Cryopreservation of cells is a critical challenge in contemporary cryobiology, particularly in the burgeoning field of tissue engineering. The method of cryopreservation significantly affects the quality of cells post-thaw, as cells are sensitive to the stress induced by freezing and thawing, leading to physical damage, loss of functionality, or cell death. It is essential to develop protocols that ensure good physiological and adherent conditions post-thaw. This study investigates the effect of cryopreservation on human keratinocytes using the intracellular cryoprotective agent dimethyl sulfoxide (DMSO), known for mitigating cell damage during freezing and thawing, although its toxicity remains debated. We evaluated the cryopreservation of human keratinocytes with low (1.8% and 2.2% v/v) and standard (5% and 10% v/v) concentrations of DMSO during short-term storage (4 days) at -80°C. Post-thaw, we examined the impact of the cryopreservation process on cell viability, plasma membrane fluidity, and identified signs of cell death depending on the concentration of DMSO used in the freezing medium. This study is the first to systematically examine the impact of various DMSO concentrations on cell viability and plasma membrane fluidity of keratinocytes in the context of 30% FBS concentration in the freezing medium, slow cooling, and − 80°C temperature, highlighting the originality and significance of our research. This research demonstrated that lower DMSO concentrations (1.8% and 2.2%) significantly reduce keratinocyte viability due to increased apoptotic activity and cellular stress. In contrast, higher concentrations (5% and 10%) provide better protection and maintain higher cell viability. The study revealed that membrane fluidity increases with higher DMSO concentrations, which may facilitate the clustering of death receptors and the formation of apoptotic signaling complexes, thereby increasing the sensitivity of keratinocytes to apoptotic stimuli. Morphological analysis showed that lower DMSO concentrations lead to significant morphological changes and apoptosis, while higher concentrations result in cell enlargement and shape alteration. Ultrastructural analysis provided detailed insights into the internal structure of cells, revealing changes in the nucleus, mitochondria, and the presence of vesicles around the plasma membrane at lower DMSO concentrations, whereas higher DMSO concentration led to significant nuclear damage. These findings have implications for cryopreservation to improve cell viability and functionality post-thaw and may enhance the success of cell preservation in biomedical applications.

BACKGROUND: Despite the widespread use of DMSO for cryopreservation, its concentration-dependent effects on epithelial cells remain insufficiently understood, particularly regarding sublethal stress responses. OBJECTIVES: To investigate how different DMSO concentrations affect cryopreservation-induced stress in human keratinocytes at both structural and functional levels. METHODS: Keratinocytes were cryopreserved using 1.8%, 2.2%, 5%, and 10% DMSO and stored at-80 degrees C. Post-thaw viability, proliferation, membrane fluidity, and transmembrane potential were assessed alongside ultrastructural analysis using SEM and TEM. RESULTS: Unexpectedly, 5% DMSO preserved optimal ultrastructure and membrane integrity, yet induced persistent hyperpolarization, suggesting latent mitochondrial stress. Although 10% DMSO yielded the highest initial viability (97.9%), it triggered depolarization, nuclear lobulation, and mitochondrial swelling, indicating early onset of oxidative stress. Low concentrations (1.8%, 2.2%) failed to maintain membrane stability, resulting in apoptosis-like morphology and a 50% drop in proliferation. A novel negative correlation (r = -0.84) between DMSO concentration and membrane fluidity was identified, redefining the assumption that higher DMSO always rigidifies membranes. CONCLUSIONS: This study provides the first evidence that cryopreservation-induced membrane potential shifts occur in a nonlinear, concentration-dependent manner, decoupled from immediate cell survival. The finding that 5% DMSO induces functional stress despite ideal morphology suggests that structural preservation does not equate to cellular homeostasis. These insights challenge current cryopreservation standards and offer a new perspective on balancing cytoprotection with post-thaw functionality in cells. The results highlight the need for next-generation cryoprotective formulations targeting mitochondrial resilience and membrane dynamics.

