Tissue Decellularization via Sequential Antigen Solubilization
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Solution Overview
Problem
Current myocardial tissue decellularization methods using harsh detergents are ineffective in removing antigenic determinants and often damage the extracellular matrix, making them unsuitable for successful recellularization and tissue transplantation.
Innovation Solution
A method involving sequential destabilization and depolymerization of cytoskeletal components, followed by solubilization and removal of water-soluble and lipid-soluble antigens using specific buffers and agents, to produce a substantially intact extracellular matrix compatible with viable cell repopulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If harsh detergents (SDS, Triton-X100, Saponin) are used for decellularization, then cellular components are solubilized and removed, but antigenic determinants remain and extracellular matrix is damaged
Solution Approach 1:
The patent divides the antigen removal process into distinct sequential stages: first removing water-soluble antigens using detergents with reducing agents, then removing lipid-soluble antigens using organic solvents. This segmentation allows each stage to target specific antigen types without the harsh conditions needed for complete cellular destruction, thereby preserving ECM integrity while effectively reducing overall antigenicity.
Solution Approach 2:
The patent changes chemical parameters by introducing reducing agents (beta-mercaptoethanol, DTT, TCEP) to break disulfide bonds in antigens, and by adjusting solvent compositions (using SDS followed by chloroform/methanol). These parameter changes enable antigen solubilization and removal at lower detergent concentrations, preventing ECM damage while maintaining antigen removal efficacy.
2Quantity of substance
If high concentration detergents are used to solubilize cellular components, then decellularization is achieved, but protein precipitation occurs instead of solubilization
Solution Approach 1:
The patent uses reducing agents as intermediaries that break disulfide bonds in proteins, preventing aggregation and precipitation. These reducing agents act as mediators between the detergent and proteins, allowing solubilization at lower detergent concentrations while maintaining protein stability and preventing the precipitation that would occur with high-concentration detergent alone.
Solution Approach 2:
The patent creates composite solubilization solutions combining detergents with reducing agents and protease inhibitors. This composite approach allows each component to contribute its specific function: detergents for membrane disruption, reducing agents for disulfide bond reduction, and protease inhibitors for protein protection. The combination achieves effective solubilization without the harsh conditions that cause precipitation.
3Quantity of substance
If detergent-based methods are used for decellularization, then nuclei and cellular components are removed, but the scaffold remains immunogenic and toxic to repopulating cells
Solution Approach 1:
The patent segments the antigen removal process into water-soluble and lipid-soluble stages, using different solvent systems for each. This segmentation allows thorough antigen removal without requiring the high detergent concentrations that cause ECM damage and cell toxicity, thereby producing a scaffold that is both non-immunogenic and biocompatible for repopulation.
Solution Approach 2:
The patent changes the chemical environment through sequential use of different solvents (aqueous detergent phase followed by organic chloroform/methanol phase) and adjusting pH and ionic strength. These parameter changes enable complete antigen removal while maintaining ECM structural integrity and biochemical composition, producing a scaffold that lacks immunogenicity and toxicity.
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 approach effectively reduces antigenicity and maintains the structural integrity and biochemical composition of the extracellular matrix, enhancing the chances of successful recellularization and reducing immune responses in tissue transplantation.
Implementation Method 1
contacting the intact tissue with one or more actin depolymerization agents, and/or one or more microtubule depolymerization or microtubule destabilizing agents
Implementation Method 2
solubilizing water-soluble antigens in the tissue, wherein the water-soluble antigens are solubilized in a first solution comprising a first buffering agent, a reducing agent, a protease inhibitor, and one or more metal halide salts
Implementation Method 3
solubilizing lipid-soluble antigens in the tissue, wherein the lipid-soluble antigens are solubilized in a second solution comprising a second buffering agent, a reducing agent, a protease inhibitor, one or more salts suitable for maintaining protein solubility and an amphiphile
Data Source
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AI summary
The present invention relates to methods for removing antigens from tissues by sequentially destabilizing and/or depolymerizing cytoskeletal components and removing and/or reducing water-soluble antigens and lipid-soluble antigens. The invention further relates to tissue scaffolding and decellularized extracellular matrix produced by such methods.