Cell Monolayer Cryopreservation With Thermal-Matched Substrates
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Solution Overview
Problem
Cryopreservation of cell monolayers is challenging due to intracellular ice formation and cell detachment during freezing and thawing processes, which leads to reduced viability and detachment, especially in tissues like corneas and endothelial cells.
Innovation Solution
A cryopreservation method using a substrate with a coefficient of thermal expansion matching that of ice, combined with specific cryoprotectants like DMSO, HES, and CS, and controlled cooling rates to minimize ice formation and cell detachment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If slow cooling is used during cryopreservation, then cell dehydration damage is reduced, but intracellular ice formation increases
Solution Approach 1:
The patent applies parameter changes by modifying the cooling rate from the conventional 1°C/min to a slower rate of 0.05-0.5°C/min. This parameter adjustment allows sufficient time for controlled intracellular ice formation while maintaining slow cooling conditions that prevent severe cell dehydration damage, thereby resolving the contradiction between avoiding dehydration and preventing intracellular ice formation
Solution Approach 2:
The patent uses a composite cryopreservation solution containing multiple components: penetrating CPAs (DMSO, glycerol), non-penetrating CPAs (HES, sugars), and buffers. This composite formulation works synergistically to protect cells during slow cooling by combining the dehydrating effect of penetrating CPAs with the protective effects of non-penetrating CPAs, allowing controlled intracellular ice formation without severe dehydration damage
2Reliability
If rapid cooling is used during cryopreservation, then intracellular ice formation is minimized, but cell dehydration damage increases
Solution Approach 1:
The patent changes the cooling rate parameter to a slow rate (0.05-0.5°C/min), which is opposite to the rapid cooling approach. This slow cooling rate allows controlled water efflux from cells while preventing severe dehydration damage through the protective cryopreservation solution, thereby avoiding both rapid cooling-induced dehydration and slow cooling-induced intracellular ice formation
3Reliability
If conventional cryopreservation protocols are used for monolayers, then cell detachment occurs during freezing and thawing, but modified protocols increase process complexity
Solution Approach 1:
The patent applies preliminary action by pre-cooling the substrate to -20°C to -80°C before adding the cryopreservation solution and cells. This preliminary cooling of the substrate creates a controlled thermal environment that prevents sudden temperature shocks and reduces cell detachment during the subsequent freezing process, while the simple cooling rate control (0.05-0.5°C/min) keeps the protocol manageable
Solution Approach 2:
The patent modifies the cooling rate parameter to 0.05-0.5°C/min, which is slower than conventional protocols. This parameter change reduces thermal stress on cell-monolayer-substrate interactions during freezing and thawing, thereby minimizing cell detachment while maintaining protocol simplicity through a single adjustable parameter
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 high post-thaw viability and attachment of cells, particularly endothelial cells, by reducing intracellular ice formation and detachment, using substrates like Rinzl™ coverslips with a thermal expansion similar to ice, and optimized cryoprotectant concentrations and cooling rates.
Implementation Method 1
the substrate has a coefficient of thermal expansion that sufficiently matches the coefficient of thermal expansion of ice that damage to the cells is avoided
Implementation Method 2
cooling the cells in the presence of a cryopreservative to freeze the medium and convert water to ice
Data Source
AI summary
Cryopreservation of endothelial cell monolayers is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (DMSO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of cell monolayers. The addition of 2% chondroitin sulfate to 5% DMSO and 6% HES and cooling at 0.2 or 1° C./min led to high membrane integrity (97.3±3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9±3.7% membrane integrity) with metabolic activity 12 hours post-thaw comparable to unfrozen control.