Inductive Cryopreservation for Viable Hepatocyte Recovery
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Solution Overview
Problem
Current cryopreservation technologies are unable to produce large quantities of high-functioning hepatocytes, leading to limitations in cell availability for therapies and drug metabolism testing, due to issues with ice crystal formation, cooling and warming rates, and the impracticality of existing methods for large tissue volumes.
Innovation Solution
A method involving loading tissues with a cryoprotective agent and susceptor particles, followed by freezing and inductive heating for thawing, which allows for the recovery of large quantities of viable hepatocytes by using perfusion to resuscitate and condition organs, ensuring homogeneous distribution and removal of cryoprotectants, and avoiding recrystallization during rewarming.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional cryopreservation technologies (freezing and vitrification) are used, then cell preservation is achieved, but the quantity of recoverable hepatocytes is limited to about 3×10^6 cells per 3 mL vial with thawing viability of 50-70%
Solution Approach 1:
The patent changes the physical state parameter from conventional freezing to vitrification (glassy state), and introduces inductive heating as a new warming method. This allows achieving both high quantity (10^11 cells or greater) and high viability by controlling the phase transition and using magnetic field-based heating instead of conventional thermal warming.
Solution Approach 2:
The patent replaces conventional thermal convection heating with inductive heating using magnetic susceptor particles. This substitution enables rapid and uniform warming throughout the tissue, achieving high recovery quantities while maintaining cell viability by avoiding the limitations of convective heating methods.
2Quantity of substance
If vitrification is used to achieve high cell quantities, then the cooling rate must be extremely fast (Critical Cooling Rate), but the warming rate requirement (Critical Warming Rate) is at least an order of magnitude higher, which is only possible for small cell and tissue systems (1-3 mL)
Solution Approach 1:
The patent replaces conventional convective heating with inductive heating using magnetic susceptor particles distributed throughout the tissue. This allows rapid and uniform warming of large tissue volumes (greater than 10 mL) without the limitations of convective heating, enabling vitrification-based cryopreservation to scale to clinically relevant organ sizes.
Solution Approach 2:
The patent introduces magnetic susceptor particles as an intermediary medium within the tissue. These particles serve as heat transfer mediators that can be activated by magnetic fields to generate internal heat throughout the tissue, enabling rapid warming of large volumes while maintaining the vitrified state during storage.
3Reliability
If addition of cryoprotectants is used to lower the Critical Cooling Rate and Critical Warming Rate, then preservation is improved, but the toxic effects of cryoprotectants increase and the process becomes complex
Solution Approach 1:
The patent extracts and removes cryoprotectants from the tissue using dialysis or diffusion-based removal systems after vitrification. This allows the tissue to benefit from the protective effects of cryoprotectants during freezing while eliminating their toxic effects before use, achieving high preservation quality without the harmful side effects.
Solution Approach 2:
The patent performs preliminary loading of cryoprotectants and susceptor particles into the tissue before freezing, then performs preliminary removal of cryoprotectants after thawing but before use. This staged approach ensures optimal preservation during freezing while eliminating toxicity before the tissue is put to use.
4Productivity
If DCD livers are used as a source of hepatocytes, then availability of organ tissue is improved, but the yield, viability, and functionality of isolated hepatocytes deteriorate due to warm ischemia injury
Solution Approach 1:
The patent performs preliminary resuscitation and conditioning of DCD livers using perfusion with nutrient-rich solutions before cryopreservation. This preliminary action reverses warm ischemia injury and prepares the tissue for successful vitrification and subsequent hepatocyte recovery, transforming previously unusable DCD organs into viable cell sources.
Solution Approach 2:
The patent changes the temperature parameter dynamically during processing - using hypothermic perfusion during initial resuscitation, then transitioning to controlled cooling for vitrification, and finally rapid warming for recovery. This dynamic temperature control enables recovery of high-quality hepatocytes from DCD livers that would otherwise be unsuitable.
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 the production of large quantities of highly functioning hepatocytes with high viability and plating efficiency, making them available on demand for therapeutic and pharmaceutical applications, while avoiding the limitations of existing methods.
Implementation Method 1
The tissue may be thawed using inductive heating
Implementation Method 2
freezing the tissue
Implementation Method 3
loading the tissue with a solution comprising a cryoprotective agent
Implementation Method 4
Vitrification involves rapid cooling to a glassy rather than crystalline state
Data Source
AI summary
A method of preserving tissue includes loading the tissue with a solution with a cryoprotective agent and susceptor particles; freezing the tissue; thawing the tissue; and after freezing and thawing, further processing the tissue. Further processing may include slicing the tissue or digesting the tissue and recovering isolated cells from the digested tissue. The tissue may include at least a portion of an organ. The tissue may include a whole organ. The loading of the solution may be done via the vasculature of the organ. The tissue may be thawed using inductive heating. The susceptor particles may include nanoparticles.