Mammalian Cell Cryopreservation Liquid Using Trehalose and Dextran
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Solution Overview
Problem
Current cell cryopreservation methods fail to effectively suppress cell death after thawing, particularly for mammalian cells like human pluripotent stem cells, which are crucial for regenerative medicine applications.
Innovation Solution
A cryopreservation liquid comprising 2.0 to 6.0% trehalose or its derivatives, 4.0 to 7.0% dextran or its derivatives, and DMSO or glycerin in an isotonic solution, such as lactated Ringer's solution, is used to cryopreserve mammalian cells, including mesenchymal stem cells, to enhance cell survival and proliferation post-thawing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cryopreservation liquids containing DMSO, FBS, and EG are used, then cell survival rate is improved, but cell death after thawing still occurs and cannot be effectively suppressed
Solution Approach 1:
The invention uses a composite cryopreservation liquid containing multiple protective components: trehalose (2.0-6.0% w/v) as a disaccharide cryoprotectant, dextran (4.0-7.0% w/v) as a polysaccharide cryoprotectant, and DMSO or glycerin as traditional cryoprotectants. This composite formulation provides synergistic protection that suppresses cell death after thawing more effectively than conventional single-component or dual-component formulations.
Solution Approach 2:
The invention optimizes the concentration parameters of each component within specific ranges: trehalose at 2.0-6.0% w/v, dextran at 4.0-7.0% w/v, and DMSO or glycerin at appropriate concentrations. These parameter optimizations ensure effective cryoprotection while maintaining cell viability and suppressing post-thaw cell death, representing a systematic approach to resolving the contradiction between survival rate improvement and death suppression.
2Ease of operation
If simple slow freezing method is used with conventional cryopreservation liquids, then ease of operation is improved, but cell death after thawing cannot be effectively suppressed
Solution Approach 1:
The composite cryopreservation liquid formulation with trehalose, dextran, and DMSO or glycerin provides robust cell protection that works effectively with the simple slow freezing method. This combination allows laboratories to use ease-of-operation simple freezing protocols without compromising cell survival, as the multi-component formulation compensates for the less controlled freezing process.
Solution Approach 2:
The trehalose and dextran components act as intermediary substances that provide additional protective mechanisms beyond traditional cryoprotectants. These intermediaries form protective complexes around cells during freezing and thawing, reducing membrane damage and protein denaturation even when using simple slow freezing methods without strict temperature control.
3Object-affected harmful factors
If vitrification method with high concentrations of DMSO, acetamide, propylene glycol, and polyethylene glycol is used, then ice crystal formation is suppressed, but device complexity and procedure complexity increase
Solution Approach 1:
The invention uses a composite formulation with trehalose, dextran, and DMSO or glycerin that provides effective cryoprotection through a combination of mechanisms including ice crystal suppression, membrane stabilization, and protein protection. This composite approach achieves ice crystal suppression similar to vitrification but with a simpler procedure that does not require ultra-rapid cooling equipment or strict temperature control protocols.
Solution Approach 2:
The cryopreservation liquid formulation uses readily available, inexpensive components (trehalose, dextran, DMSO, or glycerin) that can be easily prepared in standard laboratory settings. This eliminates the need for expensive specialized equipment required for vitrification, making the method accessible to ordinary laboratories while achieving comparable protection against ice crystal damage.
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 proposed liquid significantly reduces cell death and maintains high cell survival and proliferation rates after thawing, outperforming existing methods, making it suitable for regenerative medicine applications.
Implementation Method 1
By slowly cooling, intracellular water molecules are replaced with the cryoprotectant and dehydrated, the growth of ice crystals in and around cells is suppressed, and damage to the cell membranes and cell structures, and protein degeneration and cleavage is prevented
Implementation Method 2
suspending cells in a cryopreservation liquid containing glycerin, DMSO, keratin hydrolyzate, hydrolyzed gelatin, serum, serum albumin, and the like as a cryoprotectant
Data Source
AI summary
A liquid for cryopreserving a cell and a liquid for administration of a mammalian cell capable of cryopreserving a mammalian cell and effectively suppressing cell death after thawing, and a method for cryopreserving a mammalian cell using the cell cryopreservation liquid. The liquid is an isotonic solution that includes 2.0 to 6.0% (w/v) of trehalose or a derivative thereof, or a salt of the trehalose or the derivative, 4.0 to 7.0% (w/v) of dextran or a derivative thereof, or a salt of the dextran or the derivative, and DMSO or glycerin.