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Programmable Cooling-Free Cryopreservation via Multi-Parameter Optimization of Natural Deep Eutectic Solvents.

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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.

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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.