Hydroxyethyl starch (HES) is a synthetic, high-molecular-weight polymer derived from amylopectin that functions as a non-penetrating (extracellular) cryoprotectant (CPA). It is widely used as an alternative to DMSO, either as a partial substitute to reduce DMSO toxicity or, in some protocols, as the sole cryoprotectant.

Mechanism of Cryoprotection

As a large, hydrophilic polymer that remains outside the cell, HES protects biological material through several interrelated physical mechanisms.

Vitrification and Glass Formation. Non-penetrating polymers like HES increase solution viscosity and elevate the glass transition temperature of the unfrozen extracellular fraction. These effects reduce molecular mobility, thereby suppressing diffusion-driven damage and limiting degradative chemical reactions during cooling and storage.

Ice Recrystallization Inhibition (IRI). During rewarming, small ice crystals can merge into larger, more damaging ones—a process called recrystallization. HES plays a complex role here. While one study found that adding 6% (w/v) HES actually accelerated recrystallization during rewarming, it still protected Jurkat cells from injury, indicating that HES's protective effects extend beyond simple ice inhibition. HES is often combined with dedicated ice recrystallization inhibitors (IRIs), such as poly(vinyl alcohol) (PVA), which significantly enhance cell recovery under conditions that promote damage due to ice recrystallization.

Reduction of DMSO Toxicity. A major advantage of HES is its ability to reduce the required concentration of DMSO, a permeating CPA known to exert toxic effects on cells. By combining HES with a lower concentration of DMSO, researchers can maintain cryoprotective efficacy while minimizing the adverse effects associated with DMSO, such as heart rhythm disorders and bronchial spasms after infusion.