Dipolar aprotic solvents, including dimethyl sulfoxide (DMSO), account for 20 % of organic solvents used in organic synthesis, pharmaceutical development, and industrial processes because of their excellent solubility. However, there is a lack of sensors for the easy and real-time identification of dipolar aprotic solvents. To address this challenge, in this study, a U-shaped evanescent optical fiber (UOFE) sensor was developed by electrospinning polyacrylonitrile (PAN) combined with a Ti3C2 MXene/TiO2 hybrid to detect DMSO gas. UOFEs equipped with PAN/Ti3C2 MXene/TiO2 nanofibers demonstrated remarkable sensitivity towards DMSO, primarily owing to the dipole-dipole interactions, DMSO solubility, and refractive index changes. The cross-sensitivity of the sensor to dipolar aprotic solvents, such as N,N-dimethylacetamide, N,N-dimethylformamide, 1-methyl-2-pyrrolidone, and γ-butyrolactone, was demonstrated and the developed sensor selectively detected DMSO gas. The superior performance of the UOFEs was attributed to the synergistic effects of the electrospun PAN/Ti3C2 MXene/TiO2, thereby enhancing the gas diffusion properties and facilitating efficient interaction with DMSO. These results highlight the potential of combining electrospun nanofibers and optical fibers for gas sensing applications, such as indoor air-quality monitoring, environmental protection, and industrial safety.

Single cell transcriptomics (SCT) has revolutionized our understanding of cellular heterogeneity, yet the emergence of single cell proteomics (SCP) promises a more functional view of cellular dynamics. A challenge is that not all mass spectrometry facilities can perform SCP, and not all laboratories have access to cell sorting equipment required for SCP, which together motivate an interest in sending bulk cell samples through the mail for sorting and SCP analysis. Shipping requires cell storage, which has an unknown effect on SCP results. This study investigates the impact of cell storage conditions on the proteomic landscape at the single cell level, utilizing Data-Independent Acquisition (DIA) coupled with Parallel Accumulation Serial Fragmentation (diaPASEF). Three storage conditions were compared in 293T cells: (1) 37 °C (control), (2) 4 °C overnight, and (3) -196 °C storage followed by liquid nitrogen preservation. Both cold and frozen storage induced significant alterations in the cell diameter, elongation, and proteome composition. By elucidating how cell storage conditions alter cellular morphology and proteome profiles, this study contributes foundational technical information about SCP sample preparation and data quality.

Natural products represent one of the vital sources of anticancer drugs. However, the rapid screening of anticancer active compounds from complex extracts continues to pose a significant challenge. Although microplate-based high-resolution inhibition profiling have demonstrated their effectiveness in rapidly pinpoint individual bioactive components in extracts, the majority of these assays rely on chemical or enzymatic reactions. This study presents a new analytical screening method combining cellular assays and HPLC micro-fractionation to identify anticancer compounds in complex plant extracts. The method development involved optimizing 96-well plate configurations and DMSO transfer volumes for the cell-based assay using standard natural molecules and an artificial mixture. The optimized method was applied to profile anticancer compound in ethyl acetate extracts of Eomecon chionantha and Tacca plantaginea. Through repeated chromatographic separation and structural elucidation, we isolated two anticancer compounds from E. chionantha and eight from T. plantaginea, including three new molecules. Our method overcomes the toxicity associated with organic solvents used in HPLC fractionation on cell models and optimizes the volume of DMSO required for transferring materials into cell culture plates, enabling the profiling of anticancer molecules through the widely used MTT assay.

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Traditional treatment methods have certain limitations and it is difficult to effectively delay the disease progression. Mesenchymal stem cells (MSCs), owing to their potential for self-renewal, multidirectional differentiation, and immunomodulatory abilities, can regulate the renal immune microenvironment and repair damaged tissues, providing a new strategy for the treatment of DN. However, MSCs face problems such as immune rejection, cell inactivation, challenges in directed differentiation, insufficient homing ability, and low cell retention rate after delivery. These issues limit their clinical application in patients with DN. This review aims to propose optimization strategies targeting DN pathological features to improve MSC effectiveness and reduce their side effects. Specifically, it involves optimizing cell culture systems and cryopreservation protocols, along with pre-transplantation pharmacological conditioning to boost the functionality and viability of MSCs. Additionally, the exploration of synergistic drug-MSC combination therapies was carried out, taking advantage of diverse mechanisms of action to improve therapeutic outcomes. The integration of biomaterials and gene editing technologies to significantly enhance cell survival, target specificity, and tissue engraftment was also pursued. Concurrently, the determination of optimal therapeutic dosages and administration routes remained crucial. These multifaceted strategies not only provide a theoretical framework for overcoming existing technical limitations but also lay a robust foundation for accelerating the clinical translation of MSC-based therapies.