HYPOTHESIS:Aqueous mixtures confined in mesoporous materials exhibit solid-liquid phase behavior that departs from predictions based on bulk mixture thermodynamics and confinement effects. We investigated confined organic and salt-based aqueous mixtures to identify the molecular origins of these deviations. Organics induce weaker melting-point and enthalpy depressions, whereas salt solutions show stronger effects than predicted. We propose that this divergence originates from solute adsorption or depletion within the non-freezable δ layer coating the pore walls-organic molecules adsorbing onto silica, while ions are excluded-thereby altering local composition and water activity in the pore center. EXPERIMENTS AND SIMULATIONS:We examined binary aqueous mixtures of dimethyl sulfoxide, formamide, and sodium chloride confined in SBA-15 mesoporous silica (∼8.5 nm pores) using calorimetry, positron annihilation spectroscopy, and Raman spectroscopy to probe phase transitions and molecular organization. All-atom molecular dynamics simulations quantified solute-surface interactions. FINDINGS:The proposed hypothesis explains the divergent behavior of confined mixtures through solute-specific adsorption and exclusion at the silica interface. Simulations and Raman spectroscopy confirm that dimethyl sulfoxide and formamide adsorb onto silanol-covered pore walls, stabilized by hydrogen bonding, whereas NaCl ions are excluded. These contrasting interactions produce heterogeneous radial solute partitioning, altering local composition and phase behavior. While all mixtures exhibit eutectic crystallization in the bulk, only the confined salt solution retained a first-order eutectic transition, consistent with solute partitioning within the non-freezable δ layer. This mechanism provides a molecular basis for phase behavior in nanoconfined aqueous systems, with implications for cryobiology, prebiotic chemistry, and materials engineering.

The interaction of dimethyl sulfoxide (DMSO) with lipid membranes has been extensively studied using molecular dynamics (MD) simulations, yet discrepancies with experimental findings persist. Here, we re-evaluate the effects of low DMSO concentrations (1.5-10 vol%) on dimyristoyl phosphatidylcholine (DMPC) membranes using updated AMBER force fields (LIPID17, OPC, GAFF2) to assess its cryoprotective role. Simulations were performed in both fluid (330K) and gel (260 K) phases as well as under ice-forming conditions. Across three independent replicas, no statistically significant effects of DMSO were detected on membrane thickness, area per lipid, hydration, or acyl-chain order, indicating that low levels of DMSO do not alter bilayer structure. This represents an improvement over earlier force-field descriptions, which often exaggerated DMSO-lipid interactions, and provides results more consistent with experimental evidence. DMSO showed mild enrichment at the hydrophobic-hydrophilic interface, particularly near carbonyl and glycerol groups, but most molecules remained in the solvent. The strongest effects therefore emerged in the solvent phase: DMSO slowed ice crystal growth by about a factor of five, was excluded from the ice lattice, and accumulated at the ice-membrane boundary forming an ice-free layer. Surprisingly, even without DMSO, ice formation in contact with the bilayer did not cause structural disruption, suggesting that cryoinjury involves additional membrane components beyond lipids. DMSO also strongly inhibited the temperature-driven variation of water density in the amorphous state. These findings suggest that at low concentrations, DMSO's cryoprotective action arises mainly from modulation of water and ice behavior rather than direct bilayer perturbation.

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

The thermal deep-level transient spectroscopy (DLTS) technique was applied to study the defect states and their activation energies of a polyurethane-based polymer network (PELLE) insulator. It was demonstrated that decomposition analysis of exponential transients allows a more accurate determination of the activation energy of the defective states. Two groups of activation energies for the PELLE polymer were observed, ranging from 0.49 eV to 1.9 eV and − 7.84 eV to − 2.53 eV, which were assigned to changes in the bond properties in the hard and soft segments of the studied polymer, respectively. The applicability of the DLTS method for the characterization of polymers was demonstrated. It was shown that the DLTS technique could contribute to a more comprehensive picture of the properties of polymers.

ABSTRACT An effort is made to explain the salt loading of hypertonicity-treated human red blood cells (RBC) and their post-hypertonic lysis, which occurs upon resuspension to isotonic media. It is recognized that the salt loading is consistent with the regulatory volume increase response - a physiological response applied by cells to adapt to hypertonic conditions. It is therefore hypothesized that salt loading occurs via physiological ion transport pathways, allowing passive dissipative fluxes across the plasma membrane with the preservation of membrane integrity. It is argued that post-hypertonic lysis is essentially a hypotonic lysis occurring already in an isotonic medium due to the excess salt in the cytoplasm. A simplified physiological model accounting for transmembrane ion fluxes was applied to simulate the RBC physiological state to address the problem quantitatively. Instead of assuming the plasma membrane to be impermeable to ions, the dynamic nature of cell physiological state and capacity of cells to perform acute cell volume regulation by increasing passive cation plasma membrane permeability is recognized. It is shown that the RBC plasma membrane contains ion pathways capable of supporting a several-order increase in catio permeability, facilitating overall salt loading. The obtained picture, consistent with general physiology, predicts the experimentally observed RBC behavior. Although treated in theory, evidence supporting the proposed theory is provided, and the high explanatory power of the proposed idea is demonstrated. While focused on human RBCs, this study offers a general mechanism for the osmotic injury of cells exposed to hypertonic solutions - conditions relevant to cell cryopreservation - since many cells possess cell volume regulation mechanisms. Implications of the proposed hypothesis explaining the cryoprotective mechanism of common cryoprotectants are discussed, and additives targeting ion transport pathways are suggested as novel cryoprotectants.

