TrypLE and Dispase II RPE Cell Dissociation
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Solution Overview
Problem
Current methods for dissociating adherent retinal pigment epithelium (RPE) cells are inefficient, particularly for use in treating age-related macular degeneration (AMD), where RPE cell transplantation is a promising but challenging process due to the cells' specific characteristics.
Innovation Solution
A digestive enzyme composition comprising recombinant TrypLE™ and neutral protease Dispase II is used to dissociate adherent RPE cells, with specific concentrations and incubation times optimized for effective cell dissociation without damaging the cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional trypsin is used to dissociate adherent RPE cells, then cell dissociation efficiency is improved, but cell viability and adherent ratio deteriorate
Solution Approach 1:
The patent changes the chemical parameters of the digestive enzyme by using recombinant trypsin-like enzyme (TrypLE) instead of traditional trypsin, and optimizes the concentration and incubation time parameters of Dispase II to achieve effective dissociation while maintaining cell viability
Solution Approach 2:
The patent employs a composite digestive enzyme system combining TrypLE and Dispase II, where TrypLE provides gentle cell dissociation and Dispase II enhances the dissociation efficiency, creating a synergistic effect that resolves the contradiction between dissociation efficiency and cell viability
2Productivity
If traditional trypsin and Dispase II combination is used, then dissociation effectiveness is improved, but safety and cell damage control worsen
Solution Approach 1:
The patent substitutes traditional trypsin with recombinant TrypLE, changing the enzymatic parameters to reduce harshness and cell damage while maintaining dissociation effectiveness when combined with optimized Dispase II concentrations
Solution Approach 2:
The patent introduces Dispase II as an intermediary enzyme that works synergistically with TrypLE, where Dispase II facilitates cell dissociation through a different mechanism that is less harmful to cell integrity, thereby reducing overall cell damage
3Quantity of substance
If RPE cells are cultured for extended periods to achieve sufficient cell numbers, then cell quantity is improved, but cell contamination and culture condition control deteriorate
Solution Approach 1:
The patent applies the optimized digestive enzyme treatment (TrypLE + Dispase II) at the appropriate culture time point to efficiently dissociate cells into single cells, which then can be rapidly expanded in suspension culture format, reducing the need for extended adherent culture periods and associated contamination risks
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 provides a simple, effective, and safer way to dissociate RPE cells, maintaining high cell viability and adherent ratio, supporting their use in culturing and treating AMD, with a synergistic effect that outperforms traditional trypsin and Dispase II combinations.
Implementation Method 1
A digestive enzyme composition comprising a recombinant enzyme TrypLE™ (recombinant trypsin-like enzyme) and a neutral protease Dispase II, which can be used to dissociate adherent RPE cells
Data Source
AI summary
The present invention disclose a method for digesting RPE cells to dissociate adherent cells, comprising steps: (1) using 1× recombinant enzyme TrypLE to cover RpE cells evenly; (2) using neutral protease Dispase II solution to cover RPE cells evenly, in which steps (1) and (2) are applied step by step.