Cryopreservation techniques have been widely used, especially in biomedical applications and preservation of germplasm resources. Ideally, biological materials would maintain functional integrity as well as a normal structure and can be recovered when needed. However, this tool does not work all the time. Ice formation and growth are the key challenges. The other major reason is that the cryoprotective agents (CPAs) currently used do not meet these needs and are always accompanied by their cytotoxicity. A comprehensive and synergistic approach that focuses on the overall frozen biological system is crucial for the evolution of cryopreservation methods. In this review, we first summarize the fundamental damage mechanisms during cryopreservation, as well as common cryoprotectants and their limitations. Next, we discuss materials that interact with ice to improve cryopreservation outcomes. We evaluated natural and synthetic materials, including sugars and polymers, AFPs and mimics, ice nucleators, and hydrogels. In addition, biochemical regulation, which enhances the tolerance of biosamples to cryopreservation-induced stresses, was also mentioned. Nanotechnology, cell encapsulation, cryomesh, and isochoric freezing, such scalable approaches, are further discussed for cryopreservation. Finally, future research directions in this field for efficient cryopreservation are proposed. We emphasized the need for multidisciplinary progress to address these challenges. The combination of cryobiology mechanisms with technologies, such as synthetic biology, nanotechnology, microfluidics, and 3D bioprinting, is highlighted.

This study addressed limitations in calcein-AM-based endothelial viability assays, specifically focusing on pre-stripped DMEK grafts. Key challenges included the suboptimal calcein staining and the incompatibility of the viability assay with subsequent immunofluorescence (IF). Using human corneal grafts, we employed two strategies to optimize calcein staining. Firstly, we improved calcein staining in corneal endothelium by adjusting calcein-AM concentration and diluent, resulting in a threefold increase in fluorescence intensity with 4 µM calcein in Opti-MEM compared to the conventional 2 µM calcein in PBS. Secondly, introducing the trypan blue (TB) post-viability assay greatly reduced non-specific fluorescence, enhancing the contrast of calcein staining. This improvement significantly and importantly decreased both inter-operator's variability and the time required for viability counting. For the subsequent double IF, an extensive wash is recommended on the fixed and permeabilized graft after the viability assay, which was carried out using Hoechst-Calcein (HC) labeling. The simple technical tips outlined in this study are not only effective for pre-stripped DMEK grafts but may also prove beneficial for other types of corneal grafts, such as PK and DSAEK.

Dimethyl sulfoxide (DMSO) is an amphipathic solvent for molecules in in vitro tests for detection of anthelmintic resistance of gastrointestinal nematodes (GIN). It has been shown to have a concentration-dependent detrimental effect on Caenorhabditis elegans, a free-living nematode. If GIN are likewise affected, using DMSO in egg-hatch test and larval development test (LDT) may confound their results. Therefore, the DMSO concentration was determined at which it exerted an inhibitory effect on GIN larval development to the third stage.

A standard LDT was performed in 30 replications at DMSO concentrations of 0.0% (control), 0.6%, 1.3%, 2.6%, 5.2%, 10.4%, and 20.8%. The numbers of all developmental stages of Haemonchus contortus, Trichostrongylus spp. and Oesophagostomum spp. (unhatched eggs, larvae of the first, second and third stages (L1-L3) were determined, the proportion of L3 (the percentage of larval development - PD) was calculated and L3 were identified at the species or genus level. A five-parameter logistic curve was fitted to the observed PDs and modelled the DMSO-larval development relationship.

The PD significantly decreased with increasing DMSO concentration and was significantly reduced at the 2.6% concentration. The median inhibitory concentration (IC50) was 3.79%, the concentration for 10% inhibition (IC10) was 1.75% and for 90% inhibition (IC90) was 8.20%. The percentage of L1 and L2 followed an analogical but opposite pattern to that of PD and was complementary to it at each DMSO concentration. The unhatched egg percentage was rarely >1% and showed no pattern.

