Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

Abstract

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges

“…This result supports the transition from DMSO to non-DMSO in order to enable scale-up of the fill-and-finish process in a manufacturing setting, while adhering to a high batch consistency. Compared with DMSO whose cytotoxicity arises from dissolution, epigenetic and genomic alteration, these non-DMSO CPAs, typically with larger molecular weight than DMSO, can cause pre-freeze cell loss predominantly due to osmotic stress upon CPA introduction ( Best, 2015 ; Hornberger et al, 2021 ; Matsumura et al, 2021 ). Compared with D5 crest and D7 SN, which exhibited no significant cell loss over the course of prefreeze exposure to the non-DMSO CPA solution ( p > 0.05), D14 mSN experienced minor (10%) yet significant ( p < 0.05) decrease in viable cell count during the first 30 min of this process, suggesting that sensitivity to the osmotic stress varied by cell stage with the most mature cells being most sensitive.…”

Distribution of Sulfate-Reducing Bacteria in the Environment: Cryopreservation Techniques and Their Potential Storage Application

“…Recently, a novel cryoprotectant, carboxylated poly-L-lysine, was examined and found to indicate restricted mobility and increased solution viscosity of eukaryotic cells. In addition, intermolecular interactions facilitated the glass transition of carboxylated poly-L-lysine, which prevents intracellular ice formation and osmotic shock during freezing [88].…”

Protein hydration and its freezing phenomena: Toward the application for cell freezing and frozen food storage

“…Although mechanistic studies using synthetic polyampholytes suggested that they protect the cell membrane from freezing-induced damages [ 21 ], a conclusive mechanism was unknown. In this study, we show molecular-level investigation of the mechanism of cryopreservation using solid-state NMR [ 22 ]. We investigated the soluble state as well as the molecular mobilities of the polymer chain, water, and salts at freezing temperatures and compared it with control samples to demonstrate polyampholytes’ ability to prevent dehydration and decrease intracellular ice freezing during cryopreservation.…”