Cryopreservation method and apparatus
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Solution Overview
Problem
Current cryopreservation techniques, such as controlled cooling, result in ice crystal formation, causing damage to biological samples, especially tissues and organs, and are unsuitable for preserving larger samples due to ice damage, while vitrification methods face challenges with rapid cooling rates and cryoprotectant toxicity, limiting their effectiveness for larger biological samples.
Innovation Solution
A method involving density-assisted vitrification where the top surface of a biological sample is cooled to form an ice layer, allowing the underlying sample to solidify as glass, using a thermally conducting member to homogenize temperature and progressively form ice from the top surface towards the base, with controlled cooling rates and additional cryoprotectant addition to enhance cryopreservation, facilitated by a cryopreservation apparatus with controlled cooling and agitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If controlled cooling is used to preserve biological samples, then samples can be stored at low temperatures, but ice crystals form and cause damage to the samples
Solution Approach 1:
The patent changes the cooling rate parameter from conventional slow cooling to extremely rapid cooling (vitrification), transforming the physical state of water from crystalline ice to amorphous glass, thereby eliminating ice crystal formation while maintaining long-term storage capability
Solution Approach 2:
The patent uses composite cryoprotectant formulations combining multiple agents (e.g., DMSO, glycerol, sugars) to achieve both rapid vitrification and cellular protection, creating a composite preservation system that prevents ice damage while maintaining sample integrity
2Object-affected harmful factors
If vitrification is used to avoid ice crystal formation, then samples are preserved without ice damage, but extremely rapid cooling rates are required which are difficult to achieve for larger samples
Solution Approach 1:
The patent segments the cooling process into controlled stages and uses segmented cryoprotectant application, allowing large samples to be vitrified by treating different regions sequentially rather than requiring uniform extreme cooling across the entire sample simultaneously
Solution Approach 2:
The patent introduces cryoprotectant agents as intermediary substances that mediate the phase transition of water, allowing vitrification to occur at more manageable cooling rates by chemically modifying the water structure and preventing ice nucleation
3Object-affected harmful factors
If cryoprotectants are added to prevent ice formation, then ice damage is reduced, but cryoprotectant toxicity affects the biological samples
Solution Approach 1:
The patent optimizes cryoprotectant concentration parameters and exposure time parameters, using lower concentrations combined with rapid vitrification to achieve protection while minimizing toxic effects, and controls temperature parameters to reduce toxicity during the preservation process
Solution Approach 2:
The patent uses composite cryoprotectant formulations that combine multiple agents with different mechanisms of action, allowing lower overall concentrations to be used while achieving equivalent or superior protection, thereby reducing individual agent toxicity
4Volume of stationary object
If conventional cryopreservation is used for large samples, then samples can be preserved, but ice crystals form and cause irreparable damage
Solution Approach 1:
The patent fundamentally changes the thermal processing parameters from conventional slow cooling to rapid vitrification cooling, and adjusts chemical parameters by using optimized cryoprotectant formulations, enabling large samples to be preserved without ice crystal formation
Solution Approach 2:
The patent applies cryoprotectants to samples in advance before the vitrification process, allowing sufficient time for penetration and protection of large sample volumes, and pre-cools equipment and environment to facilitate rapid cooling when the sample is introduced
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 approach enables effective cryopreservation of larger biological samples by minimizing ice damage, achieving vitrification without extreme cooling rates or pressure, and allowing for the preservation of samples greater than 1 mm³, maintaining structural and functional integrity.
Implementation Method 1
cooling the top surface of the sample to selectively form an ice layer at the top surface of the sample
Implementation Method 2
cooling progresses through a thermally conducting member, which is preferably located centrally of the sample liquid, which homogenises the temperature in the majority of the sample's remaining liquid component
Implementation Method 3
a layer of the sample below the ice layer solidifies as a glass, thus delivering a vitrified composition of biological material in the cryopreservation medium
Data Source
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AI summary
The present invention relates to methods and apparatus for the cryopreservation of biological samples involving a density assisted vitrification wherein a sample of biological material in a cryopreservation agent is cooled from its top surface, causing an ice layer to form thereon. As cooling continues the ice layer grows downwards through the sample to provide a cryoprotectant and biological material rich layer below the ice layer that undergoes vitrification as cooling continues to below the glass transition temperature.