At ≥2.6% concentration, DMSO significantly inhibited the L3 development of all three GIN species. It had a practically important inhibitory effect (IC10) at as low concentration as 1.75%. At lower concentrations, DMSO did not appear to inhibit larval development. The compound did not seem to exert an in vitro ovicidal effect regardless of the concentration.

The global cryopreservation market size rises exponentially due to increased demand for cell therapy-based products, assisted reproductive technology, and organ transplantation. Cryoprotectants (CPAs) are required to reduce ice-related damage, osmotic cell injury, and protein denaturation. Antioxidants are needed to hamper membrane lipid peroxidation under freezing stress, and antibiotics are added to the cryo-solutions to prevent contamination. The vitrification process for sized organs requires a high concentration of CPA, which is hardly achievable using conventional penetrating toxic CPAs like DMSO. Antifreeze peptides (AFpeps) are biocompatible CPAs leveraging inspiration from nature, such as freeze-tolerant and freeze-avoidant organisms, to circumvent logistic limitations in cryogenic conditions. This study aims to introduce the advances of AFpeps with cell-penetrating, antioxidant, and antimicrobial characteristics. We herein revisit the placement of AFpeps in the biobanking of cancer cells, immune cells, stem cells, blood cells, germ cells (sperms and oocytes), and probiotics. Implementing low-immunogenic AFpeps for allograft cryopreservation minimizes HLA mismatching risk after organ transplantation. Applying AFpeps to formulate bioinks with optimal rheology in extrusion-based 3D cryobiopriners expedites the bench-to-beside transition of bioprinted scaffolds. This study advocates that the fine-tuned synthetic or insect-derived AFpeps, forming round blunt-shape crystals, are biomedically broad-spectrum, and cell-permeable AFpeps from marine and plant sources, which result in sharp ice crystals, are appropriate for cryosurgery. Perspectives of the available room for developing peptide mimetics in favor of higher activity and stability and peptide-functionalized nanoparticles for enhanced delivery are delineated. Finally, antitumor immune activation by cryoimmunotherapy as an autologous in-vivo tumor lysate vaccine has been illustrated.

Glioblastoma multiforme (GBM) is a widespread and life-threatening kind of brain cancer, which has a high mortality rate. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, irreversibly adheres to a conserved cysteine residue of two enzymes BTK and BMX, inhibiting their kinase activities, which leads to suppression of the growth of glioma cells. This study synthesised PLGA-PEG-folate (PPF) polymer and subsequently encapsulated ibrutinib within PPF nanoparticles (IBT-PPF-NPs). H NMR spectra confirmed the synthesis of PPF polymer. The efficiency of IBT-PPF-NPs was 97 ± 2.26% with 8.8 ± 0.2% drug loading. The particle size was 208 ± 4.8 nm. The IC50 value of free ibrutinib, IB-PPF-NPs and ibrutinib encapsulated in PLGA NPs (IB-P-NPs) was 10.2, 7.6 and 10.13 µM in C6 cell lines, whereas in U-87 MG cells was 24.4, 16 and 25.2 µM, respectively. The cellular uptake of FITC-PPF-NPs increased from 47.6% to 90.3% in C6 cells and from 55% to 97.3% in U-87 MG cells compared to FITC-P-NPs. The in vivo results indicate a significant reduction in tumour size in treatment groups in comparison to control groups, while the group that received the intratumoural injection of IB-PPF-NPs exhibited a greater reduction. The folate-targeting agent enhances the nanoparticles' effectiveness by promoting their uptake through the endocytosis pathway.

The objective of this work is to outline the preclinical development of ADAM®, an injectable, two-component PEG hydrogel designed for long-lasting and non-permanent vas-occlusive contraception.

A newly developed, aqueous-based hydrogel was comprehensively characterized to evaluate its potential as a male contraceptive device. The material was optimized for porosity to ensure sperm occlusion, rheological properties, injectability, and mechanical durability within the dynamic vessel. Biocompatibility of the hydrogel was assessed using a suite of ISO 10993 tests, which are critical for clinical translation and supporting a future regulatory submission. The canine model was utilized to assess the chronic tissue response of the vas deferens post-implantation, providing insights into long-term safety and functionality.

