cellular processes over time. By capturing temporal dynamics of cellular responses, the system can distinguish true signals from noise through pattern recognition across multiple time points, thereby improving measurement precision without requiring robust static fluorescence signals.
Solution Approach 2:
The patent replaces the electrostatic deflection mechanism of FACS with photochemical crosslinking and mechanical manipulation. Cells are sorted by forming photopolymer networks in response to dynamic cellular behaviors, then physically manipulated through adhesion/release mechanisms, eliminating the need for electrostatic fields and enabling detection of subtle dynamic changes.
2Adaptability or versatility
If FACS is used for cell selection, then sorting based on static fluorescence is achieved, but dynamic cellular responses such as beat rate, locomotion, and timecourse of response cannot be measured
Solution Approach 1:
The patent applies photochemical crosslinkers to cells before sorting, allowing dynamic cellular responses to be captured and fixed in time. The crosslinking process is triggered by cellular behavior itself, preserving dynamic information that would otherwise be lost in static FACS measurements.
Solution Approach 2:
The patent utilizes fluorescent reporters and colorimetric changes as readouts for dynamic cellular processes. By monitoring temporal changes in fluorescence intensity, wavelength, or pattern, the system can detect and sort based on dynamic responses such as calcium flux, gene expression changes, and cellular motility.
3Productivity
If FACS is used for cell sorting, then electrostatic deflection into collection wells is achieved, but cells must be in suspension which can damage or reduce viability of certain cell types such as neurons
Solution Approach 1:
The patent introduces photochemical crosslinkers as intermediaries between cellular behavior and sorting. These crosslinkers remain inert until activated by specific wavelengths of light, at which point they rapidly form networks that trap cells. This intermediary mechanism allows cells to remain adherent throughout the process, avoiding suspension damage while enabling controlled sorting through optical activation.
Solution Approach 2:
The patent replaces the electrostatic deflection and suspension-based collection of FACS with adhesion-based sorting on solid substrates. Cells are sorted by their ability to adhere to or be released from surfaces through photochemically controlled adhesion molecules, eliminating the need for suspension and electrostatic fields while preserving cell viability.
4Measurement precision
If laser-capture microdissection (LCM) is used to select cells, then precise selection of individual cells or tissue regions is achieved, but throughput is limited due to serial operation
Solution Approach 1:
The patent segments the cell population into multiple fields of view that can be imaged and processed in parallel. By dividing the sample into discrete regions and using high-throughput imaging to capture multiple fields simultaneously, the system achieves both precise cell-level selection and high throughput through parallel processing of segmented regions.
Solution Approach 2:
The patent uses high-throughput imaging to create optical copies or representations of cell populations, allowing computational identification and selection criteria to be applied to many cells simultaneously. Digital image processing and machine learning algorithms analyze copied cellular images to identify target cells across the entire population, enabling parallel selection rather than serial processing.
5Reliability
If LCM is used for cell selection, then selection on fixed samples is achieved, but compatibility with dynamic cellular properties and subsequent cell growth is limited
Solution Approach 1:
The patent applies photochemical crosslinkers to living cells before sorting, allowing dynamic cellular responses to be captured and fixed in time. The crosslinking process is triggered by cellular behavior itself, preserving dynamic information while keeping cells alive and capable of subsequent growth and analysis.
Solution Approach 2:
The patent changes the physical state of the sorting mechanism from fixed-sample-based (LCM) to living-cell-compatible photochemical crosslinking. By using reversible adhesion molecules and controllable photochemical reactions, the system maintains cells in a viable state throughout sorting, enabling subsequent cell growth and functional analysis that would be incompatible with fixed-sample methods.
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 efficient selection of cells with complex features of interest, maintaining cell viability and compatibility with diverse cell types, and allowing for further genetic or biochemical analysis, overcoming the limitations of existing methods by using patterned illumination and photochemical processing.
Implementation Method 1
photochemical crosslinkers and photocleavable crosslinkers to selectively attach or release cells from a surface
Implementation Method 2
photocleavable crosslinkers to selectively attach or release cells from a surface
Data Source
AI summary
Provided herein are photochemical crosslinkers and photocleavable crosslinkers and their uses in methods for cell selection from cell cultures. The photochemical crosslinkers comprise a fluorescent dye and a radical generator. The photocleavable crosslinkers comprise a photocleavable linker linking two electrophilic groups to each other. Also provided are systems for imaging cells comprising a plurality of cells crosslinked to extracellular matrix proteins using a crosslinker as described, an imaging apparatus, an illuminating apparatus, and software for image processing.