Organ Cryopreservation Using Two-Step Vitrification Freezing
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Solution Overview
Problem
Current methods for cryopreservation of large tissues and organs, such as limbs and organs, face challenges in preventing ice crystal formation, tissue toxicity from high CPA concentrations, and fracturing due to rapid cooling, limiting their long-term survival and viability for transplantation.
Innovation Solution
A two-step cryopreservation method involving a rapid freezing step followed by a gradual freezing step, using a vitrification solution at controlled temperatures to achieve long-term survival, including a first seconds-long freezing at -210 °C to -197 °C and a second minutes-long freezing at or above the glass-transition temperature (Tg') of -140 °C to -100 °C.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If conventional cryopreservation methods are used to preserve large tissues and organs, then long-term preservation is achieved, but ice crystal formation and tissue damage occur
Solution Approach 1:
The patent changes the physical-chemical parameters of the preservation medium by using high concentration cryoprotectants (40% v/v) to alter the freezing behavior of water in tissues. This parameter change enables vitrification instead of conventional freezing, preventing ice crystal formation while achieving long-term preservation at temperatures above -196°C
Solution Approach 2:
The patent utilizes the phase transition from liquid to glassy state (vitrification) instead of the conventional liquid-to-crystalline ice transition. By controlling cooling rates and using cryoprotectants, the tissue water transforms into a glassy phase that prevents ice crystal formation while maintaining molecular mobility restrictions for long-term preservation
2Reliability
If high concentrations of cryoprotectants are used to prevent ice formation, then vitrification is achieved, but tissue toxicity increases
Solution Approach 1:
The patent applies preliminary action by pre-equilibrating tissues with lower concentrations of cryoprotectants before the final high-concentration vitrification step. This staged approach allows cells to gradually adapt to increasing CPA concentrations, reducing osmotic shock and toxicity while ensuring complete vitrification protection
Solution Approach 2:
The patent segments the cryopreservation process into multiple stages with progressively increasing cryoprotectant concentrations. This segmentation allows the tissue to undergo gradual adaptation to CPA toxicity, with each stage building tolerance for the next, ultimately achieving high-concentration vitrification with minimized overall toxic exposure
3Reliability
If rapid cooling is applied to achieve vitrification, then ice nucleation is minimized, but tissue fracturing occurs
Solution Approach 1:
The patent changes the mechanical parameters of the tissue by modifying its viscoelastic properties through cryoprotectant infiltration. This parameter change increases tissue ductility and reduces brittleness during the rapid cooling phase, preventing fracture while maintaining the rapid cooling rates necessary for vitrification
Solution Approach 2:
The patent applies beforehand cushioning by infusing tissues with cryoprotectants that act as molecular cushioning agents during rapid cooling. These cryoprotectants fill interstitial spaces and stabilize cellular structures, cushioning against the mechanical stresses and thermal shocks that would otherwise cause tissue fracturing during rapid cooling
4Loss of time
If the limb ischemia window is extended for reconstruction, then autologous tissue availability improves, but tissue viability decreases
Solution Approach 1:
The patent uses vitrification phase transition to preserve tissue in a metabolically inactive but structurally intact state. This phase transition allows tissues to be stored for extended periods without degradation, then rapidly transitioned back to viable state through controlled warming, effectively extending the reconstruction time window while maintaining tissue viability
Solution Approach 2:
The patent applies preliminary action by performing cryopreservation immediately after tissue harvest, placing the tissue in a protected vitrified state before any degradation can occur. This preliminary preservation action maintains tissue viability indefinitely, allowing reconstruction to be performed at any convenient time rather than within a narrow ischemic window
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 method enables long-term survival and viability of cryopreserved tissues and organs, allowing for successful transplantation with minimal damage, as demonstrated by the successful cryopreservation and re-transplantation of vascularized tissues and limbs.
Implementation Method 1
Vitrification is the solidification of liquid into a solid glassy phase. Tissue vitrification is achieved by rapid cooling and rapid warming of tissues in the presence of high concentrations of cryoprotectants (CPAs).
Implementation Method 2
The main challenge of cryopreservation lies in preventing the damage induced by the formation of intra and extracellular ice crystals produced by conventional cryopreservation methods.
Data Source
Figure 1A~1F
Figure 2AI
Figure 2AII~2CII
AI summary
The present invention is directed to a method and a device for long-term cryopreservation of a biological sample, including a vascularized tissue, an innervated tissue, or both, such, but not limited to a limb.