Thin-Walled Capillary Tube for Rapid Cryopreservation
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Solution Overview
Problem
Current cryopreservation methods for mammalian cells, such as slow freezing and vitrification, face challenges including cell injury from ice formation and high concentrations of cryoprotective agents, which are toxic to many cell types, limiting their effectiveness for long-term storage and viability.
Innovation Solution
A novel method using a thin-walled capillary tube made of thermally conductive material for rapid cooling of mammalian cells to a vitrified state without toxic levels of cryoprotectants, allowing cells to transition from a liquid to a super-cooled state without ice formation, using a vitrification solution with reduced CPA concentrations and optional nanoparticles or microparticles for enhanced thermal conductivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If slow freezing is used to cryopreserve cells, then ice formation is controlled and cell dehydration is achieved, but cell injury occurs due to highly concentrated solutions and mechanical interactions with ice
Solution Approach 1:
The patent applies vitrification, a phase transition process where the cell contents are cooled rapidly enough to form a glassy state without ice crystal formation. This eliminates the harmful mechanical interactions between ice crystals and cell structures that occur in slow freezing, while still achieving the temperature reduction necessary for cryopreservation
Solution Approach 2:
The patent changes the cooling rate parameter from slow (controlled freezing) to extremely rapid (vitrification), and adjusts CPA concentration parameters to optimize the balance between preventing ice formation and minimizing toxicity. This parameter optimization resolves the contradiction by finding conditions where rapid cooling achieves vitrification with reduced CPA levels
2Reliability
If vitrification is used to avoid ice formation, then cell injury from ice is prevented, but extremely high cooling rates or toxic concentrations of CPA are required
Solution Approach 1:
The patent optimizes multiple parameters simultaneously: cooling rate, CPA concentration, and exposure time. By finding the optimal combination where moderate CPA concentrations (avoiding toxicity) are used with controlled rapid cooling rates, the patent achieves vitrification without the extreme conditions that cause harm
Solution Approach 2:
The patent uses a partial approach to vitrification by applying rapid cooling for a sufficient but not excessive duration to achieve the glassy state, then immediately warming. This partial application of the vitrification process avoids the need for toxic CPA concentrations while still preventing ice formation
3Reliability
If high concentrations of CPA are used to achieve vitrification, then ice nucleation is avoided, but cell toxicity increases significantly
Solution Approach 1:
The patent changes the CPA concentration parameter from high (toxic) to optimized moderate levels, and compensates by adjusting the cooling rate parameter. This parameter trade-off allows achieving ice-free vitrification with reduced CPA concentrations that are less toxic to cells
Solution Approach 2:
The patent applies preliminary cooling at controlled rates before the final rapid vitrification step, allowing cells to adapt and reducing the CPA concentration needed for the subsequent vitrification phase. This preliminary action reduces the overall CPA requirement and associated toxicity
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 method effectively cryopreserves mammalian cells, including stem cells and oocytes, maintaining viability and pluripotency, while minimizing adverse effects, enabling long-term storage with rapid and efficient cooling rates, thus improving their usability in clinical and research settings.
Implementation Method 1
a thin-walled capillary tube made of a thermally conductive wall material that allows for rapid cooling of mammalian cells to a vitrified state
Implementation Method 2
rapid cooling to achieve vitrification in the absence of high concentration of CPAs and in the absence of ice formation
Implementation Method 3
allowing for the passage of cells suspended in the media from a liquid to a super-cooled liquid to a vitrified state
Data Source
AI summary
The present invention features novel methods for the cryopreservation of mammalian cell that combine the advantages of the slow-freezing and vitrification approaches while avoiding their shortcomings. Generally, the methods include the use of a capillary tube made of a thermally conductive wall material and a thin wall such that the ratio of the thermal conductivity of the wall material to the wall thickness is at least 1,000-500,000. The solution is then exposed to temperatures equal to or less than −80° C. and the vitrification solution containing the mammalian cells is cooled at a rate equal to or greater than 30,000-100,000,000° C./minute. The exposure of the capillary tube with a thermally conductive and thin wall allows for vitrification of the solution in the absence of ice formation. Cryoprotectants can also be added to the vitrification solution to further prevent ice formation.