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Programmable Cooling-Free Cryopreservation via Multi-Parameter Optimization of Natural Deep Eutectic Solvents.
Hengxin Han1,2,3, Ying Wang1,2,3, Taijie Zhan1,2,3
1Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, Shanghai 200093, China.
Langmuir : the ACS Journal of Surfaces and Colloids
|April 17, 2026
Summary
Natural deep eutectic solvents (NADES) offer a sustainable, low-toxicity alternative for cryopreservation. This study developed an efficient NADES-based protocol, achieving high cell viability without specialized cooling equipment.
Area of Science:
- Biophysics
- Materials Science
- Cell Biology
Background:
- Natural deep eutectic solvents (NADES) are emerging as low-toxicity, sustainable cryoprotectants.
- Conventional cryoprotective agents often exhibit significant cytotoxicity.
- NADES offer potential as eco-friendly alternatives in cryopreservation.
Purpose of the Study:
- To evaluate the cryoprotective efficacy of two NADES formulations (l-proline and glucose).
- To develop an optimized, equipment-free cryopreservation protocol using NADES.
- To investigate the biophysical properties influencing NADES cryoprotection.
Main Methods:
- Systematic evaluation of NADES cytotoxicity, ice inhibition, viscosity, and cellular permeability.
- Fourier-transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy to analyze hydrogen-bonding networks.
- Optimization of cooling rate and loading time for NADES-based cryopreservation.
Main Results:
- NADES demonstrated extensive hydrogen-bonding networks, leading to high ice recrystallization inhibition and unfrozen water content.
- An optimized protocol using 5% NADES at 5.28 °C/min achieved cell viability comparable to DMSO.
- The developed NADES protocol significantly reduced cryopreservation cytotoxicity.
Conclusions:
- NADES are highly efficient and biocompatible cryoprotectants.
- A straightforward, equipment-free cryopreservation protocol was established using NADES.
- This study validates NADES as a promising sustainable alternative in cryobiology.
