Sequential Crosslinking for Low-Leakage Cell and Tissue Preservation
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Solution Overview
Problem
Existing methods for sample preservation and storage result in significant leakage of biological components from cells, which compromises the integrity and analysis of nucleic acids and proteins.
Innovation Solution
A method involving sequential use of hydrophobic and hydrophilic crosslinkers, such as DSP and DTSSP, followed by a storage buffer, to stabilize samples, reducing leakage by 2-10-fold.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing methods for sample preservation and storage are used, then sample storage is simple and cost-effective, but significant leakage of biological components from cells occurs, compromising integrity and analysis
Solution Approach 1:
The patent applies parameter changes by transitioning from conventional single-crosslinker methods to a sequential two-step crosslinking process using hydrophobic followed by hydrophilic crosslinkers. This changes the chemical parameters of the fixation process, resulting in reduced biological component leakage (2-10 fold reduction) while maintaining cell structural integrity for downstream analysis
Solution Approach 2:
The patent employs composite materials by combining two different types of crosslinkers with distinct properties: hydrophobic crosslinkers (e.g., DSP, EGS, DSS) and hydrophilic crosslinkers (e.g., DTSSP, sulfo-EGS, BS3). This composite approach leverages the complementary characteristics of both crosslinker types to achieve superior sample preservation compared to using either crosslinker alone
2Reliability
If sequential crosslinking with hydrophobic and hydrophilic crosslinkers is used, then leakage of biological components is reduced by 2-10-fold, but the sample preparation process becomes more complex
Solution Approach 1:
The patent applies segmentation by dividing the crosslinking process into two distinct sequential steps: first applying hydrophobic crosslinkers, then applying hydrophilic crosslinkers. Each step targets different aspects of cell structure stabilization, and the processes are clearly separated with defined incubation and washing steps, making the complexity manageable and systematic
Solution Approach 2:
The patent manages process complexity through parameter changes by optimizing incubation times (e.g., 15-60 minutes per crosslinking step), temperatures (4-25°C), and crosslinker concentrations (0.05-10 mM). These standardized parameter ranges allow the enhanced two-step process to be performed reliably without excessive complexity
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 effectively minimizes the leakage of biological components, allowing for accurate nucleic acid and protein analysis by maintaining sample integrity during preservation and storage.
Implementation Method 1
contacting a sample comprising at least one cell or tissue with a hydrophobic crosslinker
Implementation Method 2
contacting the sample with a hydrophilic crosslinker
Data Source
AI summary
Methods and kits for sample preparation are provided, wherein a sample comprising at least one cell or tissue is contacted sequentially with a hydrophobic crosslinker, and then a hydrophilic crosslinker. The method prevents cell and tissue analytes from diffusing out of the at least one cell or tissue, such that the cell and tissue analytes can be analyzed and/or imaged.