Chop-Fix Method for Biological Tissue Preservation
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Solution Overview
Problem
Biological samples, especially tissue samples, are unstable and prone to degradation when removed from their natural environment, making it challenging to use them in partition-based assays, as standard fixation and preservation methods fail to accurately preserve the native state of all cell types within the tissue.
Innovation Solution
A method involving chopping biological tissue into fragments and treating them with a fixing reagent, followed by cell dissociation, which can include generating barcoded nucleic acid molecules from crosslinked nucleic acid molecules, allowing for the analysis of fixed or un-fixed cells or nuclei in bulk or single-cell assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If standard fixation and preservation methods are used on tissue samples, then sample stability is improved, but the native state of certain cell types is altered or destroyed
Solution Approach 1:
The tissue sample is segmented into small fragments through mechanical chopping before fixation. This segmentation allows the fixing reagent to rapidly penetrate all cell types throughout the tissue, preserving the native state of rare and hard-to-reach cells while maintaining overall sample stability. The chopping creates uniform small pieces that enable consistent and complete fixation across all cell populations.
Solution Approach 2:
The tissue sample undergoes preliminary chopping into fragments before the fixation process. This preliminary mechanical preparation increases the surface area and accessibility of cells to the fixing reagent, ensuring that fixation occurs rapidly and uniformly across all cell types, including rare populations that would otherwise be difficult to reach in intact tissue.
2Ease of operation
If tissue samples are dissociated using heat and enzymes, then cell separation is achieved, but the native state of cell types is greatly altered or destroyed
Solution Approach 1:
The conventional sequence is inverted: instead of dissociating tissue first and then fixing cells, the invention fixes the chopped tissue fragments first and then dissociates them. This reversal ensures that cells are preserved in their native state during fixation before any dissociation occurs, preventing the loss of native state information that occurs when dissociation is performed first.
Solution Approach 2:
Fixation is performed as a preliminary step before dissociation. By fixing the chopped tissue fragments first, the native state of all cell types is captured and preserved before any enzymatic or mechanical dissociation takes place. This preliminary fixation protects cellular structures and molecules from degradation during subsequent dissociation procedures.
3Stability of the object's composition
If standard preservatives and fixatives are used on tissue samples, then sample preservation is achieved, but measurements are highly skewed due to inability to reach all cell types
Solution Approach 1:
The tissue is segmented into small fragments through chopping, which dramatically increases the accessibility of fixing reagents to all cell types including rare populations. This segmentation ensures that no cell type is missed during fixation, enabling accurate and unbiased measurements from the complete cellular composition of the tissue sample.
Solution Approach 2:
The chopping process creates local uniformity in fragment size and structure, ensuring that the fixing reagent can uniformly access and preserve all cell types throughout the sample. This local quality control through mechanical fragmentation eliminates the heterogeneity and accessibility issues that plague standard fixation methods, allowing all cell populations to be equally well-preserved and measured.
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 preserves the cellular analytes from tissue samples, enabling accurate and reproducible analysis of nucleic acid sequences by maintaining the native state of cell types and reducing sample degradation, thus improving the reliability of assays.
Implementation Method 1
treating the composition of tissue fragments with a fixing reagent, thereby providing a composition of fixed tissue fragments
Data Source
AI summary
The present disclosure provides methods for biological sample preparation and analysis that use a chopping and fixing treatment in the preparation of dissociated fixed cells from biological tissue for use in either bulk assays, or in single-cell/single-nucleus assays, such as partition-based assays. The disclosure also provides assay methods, including partition-based methods, for use of the fixed cells prepared from biological tissue that can optionally be used in combination with un-fixing treatments. Kits comprising dissociation reagents, un-fixing agents, and other assay reagents for use in the methods are also provided.