The vas-occlusive hydrogel demonstrates selective permeability with a pore size of 10-20 nm, effectively preventing sperm passage while allowing diffusion of small molecules. Its mechanical properties facilitated injection into the vas deferens with minimal force (3.1 N), forming a viscoelastic material within 30 seconds, with the ability to resist ejaculatory pressures. The implant and its degradation products were found to be non-cytotoxic, non-irritating, non-sensitizing, and non-genotoxic. Moreover, a two-year in vivo study in a canine model showed minimal tissue reaction following implantation, with no adverse effects reported.

These findings indicate that the hydrogel's chemical and mechanical properties align with the requirements for a safe, effective, and non-permanent vas-occlusive contraceptive.

Currently, men lack options for effective, long-lasting, and reversible contraception. The development and evaluation of the study hydrogel described here suggests it would make a safe and effective vas-occlusive contraceptive and should be researched further in a clinical setting.

Dimethyl sulfoxide (DMSO) alters DNA methylation in vitrified-warmed embryos and potentially affects subsequent development. This study aimed to examine possible countermeasures against DMSO-induced demethylation.

In vitro-produced bovine embryos (8-cell stage) were vitrified using a combination of DMSO and ethylene glycol (EG) or propylene glycol (PG) + EG. After warming, the lipid content and expression levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), DNMTs, and TETs were examined. In addition, RNA-sequencing was performed on blastocysts derived from the vitrified embryos. Furthermore, the effect of supplementation with a vitrification medium containing DMSO and N-acetyl-L-cysteine (NAC, 5 mM) on the levels of 5mC in embryos was examined.

Vitrification decreased the levels of 5mC and increased the levels of 5hmC in 8-cell stage embryos. Low levels of 5mC persisted until the blastocyst stage in the DMSO group but increased in the PG group. The expression level of TET3A was higher in the DMSO group than in the fresh group, but not in the PG group. Both cryoprotectants reduced the lipid levels in post-warmed 8-cell stage embryos. The addition of NAC ameliorated DMSO-induced demethylation at both the 8-cell and blastocyst stages. RNA-seq analysis revealed that PG-specific pathways included ribosomes and mitochondria and that both DMSO and PG affected cGMP-PGK, MAPK, Wnt, and insulin secretion-related signaling. The K-medoids method predicted that DMSO affected cell adhesion molecules and that MAPK signaling was affected the most.

PG and NAC may antagonize DMSO-induced demethylation; however, PG exerts adverse effects on embryos.

Oocyte vitrification has a wide range of applications in assisted reproduction and fertility preservation. It requires precise cryoprotectant agents (CPAs) loading and removal sequences to alleviate osmotic shock, which requires manual manipulation by an embryologist. In this study, a microfluidic system was developed to facilitate the precise adjustment of the CPA concentration around the oocyte by linear loading and removal of CPA. In addition, the microfluidic-based automated vitrification (MAV) device combines CPA loading/removal process, with vitrification process, thereby achieving automated oocyte vitrification. Oocytes were vitrified by Cryotop/QC manual method and MAV method. The results showed that the survival, cleavage, and blastocyst rates of oocytes were 80.44, 54.17, and 32.95% for the MAV method, which were significantly higher than Cryotop manual method (73.35, 43.73, and 23.67%) (p < 0.05). In MAV, solution injection rate during CPA loading/removal process was designed as a 1-segment, 2-segment, and 4-segment function. Accordingly, three concave loading and convex removal protocols were adopted to vitrify oocytes. Oocytes vitrified using the 4-segment function group exhibited increased survival (86.18%), cleavage (63.29%), and blastocyst (45.58%) rates compared to those vitrified using the 1-segment and 2-segment groups. The oocytes vitrification with the highest concentration of CPA, denoted as VS1-TS1, exhibited the highest survival rate after rewarming (86.18%). In contrast, the VS3-TS3 group, characterized by a CPA concentration half that of VS1-TS1, exhibited lower survival (74.14%) and cleavage (59.31%) rates, but displayed the higher blastocyst rate (50.79%) following oocyte activation. Our study demonstrates potential of the MAV device for oocyte or embryo vitrification.

