Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency.

We describe the development and application of a novel rapid sample-mixing technique for real-time NMR (nuclear magnetic resonance) spectroscopy. The apparatus consists of an insert inside a conventional NMR tube coupled to a rapid injection syringe outside the NMR magnet. Efficient and homogeneous mixing of solutions in the NMR tube is achieved with a dead time of tens of milliseconds, without modification of the NMR probe or additional hardware inside the magnet. Provision is made for the inclusion of an optical fiber to allow in situ laser irradiation of samples, for example to generate photo-CIDNP (chemically induced dynamic nuclear polarization). An NMR water suppression method has been implemented to allow experiments in H(2)O as well as in deuterated solvents. The performance of the device has been tested and optimized by a variety of methods, including sensitive detection of residual pH gradients and the use of NMR imaging to monitor the extent of mixing in real time. The potential utility of this device, in conjunction with the sensitivity and selectivity of photo-CIDNP, is demonstrated by experiments on the protein hen lysozyme. These measurements involve the direct detection of spectra during real-time refolding, and the use of CIDNP pulse labeling to study a partially unfolded state of the protein under equilibrium conditions. Magnetization transfer from this disordered state to the well-characterized native state provides evidence for the remarkable persistence of nativelike elements of structure under conditions in which the protein is partially denatured and aggregation prone.

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Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs can be used for a variety of regenerative medicine therapies, in fact, hBMC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or dimethyl sulfoxide (DMSO) by using a novel polyampholyte cryoprotective agent (CPA). This CPA is based on carboxylated poly-l-lysine (COOH-PLL) and it was produced by a reaction between ε-poly-l-lysine and succinic anhydride. (1)H-NMR and two-dimensional correlation ((1)H-(13)C HSQC) spectroscopy revealed that COOH-PLL did not have a special structure in solution. The hBMSCs can be cryopreserved for 24 months at -80 °C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL, which introduces carboxyl groups that result in > 90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small molecule CPAs. These results also suggest that using COOH-PLL as a CPA for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine.

Three extracts, namely hot-water fraction (HWF), water-soluble fraction (WSF), and ethanol-soluble fraction (ESF), were prepared from fresh bee bread imported from Lithuania. The protein and total phenolic contents of these samples were very high. Among them, WSF at 100% concentration showed the highest antioxidative ability and scavenging ability. On the other hand, ESF at 10% concentration possessed the highest ability against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals. Bee bread will apply more and more as health food and medicine due to its functional properties such as antioxidative ability and scavenging activities of reactive oxygen species.

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.