Frozen Eukaryotic Cell Assay Variability Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Assay-ready frozen cells exhibit high intra- and inter-assay variability, which affects the reliability of cell-based assays, and the treatment with vinblastin can lead to apoptosis and oxidative stress, impairing bioluminescent enzyme activity.
Innovation Solution
Incorporating a cryopreservative and an inhibitor of xanthine oxidase, such as allopurinol, into the freezing composition to minimize variability, along with scavengers like N-acetylcystine or glutathione SH, to stabilize the cells and maintain assay performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cells are treated with vinblastin to prevent proliferation, then commercialization is enabled, but apoptosis and cell lysis occur resulting in activation of proteases and oxidative stress that impair bioluminescent enzyme activity
Solution Approach 1:
The patent applies preliminary anti-action by pre-treating cells with protease inhibitors and antioxidants before freezing to prevent the harmful effects of apoptosis. The cells are prepared with protective agents already in place, so when apoptosis occurs during freezing, the proteases are blocked and oxidative stress is minimized, preserving bioluminescent enzyme activity.
Solution Approach 2:
The patent uses protease inhibitors and antioxidants as intermediary substances that mediate between the apoptotic process and the bioluminescent enzyme. These intermediaries block the harmful pathways without interfering with the intended assay function, allowing the enzyme to remain active despite apoptosis occurring in the cells.
2Duration of action of stationary object
If cells are frozen using conventional cryopreservatives, then storage is enabled, but ice crystal formation causes cell lysis and activates proteases
Solution Approach 1:
The patent converts the harmful effect of ice crystal formation into a beneficial outcome by using it to deliver protective agents into the cells. The freezing process itself is harnessed to force the entry of protease inhibitors and antioxidants, which then protect against the damaging effects of the ice crystals and subsequent thawing.
Solution Approach 2:
The patent employs a composite cryopreservation medium that combines conventional cryopreservatives with protease inhibitors and antioxidants. This composite formulation provides both the freezing protection needed for storage stability and the biochemical protection needed to prevent protease activation and oxidative stress during the freezing and thawing process.
3Measurement precision
If firefly luciferase is used for reporting, then assay sensitivity is achieved, but the enzyme is less stable and more sensitive to inactivation in apoptotic cells compared to Renilla luciferase
Solution Approach 1:
The patent uses protease inhibitors and antioxidants as intermediary substances that specifically protect firefly luciferase from degradation during apoptosis. These intermediaries block the proteolytic and oxidative pathways that would otherwise inactivate the enzyme, allowing firefly luciferase to maintain both its high sensitivity and stability in apoptotic 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
The solution significantly reduces intra- and inter-assay variability, extending the shelf life of treated cells and maintaining their signal transduction activity, ensuring consistent assay results.
Implementation Method 1
Treatment of cells with vinblastin and subsequent freezing can lead to apoptosis and cell lysis resulting from vinblastin treatment and the formation of ice crystals respectively
Implementation Method 2
Reactive oxygen species (ROS) are also released and can create a state of oxidative stress
Data Source
AI summary
The invention provides an assay-ready frozen eukaryotic cell (stored frozen) in a composition that includes a cryopreservative and an inhibitor of xanthine oxidase, a kit containing a plurality of the assay-ready frozen eukaryotic cell, and a method of preparation thereof to minimize the variability in the performance of the cell, particularly in terms of undesirable intra- and inter-assay variability.