High Aspect Ratio Vitrification Cassettes for Cryopreservation
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Solution Overview
Problem
Current methods for cryopreserving Cryptosporidium oocysts are limited by small sample volumes, toxicity of cryoprotective agents, and inefficiencies in CPA loading, which hinder research and clinical applications due to low viability and infectivity post-thawing.
Innovation Solution
Development of high aspect ratio vitrification cassettes and step-wise addition of cryoprotective agents to increase intracellular concentrations while minimizing toxicity, enabling large sample volume cryopreservation with high viability and infectivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If conventional microcapillary methods are used for vitrification, then cooling rate is maintained, but sample volume is limited to 2 μL
Solution Approach 1:
The patent transitions from conventional microcapillary geometry to a high aspect ratio cassette design with dimensions of 40-65 mm by 15-45 mm. This dimensional change allows the sample chamber to hold 50-500 μL (up to 100-fold volume increase) while maintaining high surface area to volume ratio for rapid cooling at rates ≥100,000°C/minute
Solution Approach 2:
The patent modifies the geometric parameters of the containment device by increasing the length and width dimensions while controlling thickness, creating a high aspect ratio structure. This parameter optimization enables simultaneous achievement of large sample volume and rapid cooling rate, resolving the traditional trade-off between these two parameters
2Reliability
If cryoprotective agents are added all at once, then vitrification is achieved, but toxicity increases and intracellular concentration is insufficient
Solution Approach 1:
The patent divides the cryoprotective agent addition process into multiple sequential steps: (1) initial addition of CPA to achieve first concentration, (2) removal and replacement with second CPA solution at higher concentration, (3) optional third addition. This segmentation allows progressive intracellular accumulation of CPAs while minimizing acute toxicity effects
Solution Approach 2:
The patent performs preliminary dehydration of cells using dehydrating agents (sucrose, trehalose, raffinose, stachyose, dextran, or salts) before adding cryoprotective agents. This preliminary action prepares cells to better tolerate and accumulate CPAs, reducing toxicity while ensuring adequate intracellular concentrations for effective vitrification
3Reliability
If high concentration of cryoprotective agents is used, then vitrification is achieved, but toxicity increases
Solution Approach 1:
The patent segments the CPA concentration increase into steps, starting with lower concentrations and progressively increasing to higher concentrations in subsequent additions. This allows the cell membrane to adapt gradually to increasing CPA levels, achieving effective vitrification concentrations while minimizing toxic effects
Solution Approach 2:
The patent employs periodic addition and removal of CPA solutions with incubation periods between steps. This periodic action allows cells to adjust to each concentration level before the next addition, maintaining viability while achieving the necessary intracellular CPA concentrations for successful vitrification
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 achieves a 100-fold increase in sample volume with maintained viability and infectivity, overcoming previous limitations and enabling multiple inocula for research and clinical trials.
Implementation Method 1
cooling the cells in the vitrification solution in the vitrification cassette to a temperature less than or equal to a glass transition temperature of the vitrification solution at a rate equal to or greater than 100,000° C./minute, wherein the cooling causes vitrification of the plurality of cells
Implementation Method 2
cooling the cells in the vitrification solution in the vitrification cassette to a temperature less than or equal to a glass transition temperature
Data Source
AI summary
Methods of bulk cryopreservation of C. parvum oocysts by vitrification using high aspect ratio cryopreservation devices are disclosed. Cryopreserved oocysts exhibit high viability, maintain infectivity in vitro, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to that observed with unfrozen oocysts.