Non-Freezing Stem Cell Storage Liquid for DMSO-Free Preservation
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Solution Overview
Problem
Existing cryopreservation methods for human iPS cells, such as using UW solution, face challenges with low survival rates, cytotoxicity from cryoprotective agents like DMSO, and differentiation inducing capability, while non-freezing low-temperature storage methods are inadequate for pluripotent stem cells.
Innovation Solution
A non-freezing refrigerated storage liquid is developed by mixing MEM-alpha with UW solution at a ratio of about 1/1 to 1/2, incorporating arousal periods at 37°C during storage to mimic hibernation, with specific ion concentrations and additives to maintain cell viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If UW solution is used for refrigerated storage of human iPS cells, then the storage method is simple and widely applicable, but the cell viability and proliferative ability deteriorate significantly
Solution Approach 1:
The patent modifies the chemical composition parameters of the storage solution by replacing UW solution with a new formulation containing MEM-alpha medium, b2 supplement, and specific amino acids (L-alanyl-L-glutamine, L-proline, L-ornithine) to achieve both high cell viability and ease of use
Solution Approach 2:
The patent creates a composite storage solution by combining multiple components (MEM-alpha medium, b2 supplement, and specific amino acids) to achieve synergistic effects that maintain cell viability while simplifying the storage process
2Duration of action of stationary object
If cryopreservation with DMSO is used, then long-term storage is achieved, but cytotoxicity and differentiation inducing capability increase
Solution Approach 1:
The patent extracts and eliminates DMSO and other harmful cryoprotective agents from the storage system, replacing them with a non-toxic refrigerated storage solution that achieves equivalent preservation without cytotoxic side effects
Solution Approach 2:
The patent changes the temperature parameter from cryopreservation (below -80°C) to refrigerated storage (2-15°C) and modifies the chemical composition to eliminate DMSO, achieving a balance between storage duration and cell health
3Reliability
If slow-freezing cryopreservation method is used, then cell preservation is attempted, but survival rate after storage decreases
Solution Approach 1:
The patent avoids phase transition (freezing) entirely by using refrigerated storage at 2-15°C, eliminating ice crystal formation and associated mechanical damage to cells, thereby improving survival rate while maintaining preservation quality
Solution Approach 2:
The patent changes the temperature parameter from freezing conditions to refrigerated conditions and modifies the solution composition to prevent ice crystal formation, achieving high survival rates through non-freezing preservation
Data Source
AI summary
Provided are a cold storage method and a cold storage liquid that are suitable for non-freezing refrigerated storage of human or animal stem cells such as iPS cells or embryos in a non-frozen state. According to an embodiment, the non-freezing refrigerated storage liquid is: a mixture liquid (HTMα, HTMx2c13), in which MEM-alpha (Minimum Essential Medium Eagle (MEM) Alpha Modification) is mixed with a UW solution (UW_sln; BELZER UW (registered trademark) COLD STORAGE SOLUTION), or a modified UW solution (polymer component being changed to PVA), are mixed in a ratio of about 1/1 to 1/2; or a storage liquid having a composition equivalent to the mixture liquid, especially in respect of concentrations of potassium ions and sodium ions. And, arousal period(s) of maintaining a temperature of 30° C. to 37° C. is inserted at each of two to five days, into a non-freezing refrigerated storage period, which is made at 2° C. to 8° C. for 2 to 10 days or 4 to 20 days.