Adipose Tissue Enzyme Ratio for Vascular Cell Yield
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Solution Overview
Problem
Current methods for preparing stromal vascular fraction (SVF) from adipose tissue, particularly for isolating vascular endothelial cells and progenitor cells, are inefficient and damage cells due to the use of thermolysin, making it difficult to achieve high-purity and high-yield preparations.
Innovation Solution
A method involving the use of a combination of collagenase and neutral protease enzymes, with a specific activity ratio, to effectively disintegrate adipose tissue, enhancing the yield and purity of vascular endothelium-related cells in the SVF, without the need for clostripain or thermolysin, which can be harmful.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If thermolysin is used for enzymatic disintegration of adipose tissue, then the disintegration efficiency is improved, but the cell activity and purity of vascular endothelium-related cells deteriorate due to great damage to cells
Solution Approach 1:
The patent changes the enzymatic parameters by replacing thermolysin with a specific combination of collagenase (10,000 U) and neutral protease (at least 1 U, preferably at least 2 U) per gram of adipose tissue. This parameter change maintains disintegration efficiency while preserving cell activity and vascular endothelium-related cell purity, directly resolving the technical contradiction between productivity and reliability.
2Productivity
If crude collagenase preparation containing multiple proteases is used, then the disintegration capability is improved, but the specificity and purity of isolated cells deteriorate due to contamination with other proteases like Clostripain
Solution Approach 1:
The patent extracts and isolates only the necessary enzymes (collagenase and neutral protease) from crude collagenase preparations, eliminating contaminating proteases such as Clostripain. This selective extraction maintains effective disintegration capability while achieving high cell purity, specifically preserving vascular endothelium-related cells without contamination from other cell types.
3Speed
If high concentration of collagenase is used to improve disintegration speed, then the processing time is reduced, but the cell damage increases and affects the yield of viable cells
Solution Approach 1:
The patent uses a composite enzymatic system combining collagenase (10,000 U/g) and neutral protease (at least 1 U/g, preferably at least 2 U/g) to achieve effective adipose tissue disintegration. This composite approach provides synergistic action that maintains rapid disintegration speed while preserving cell viability and reducing cell damage, thereby improving the yield of viable cells including vascular endothelium-related cells.
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 significantly improves the yield and purity of vascular endothelium-related cells, making it a more efficient and effective method for regenerative medicine applications by optimizing the enzyme ratio and eliminating harmful components.
Implementation Method 1
Enzymes are generally used to prepare SVF containing vascular endothelial cells/vascular endothelial progenitor cells from an adipose tissue. For dispersing adipose tissue, that is, enzymatic disintegration of adipose tissue
Implementation Method 2
an enzyme preparation in which a protease is mixed with collagenase (crude collagenase preparation, for example, collagenase contaminated with Clostripain, Neutral protease, etc.)
Data Source
AI summary
A method for preparing a stromal vascular fraction from an adipose tissue, including a step of treating an adipose tissue with an enzyme solution containing a collagenase and a neutral protease, preferably an enzyme solution free of clostripain and thermolysin, and showing not less than 1 U neutral protease activity with respect to 10,000 U collagenase activity, and recovering cells is provided by the present invention.