Migrasome Filtration and Reverse Elution for High-Purity Separation
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Solution Overview
Problem
Current methods for separating migrasomes are cumbersome, time-consuming, and require high equipment, making them unsuitable for large-scale extraction and subsequent applications.
Innovation Solution
A method involving filtration and reverse filtration using a filter with specific pore sizes to intercept and elute migrasomes from macrophages, followed by ultrafiltration to obtain purified migrasomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If low-speed stepwise centrifugation is used to extract migrasomes, then the extraction process is simple, but only a crude extract is obtained with low purity
Solution Approach 1:
The patent divides the extraction process into two distinct stages: first using low-speed stepwise centrifugation to obtain a crude extract, then applying gradient density centrifugation to further purify the migrasomes. This segmentation allows each method to perform its optimal function - the first stage for simplicity and the second for precision.
Solution Approach 2:
The patent applies different centrifugation conditions to different stages of the extraction process. The first stage uses low-speed centrifugation suitable for crude extraction, while the second stage uses gradient density centrifugation with specific density gradients (1.063, 1.085, 1.107 g/mL) to achieve high purity. Each stage is optimized for its specific purpose.
2Manufacturing precision
If gradient density centrifugation followed by ultra-high-speed centrifugation is used to purify migrasomes, then high purity is achieved, but the operation steps become cumbersome and time-consuming
Solution Approach 1:
The patent combines gradient density centrifugation and ultra-high-speed centrifugation into a single integrated purification step. After the low-speed centrifugation obtains a crude extract, the supernatant is directly subjected to gradient density centrifugation, and the migrasomes are purified in one continuous process rather than separate discrete steps.
Solution Approach 2:
The patent performs preliminary low-speed stepwise centrifugation to obtain a crude extract and remove large debris before the main purification step. This preliminary action simplifies the subsequent gradient density centrifugation by reducing the complexity of the sample, allowing the main purification to focus only on separating migrasomes from smaller contaminants.
3Manufacturing precision
If gradient density centrifugation and ultra-high-speed centrifugation are used for separation, then migrasomes are purified effectively, but the separation time is extended
Solution Approach 1:
The patent maintains continuous centrifugation action throughout the purification process. The low-speed centrifugation, gradient density centrifugation, and ultra-high-speed centrifugation are performed in continuous sequence without interruption, keeping the sample in motion and processing throughout, thereby minimizing idle time while achieving high purity.
4Manufacturing precision
If conventional centrifugation methods are used, then migrasomes can be separated, but high equipment requirements are needed
Solution Approach 1:
The patent uses a range of centrifugation speeds (low-speed stepwise centrifugation at lower g-forces, then gradient density centrifugation, and ultra-high-speed centrifugation at higher g-forces) to achieve separation. By varying the centrifugal force parameters across different stages, the method achieves effective separation using standard centrifugation equipment rather than requiring specialized high-end devices.
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 achieves rapid, efficient, and cost-effective separation of migrasomes with high integrity of RNA, maintaining their unique vesicular structure and protein expression.
Implementation Method 1
filtering the supernatant with a filter to obtain intercepted migrasomes
Implementation Method 2
reversing a direction of the filter for reverse filtration to elute the intercepted migrasomes
Implementation Method 3
ultrafiltering the eluted migrasomes using an ultrafiltration tube with a molecular weight cutoff of 100 kilodaltons (KD)
Implementation Method 4
adding 2.5% glutaraldehyde to completely cover cell surfaces of the macrophages
Implementation Method 5
fixing the rinsed macrophages for 1 hour to 2 hours by using a pre-cooled osmic acid solution
Implementation Method 6
drying the dehydrated macrophages by using a freeze dryer in vacuum
Implementation Method 7
spraying a gold coating on the dried macrophages by using an ion sputtering device
Data Source
AI summary
A simple and efficient method for separating migrasomes from macrophages is provided. The method separates migrasomes having a diameter range of 0.5 micrometers (μm) to 3 μm by intercepting through a filter and eluting through reverse filtration successfully. The separated migrasome has a vesicle-liked structure and wrinkles on its surface, and the separated migrasome has a diameter over 500 nanometers (nm). The separated migrasomes express their characteristic proteins PIGK, EOGT, and TSPAN4, but do not express specific markers TSG101 and ALIX of EVs, indicating that the separated migrasomes are a unique type of vesicles distinct from extracellular vesicles (EVs). The integrity of ribonucleic acids (RNA) carried by the migrasomes is not affected. The method for separating migrasomes from macrophages has the characteristics of simplicity, high efficiency, good controllability, good repeatability, and low cost, and large special equipment is not needed.