An effort was made to design a simple and efficient protocol for the cryopreservation of human keratinocytes, a cell line relevant to tissue engineering. A possibility of simultaneously preventing the injury from intracellular ice formation and slow-freezing injury by introducing a macromolecular nonpermeant polyethylene glycol 400 as a sole cryoprotectant with rapid freezing was recognized. Thermoanalytical and microstructural analysis of the potential cryoprotective mixture has been performed to test its efficacy in preventing cell cryoinjury mechanisms. The post-thaw cell recovery indicated successful cryopreservation with a similar recovery to the standard slow-freezing protocol with permeant dimethyl sulfoxide. The novel approach represents an alternative cryopreservation strategy that avoids intracellular cryoprotectant toxicity and offers a simple freezing procedure (plunging into liquid nitrogen) and a more practical cryoprotectant wash-out after the thawing. The cryopreservation protocol also brings up new possibilities for applying macromolecular additives as novel cryoprotectants. & COPY; 2023 Elsevier B.V. All rights reserved.

In this work, the phase behavior of cryoprotective mixtures based on dimethyl sulfoxide (DMSO) mixed with a lipid bilayer consisting of dimyristoyl phosphatidylcholine (DMPC) was studied. This system represented a model of a biological cell and its membrane. The aim of the work was to clarify the origin of the cryoprotective action of low-concentrated mixtures (1-10 vol%) DMSO in water, representing mixtures used in cryopreservation in cell therapy. The combination of experimental techniques of differential scanning calorimetry (DSC) and positron annihilation lifetime spectroscopy (PALS) allowed a study of crystallization behavior of water confined in liposomes imitating the intracellular environment. The ability of liposomes to show the fundamental aspects of water phase behavior seen during freezing of biological cells was proved. The presence of an amorphous freeze-concentrated phase of DMSO in the frozen state was confirmed and its possible crystallization into the DMSO trihydrate and ice during thawing was demonstrated. Correlation between the critical temperature range for the loss of cell viability during slow thawing and the temperatures of freeze-concentrated phase crystallization was found. Based on this finding, possible mechanisms of DMSO cryoprotection are discussed with support brought by results for the studied model system. Quantification of the ice phase fraction in the frozen mixtures revealed that even low concentrations of DMSO can induce a considerable decrease in the amount of ice present.

Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.

