Polyethylene glycol (PEG) is a synthetic, water-soluble polymer that functions primarily as a non-penetrating (extracellular) cryoprotectant (CPA). Its cryoprotective efficacy is highly dependent on its molecular weight, with low-molecular-weight formulations exhibiting unique properties that can even allow for cellular internalization. PEG is widely investigated as an alternative to dimethyl sulfoxide (DMSO) to mitigate toxicity concerns in cell and tissue preservation.
Mechanism of Cryoprotection
PEG protects biological material through a combination of extracellular and, for low-molecular-weight variants, intracellular mechanisms.
- Osmotic Dehydration and Ice Inhibition: As a non-penetrating CPA, PEG accumulates in the extracellular space, elevating osmotic pressure. This draws water out of the cell, reducing intracellular water content and thereby lowering the risk of damaging intracellular ice formation. For low-molecular-weight PEGs (e.g., PEG200), simple diffusion into cells or 3D structures like spheroids can suppress internal osmotic pressure development and directly inhibit ice formation.
- Membrane Stabilization: PEG is classified among non-permeating CPAs that help maintain membrane stability, protecting cells from the high osmotic stresses of freezing and thawing.
- Hydrogen Bonding and Water Structure Modification: PEG molecules contain hydroxyl groups that can interact with water through hydrogen bonding, altering water's hydrogen-bonding network. This can depress the freezing point and disrupt the ordered arrangement of water molecules required for ice crystallization.
- Ice Recrystallization Inhibition (IRI) and Ice Nucleation Inhibition (INI): The role of PEG in IRI is complex. Some cryomicroscopic studies have found that PEG did not exhibit IRI activity, unlike poly(vinyl alcohol) (PVA), yet it still reduced freezing-induced membrane leakage. Other research indicates that the effectiveness of low-molecular-weight PEGs like PEG200 may partly be attributed to INI, preventing the formation of initial ice nuclei.
