Reversible Cell Staining via Fab Fragments for High-Purity Isolation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current cell therapy methods are laborious, time-consuming, and expensive due to the need for high-purity cell preparations, and existing cell purification techniques face challenges in achieving purity and functionality due to residual labeling and receptor blockade issues.
Innovation Solution
A method for reversibly staining target cells using a receptor binding reagent with a dissociation rate constant of 0.5 x 10^-4 sec^-1 or greater, allowing for the formation of multivalent binding complexes that can be easily removed, thereby enabling efficient cell isolation and purification without affecting cell functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If paramagnetic beads and labels are used for cell purification, then cell purity is improved, but cell functionality is compromised due to receptor blockade and residual labeling
Solution Approach 1:
The invention extracts the labeling function from the purification process by using reversible staining with Fab fragments that can be completely removed. The Fab fragments bind to surface markers during sorting but are subsequently eliminated through protease treatment or natural shedding, leaving no residual labels on the purified cells. This resolves the contradiction by achieving high purity through temporary labeling while preserving cell functionality through complete label removal.
Solution Approach 2:
The invention introduces Fab fragments as an intermediary staining reagent that temporarily mediates cell identification during purification. These Fab fragments serve as a transient mediator that binds to surface markers for sorting purposes but does not permanently remain on the cells. The intermediary nature of Fab fragments allows them to facilitate purification while being completely removable, thus resolving the contradiction between purity achievement and functionality preservation.
2Measurement precision
If multiple surface markers are used for combinatorial staining to identify specific cell subsets, then cell identification precision is improved, but process complexity increases
Solution Approach 1:
The invention merges multiple Fab fragment staining steps into a unified reversible staining protocol. By using Fab fragments against different surface markers in sequence or combination, all stains can be simultaneously removed through a single protease treatment step. This merging approach maintains the precision of combinatorial marker identification while simplifying the removal process, thus resolving the contradiction between identification precision and process complexity.
Solution Approach 2:
The invention changes the chemical nature of staining reagents from irreversible antibodies to reversible Fab fragments with specific dissociation rate constants. This parameter change allows the staining to be highly specific for precise cell identification while enabling complete removal through protease treatment or natural shedding. The reversible binding parameter resolves the contradiction by allowing precise identification during sorting while facilitating complete removal afterward.
3Stability of the object's composition
If high affinity binding reagents are used for cell staining, then staining stability is improved, but reversibility is compromised
Solution Approach 1:
The invention optimizes the dissociation rate constant parameter of Fab fragments to achieve a specific range (k off ≥ 0.5 x 10^-4 sec^-1) that balances stability and reversibility. This parameter optimization ensures that Fab fragments bind stably enough for accurate cell sorting while dissociating rapidly enough for complete removal. The controlled parameter change resolves the contradiction by achieving both staining stability during sorting and reversibility for complete removal.
Solution Approach 2:
The invention introduces dynamic control over binding stability through the use of Fab fragments with optimized dissociation rates. The binding is dynamically stable during the sorting process but becomes dynamically reversible when protease is added or when natural shedding occurs. This dynamic behavior resolves the contradiction by providing stability when needed and reversibility when needed, rather than a fixed binding characteristic.
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
This method allows for the effective isolation of target cells with high purity and maintains cell functionality by completely removing staining reagents, reducing the risk of side effects and improving the predictability of cell therapy outcomes.
Implementation Method 1
a receptor binding reagent, the receptor binding reagent comprising at least one (any) binding site B, wherein the binding site B specifically binds to said receptor molecule
Implementation Method 2
allowing for the formation of multivalent binding complexes that can be easily removed
Implementation Method 3
wherein the dissociation rate constant (k off ) as determined by surface plasmon resonance for the binding between said receptor binding reagent via the binding site B and said receptor molecule has a value of 0.5 x 10 -4 sec -1 or greater
Data Source
Figure 1a~1b
Figure 1c~1d
Figure 2
AI summary
The present invention relates to methods of reversibly staining a target cell. The invention also relates to methods of isolating a target cell or a target cell population that is defined by the presence of at least one common specific receptor molecule. The invention also provides kits that can be used to carry out the methods of the invention.