Microfluidic Vitrification Gradient for Oocyte Cryopreservation
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Solution Overview
Problem
Conventional cryopreservation methods, such as vitrification, cause osmotic shock and sub-lethal damage to oocytes and zygotes due to sudden changes in osmolality, leading to reduced viability and increased costs in assisted reproductive techniques.
Innovation Solution
A microfluidic device and method that control the rate of cryoprotectant exchange using a continuous temporal gradient of CPA concentrations, minimizing osmotic stress and cell shrinkage rate, as predicted by the Kedem-Katchalsky equations, to preserve the health and fertility of oocytes and embryos.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If high concentrations of cryoprotectant agents are used to avoid ice formation during vitrification, then ice crystal formation is prevented, but osmotic stress and cell shrinkage increase causing sub-lethal damage
Solution Approach 1:
The vitrification process is divided into multiple sequential equilibration steps with progressively higher CPA concentrations (e.g., 15% EG/15% DMSO for 5 min, then 20% EG/20% DMSO for 5 min, then 25% EG/25% DMSO for 5 min). This segmentation allows gradual osmotic adaptation, preventing sudden cell shrinkage while ultimately achieving sufficient CPA concentration to prevent ice crystal formation during vitrification.
Solution Approach 2:
Equilibration solutions containing lower concentrations of CPA are applied before the actual vitrification step. This preliminary exposure allows cells to gradually adapt to increasing osmolality and accumulate CPA inside cells in a controlled manner, preparing them for the final high-concentration vitrification step without experiencing sudden osmotic shock.
2Ease of operation
If manual pipetting methods are used for CPA exchange, then the process is simple to perform, but osmotic shock and cell damage increase due to sudden concentration changes
Solution Approach 1:
A microfluidic device is used to deliver CPA solutions to oocytes/zygotes through controlled fluid flow. The device uses hydraulic principles to maintain continuous exposure to gradually increasing CPA concentrations, eliminating the sudden concentration changes associated with manual pipetting while requiring minimal operator intervention.
Solution Approach 2:
The microfluidic device maintains continuous exposure of oocytes/zygotes to CPA solutions with gradually increasing concentrations, rather than the discontinuous step-wise addition of manual pipetting. This continuous exposure ensures smooth osmotic adaptation and prevents sudden cell shrinkage, improving cell viability while simplifying operator tasks.
3Reliability
If the number of equilibration steps is increased to reduce osmotic stress, then cell viability improves, but the complexity and time required for the procedure increases
Solution Approach 1:
Multiple equilibration steps with progressively higher CPA concentrations are merged into a single continuous microfluidic exposure process. The device automatically delivers a continuous gradient of increasing CPA concentrations, combining what would traditionally require multiple separate manual pipetting steps into one integrated operation, thereby maintaining high cell viability while reducing procedural complexity.
Solution Approach 2:
The microfluidic device dynamically changes the concentration parameter of CPA solutions delivered to oocytes/zygotes, creating a continuous gradient from low to high concentrations. This parameter change approach replaces the discrete step-wise concentration changes of manual methods, achieving gradual osmotic adaptation with fewer operational steps.
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 technology improves cell health, fertility, and developmental competence by reducing osmotic stress and cell damage, resulting in higher survival rates and better morphology of cryopreserved oocytes and embryos compared to manual pipetting methods.
Implementation Method 1
the osmotic stress produced by the sudden volume change due to loss of water caused by these agents
Implementation Method 2
transfer of oocytes and zygotes from culture media (CM) conditions at physiologic osmolality to a vitrification solution (VS) with high osmolality produces deleterious osmotic stress on the oocytes and zygotes
Implementation Method 3
the non-equilibrium phase change from liquid to glass phase
Implementation Method 4
Vitrification is a process of freezing used to preserve oocytes (e.g., eggs) and embryos used for assisted reproductive therapies
Implementation Method 5
conventional methods of manual vitrification, the oocytes and/or embryos are exposed to solutions with increasing concentrations of cryopreservative agents that prevent the formation of ice crystals
Data Source
AI summary
Provided herein is technology relating to cryopreservation and particularly, but not exclusively, to devices, systems, and methods for cryopreservation of biological materials such as oocytes, zygotes, and embryos. In particular, provided herein are methods of using microfluidic devices to exposing biological material to a vitrification solution having a time dependent concentration of a cryoprotectant agent.