Frozen Biological Sample Preparation with Non-Crosslinking Fixation
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Solution Overview
Problem
Existing methods for preserving biological samples for analysis, such as paraffin-embedding and snap-freezing, often result in chemical modifications and degradation of biomolecules, compromising morphology and the integrity of biological components like RNA and proteins, and require laborious processing steps.
Innovation Solution
A method involving fixation with a non-crosslinking fixative solution followed by incubation in an aqueous cryoprotectant solution and freezing, which preserves morphology and integrity of biological components without the need for paraffin-embedding or immediate freezing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If cross-linking agents such as formaldehyde are used for fixation, then sample morphology is well preserved, but severe chemical modifications and degradation of biomolecules occur which prevents purification of biological components suitable for molecular analysis
Solution Approach 1:
The patent changes the chemical parameters of the fixative solution by using non-crosslinking agents (alcohols, acids, acetone) instead of cross-linking agents like formaldehyde. This parameter change preserves morphology through precipitation mechanisms while avoiding the harmful crosslinking reactions that degrade biomolecules.
Solution Approach 2:
The patent extracts and removes the harmful cross-linking function from the fixation process. By eliminating cross-linking agents entirely and using only precipitating agents, the method separates the morphology-preserving function from the biomolecule-damaging function, achieving both goals without compromise.
2Shape
If paraffin-embedding is performed to preserve tissue morphology, then morphology is retained, but the process is laborious and takes up to two days
Solution Approach 1:
The patent extracts and eliminates the time-consuming paraffin-embedding step from the workflow. By using non-crosslinking fixatives that are compatible with frozen sectioning, the method removes the intermediate paraffin processing steps while still achieving good morphology preservation through the freezing process.
Solution Approach 2:
The patent performs preliminary fixation with non-crosslinking agents that prepare the tissue for direct freezing without requiring subsequent paraffin embedding. This preliminary action with compatible fixatives enables the tissue to withstand freezing and sectioning while maintaining morphology, thereby eliminating later time-consuming steps.
3Stability of the object's composition
If immediate freezing of the sample is performed to preserve biological components, then biomolecule integrity is maintained, but the sample morphology may be compromised and immediate freezing tools must be available
Solution Approach 1:
The patent performs preliminary fixation with non-crosslinking agents before freezing. This preliminary action stabilizes the tissue structure and prevents morphological degradation during the freezing process, enabling morphology preservation without requiring immediate freezing tools while still maintaining biomolecule integrity.
Solution Approach 2:
The patent uses non-crosslinking fixatives as a protective cushioning agent before freezing. These fixatives prevent the formation of ice crystals and structural damage that would otherwise occur during freezing, thereby cushioning the tissue against morphological damage while preserving biomolecules.
4Stability of the object's composition
If non-crosslinking fixative solutions are used to avoid crosslinks, then biomolecule preservation is improved, but the solubility of protein molecules is reduced through precipitation
Solution Approach 1:
The patent changes the physical parameters of the fixative solution by using aqueous-based non-crosslinking fixatives with controlled composition. These solutions achieve protein precipitation through controlled parameter changes that preserve biomolecule integrity while maintaining sufficient protein solubility for subsequent analysis after the fixation process.
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 method allows for high-quality preservation of DNA, RNA, and proteins, enabling excellent performance in molecular analysis and maintaining sample morphology, even in clinical settings where immediate freezing tools are not available.
Implementation Method 1
one important mechanism being the precipitation of proteins. Precipitating fixative solutions represent an important group of non-crosslinking fixative solutions and act by reducing the solubility of protein molecules. They may also disrupt the hydrophobic interactions that give many proteins their tertiary structure.
Implementation Method 2
it is also required to remove the paraffin from the sample in a series of steps for most downstream applications. A disadvantage of freezing tissue samples slowly is that an aggregation of water molecules into ice crystals occurs and artefacts are produced
Data Source
AI summary
The present invention pertains to a method of preparing a frozen biological sample, comprising the steps of fixing the biological sample with a non-crosslinking fixative solution, incubating the fixed biological sample in an aqueous solution comprising a cryoprotectant, and freezing the cryoprotected biological sample. The method advantageously allows to preserve both, morphology of the sample as well as biological components such as nucleic acids and proteins, in high quality for subsequent analysis. The method is robust, simple and neither requires laborious steps associated with paraffin-embedding nor immediate freezing of the sample. Also provided are advantageous uses and kits.