Sarcoidosis is a Th1-mediated chronic inflammatory disease characterized by non-caseating granulomas. Its pathogenesis is not yet clear, but the possible role of various proinflammatory cytokines is being discussed.This study aims to determine serum cytokine (IL-6, IL-12, IL-17, and IL-23) levels in patients with sarcoidosis, and to determine a possible correlation with clinical and laboratory findings of the disease.Forty-four biopsy-proven sarcoidosis patients followed up at a single centre and 41 healthy volunteers were included in the study. Demographic, clinical, laboratory, and radiological data of all patients were recorded. Serum samples from the patients and the control group were taken and IL-6, IL-12, IL-17, IL-23 were measured by ELISA method.Of the 44 sarcoidosis patients, 13(29.5%) were male and 31(70.5%) were female. Average patient age was 47.4 years, mean disease duration was 3.2 years. Twenty-one (47.7%) patients had erythema nodosum, three (6.8%) had uveitis, 40(90.9%) had arthralgia, 23(52.3%) had ankle arthritis, 15(34.1%) had enthesitis. Laboratory evaluation showed increased serum ACE levels in 24(54.5%) patients, increased serum calcium levels in 11 (25%) patients, increased serum D3 levels in 5(11.4%) patients, increased ESR and CRP levels in 22(50%) and 23(52.3%) patients, respectively. Compared with the control group higher serum IL-23 levels were found in the patients with sarcoidosis (p = .01). Serum IL-23 was associated with ankle arthritis (p = .02). Serum IL-6, IL-12, and IL-17 levels were similar in the sarcoidosis patients and the control group (p = .128, p = .212, p = .521 respectively).In our study, we found increased serum IL-23 in patients with sarcoidosis, while serum IL-6, IL-12, and IL-17 were detected as normal. Although our results are somewhat contradictory to other studies in the literature, the question should still be whether sarcoidosis is a Th1/Th17 disease. Multicentre studies are needed in this regard.La sarcoidosis es una enfermedad inflamatoria crónica mediada por Th1, caracterizada por granulomas no caseificantes. Su patogenia no está clara todavía, aunque se está debatiendo el posible rol de las diversas citocinas proinflamatorias.El objetivo de este estudio es determinar los niveles de citocinas séricas (IL-6, IL-12, IL-17 e IL-23) en los pacientes con sarcoidosis, así como establecer una posible correlación con los hallazgos clínicos y de laboratorio de la enfermedad.Se incluyó en el estudio a 44 pacientes con sarcoidosis verificada mediante biopsia, cuyo seguimiento se realizó en un único centro, y 41 voluntarios sanos. Se registraron los datos demográficos, clínicos, de laboratorio y radiológicos de todos los pacientes. Se tomaron muestras séricas de los pacientes y el grupo control, midiéndose los niveles de IL-6, IL-12, IL-17 e IL-23 mediante el método ELISA.De los 44 pacientes con sarcoidosis, 13 (29,5%) fueron varones y 31 (70,5%) fueron mujeres. La edad media de los pacientes fue de 47,4 años, y la duración media de la enfermedad fue de 3,2 años. Veintiún (47,7%) pacientes tenían eritema nudoso, 3 (6,8%) tenían uveítis, 40 (90,9%) tenían artralgia, 23 (52,3%) tenían artritis de tobillo y 15 (34,1%) tenían entesitis. La evaluación de las pruebas de laboratorio reflejó un incremento de los niveles séricos de ECA en 24 (54,5%) pacientes, de los niveles séricos de calcio en 11 (25%) pacientes, de los niveles séricos de D3 en 5 (11,4%) pacientes y de los niveles de ESR y PCR en 22 (50%) y 23 (52,3%) pacientes, respectivamente. En comparación con el grupo control, se encontraron niveles séricos de IL-23 más elevados en los pacientes con sarcoidosis (p = 0,01). Los niveles séricos de IL-23 estuvieron asociados a artritis de tobillo (p = 0,02). Los niveles séricos de IL-6, IL-12 e IL-17 fueron similares en los pacientes con sarcoidosis y en los sujetos del grupo control (p = 0,128, p = 0,212 y p = 0,521, respectivamente).En nuestro estudio encontramos un incremento de los niveles séricos de IL-23 en los pacientes con sarcoidosis, mientras que se detectaron niveles normales de IL-6, IL-12 e IL-17. Aunque nuestros resultados son ligeramente contradictorios con respecto a otros estudios de la literatura, persistiría la cuestión de si la sarcoidosis es una enfermedad de Th1/Th17. Son necesarios estudios multicéntricos a este respecto.

This work investigates the free-volume properties of the dimethyl sulfoxide (DMSO)-water mixtures by positron annihilation lifetime spectroscopy over a wide temperature range of 20-320 K. The processes of melting and solidification of the water, DMSO and the DMSO-water mixtures at 1.8, 2.0 and 10% vol. DMSO respectively were studied. It was found that the recrystallization during heating of the water-DMSO cryoprotective mixtures above 160 K at low DMSO concentrations is affected by the amount of DMSO in the mixture. The amount of amorphous phase formed during cooling influences the hysteresis between cooling and heating cycles which could be crucial for cell survival. Experiments also show the time dependence of crystallization which indicates that rapid heating can suppress this secondary crystallization which is undesirable during the cell thawing process. Similar concentrations of DMSO (1.8% and 2% vol. DMSO in water) where a 2% vol. DMSO mixture secures cell survival but 1.8 vol% does not, showed differences in structural and dynamic properties that are key factors in cell survival. These results were supported by differential scanning calorimetry and low frequency dielectric spectroscopy measurements. The obtained data are in strong agreement with the observed cryoprotective efficacy of the DMSO-water mixtures on living cells.