Cryopreservation Vessel with Mechanical Ice Nucleator
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Solution Overview
Problem
Current methods for cryopreservation of cells and tissues result in significant cell loss during the freeze-thaw process, especially in complex systems like tissues and organs, and are inadequate for large-scale cryopreservation in multi-vessel formats, leading to high well-to-well variability and unsatisfactory post-thaw viability.
Innovation Solution
The use of specially configured vessels with a mechanical ice nucleating device and an optimized preservation medium, such as CRYOSTOR™, to facilitate uniform ice nucleation and reduce osmotic stress, combined with insulating materials for controlled cooling and warming, enabling consistent cell survival and viability across multiple wells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current cryopreservation methods are used, then cells can be frozen and stored, but significant cell loss occurs during the freeze-thaw process
Solution Approach 1:
The patent applies preliminary action by pre-cooling the multiwell plate to 4°C before adding the cryopreservation medium, and pre-chilling the medium itself to 4°C. This preliminary temperature preparation ensures uniform cooling throughout the freezing process, preventing temperature gradients that cause differential freezing rates and subsequent cell loss. The insulating material is also pre-positioned in the plate wells to maintain thermal uniformity during the actual freezing cycle.
Solution Approach 2:
The patent changes critical parameters including the temperature profile (cooling rate of 1-2°C per minute to 4°C, then to -80°C), the composition of the cryopreservation medium (containing specific concentrations of cryoprotectants, salts, and buffering agents), and the physical configuration (insulating material placement, ice nucleating device). These parameter optimizations work together to minimize osmotic shock and intracellular ice crystal formation, thereby reducing cell loss while maintaining viability.
2Reliability
If conventional freezing methods are used, then cells can be preserved, but high well-to-well variability occurs in multi-vessel formats
Solution Approach 1:
The patent applies local quality by placing insulating material specifically in the peripheral wells of the multiwell plate, recognizing that these wells experience greater heat loss than central wells. This localized insulation compensates for the thermal gradient across the plate, ensuring uniform cooling rates throughout. Additionally, ice nucleating devices are strategically positioned in specific wells to promote synchronized freezing initiation, thereby eliminating well-to-well variability in freezing timing and outcome.
Solution Approach 2:
The patent performs preliminary cooling of the entire multiwell plate to 4°C before adding the cryopreservation medium to all wells. This preliminary action ensures that temperature uniformity is established before the freezing process begins, preventing differential cooling rates that would cause well-to-well variability. The insulating material is pre-installed in peripheral wells to maintain this thermal uniformity throughout the subsequent freezing cycle.
3Reliability
If standard cryopreservation protocols are used, then cells can be frozen, but osmotic shock and intracellular ice crystal formation cause cell death
Solution Approach 1:
The patent optimizes the chemical composition parameters of the cryopreservation medium, including the concentrations of cryoprotectants (e.g., DMSO, glycerol), salts (e.g., NaCl, KCl), buffering agents (e.g., HEPES), and other additives. These parameter adjustments create an osmotically balanced environment that minimizes osmotic shock during freezing and thawing, while the controlled cooling rate prevents rapid ice crystal formation. The insulating material and controlled thermal profile further prevent thermal shock that could exacerbate cellular damage.
Solution Approach 2:
The patent applies preliminary cooling to 4°C before initiating the freezing process, allowing gradual temperature reduction that prevents thermal shock and enables controlled ice nucleation. This preliminary action gives cells time to adapt to changing temperatures, reducing the severity of osmotic stress and ice crystal formation that would occur with rapid freezing. The insulating material is pre-positioned to maintain this gradual, uniform cooling throughout the sample.
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 significantly reduces well-to-well variability and improves post-thaw viability and shelf-life of cryopreserved cells, making large-scale cryopreservation more viable and reliable for applications like disease diagnosis and pharmaceutical analysis.
Implementation Method 1
an ice nucleating device which facilitates consistent well to well ice nucleation, a step necessary for the uniform survival of cells during the cryopreservation process
Implementation Method 2
The insulating material is a mechanical component, which provides a means of thermal insulation to the exterior (e.g., periphery) or interior of the vessel
Implementation Method 3
a nutrient-rich biopreservation medium that is configured to optimally maintain cellular osmotic and ionic balances, control free radical accumulation, and reduce the stress responses under non-normothermic conditions
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E~1G
AI summary
Provided are apparatuses for cryopreserving cells which include a vessel comprising a biocompatible substrate, wherein the vessel further comprises an interior and an exterior, and a mechanical ice nucleating device disposed in or on the vessel interior for initiating ice crystal formation. Also provided are kits comprising one or more apparatuses for cryopreserving cells and a biopreservation medium. Further provided are compositions comprising a vessel for holding cells, a mechanical ice nucleating device, a biopreservation medium, and cells disposed in the vessel. The apparatuses, kits, and compositions of the invention can optionally include an insulating material which is disposed on all or a portion of the vessel.