In this study, a novel phenomenon is identified where precise control of topology and generation of polyglycerol induce the retention of Na+ ions in biological buffer systems, effectively inhibiting ice crystal growth during cryopreservation. Unlike linear and hyperbranched counterparts, densely-packed hydroxyl and ether groups in 4th-generation dendritic polyglycerol interact with the ions, activating the formation of hydrogen bonding at the ice interface. By inhibiting both intra- and extracellular ice growth and recrystallization, this biocompatible dendritic polyglycerol proves highly effective as a cryoprotectant; hence, achieving the cell recovery rates of ≈134-147%, relative to those of 10% dimethyl sulfoxide, which is a conventional cryoprotectant for human tongue squamous carcinoma (HSC-3) cell line and human umbilical vein endothelial (HUVEC) cells. Further, it successfully recovers the network-forming capabilities of HUVEC cells to ≈89% in tube formation after thawing. The Na+ ion retention-driven ice-growth inhibition activity in biological media highlights the unique properties of dendritic polyglycerol and introduces a new topological concept for cell-cryoprotectant development.

A primary factor in the pathogenesis of aging is oxidative stress, with cardiac inflammation and fibrosis being contributed to by increased oxidative stress as organisms age. Oxidative stress enhances the cardiac fibrotic signaling pathway, with reactive oxygen species inducing cardiac fibrosis through increased expression of the profibrotic factor transforming growth factor-beta 1 (TGF-β1). Furthermore, Wnt/β-catenin signaling pathway is implicated in interstitial fibrosis, which is associated with TGF-β. Sirtuin 2 (SIRT2) is expressed in heart tissue, with protective effects in pathological cardiac hypertrophy. We aimed to investigate the mechanisms of cardiac fibrosis in D-Galactose (D-Gal)-induced accelerated aging, focusing on TGF-β1, β-catenin, and SIRT2.

A total of 30 young male Sprague-Dawley rats were randomly divided into 4 groups: control group, D-Gal group, D-Gal + 4% dimethyl sulfoxide (DMSO) group, and D-Gal + the SIRT2 inhibitor (AGK2) group. After 10 weeks, the rats were sacrificed, and their hearts were removed. SIRT2 expression levels were measured by western blot and gene expression levels of TGF-β1 and β-catenin by quantitative real-time polymerase chain reaction.

Transforming growth factor-beta 1 (TGF-β1) mRNA expression in heart tissue was higher in the D-Gal group compared to all other groups. β-catenin mRNA expression was higher in the D-Gal group than in the D-Gal + AGK2 group. SIRT2 protein expression was higher in the D-Gal + DMSO group compared to the control group. Sirtuin 2 expression was lower in the D-Gal + AGK2 group compared to the D-Gal and D-Gal + DMSO groups.

Sirtuin 2 inhibition attenuates fibrosis, as evidenced by the downregulation of TGF-β1 and β-catenin. Thus, targeting SIRT2 may represent a potential therapeutic strategy for diseases characterized by cardiac fibrosis in the future.

Dimethyl sulfoxide (DMSO) is a solvent used to dissolve a variety of organic compounds. It is presumed to be non-toxic at concentrations below 1 % v/v, although several studies have demonstrated that low dose DMSO exposure can alter cellular biochemistry. This study evaluated the toxicity of DMSO at 0.0002 % v/v to the Sydney Rock oyster, Saccostrea glomerata, following 7d of exposure. Metabolites were chosen as the toxicity endpoints because they can be used as energy sources and counteract contaminant-induced stress. Relative to seawater controls, exposure to DMSO caused a 74 % significant change in metabolites in the female digestive gland, including decreases in most amino acids, carbohydrates, nicotinamides, and lipids. The female gonad showed a 43 % significant change in metabolites, with decreases in amino acids and carbohydrates, but increases in lipids. The male digestive gland showed a 29 % significant change in metabolites, with increases in lipids. The decline in metabolites in the female digestive gland, but not in the male digestive gland, may be due to their differential metabolic demands. Furthermore, pathway impact analysis revealed that DMSO exposure altered energy metabolism, disturbed osmotic balance, and induced oxidative stress in oysters. Because the effects of DMSO are not uniform across gender and tissue, use of DMSO as a solvent will confound metabolomic experimental results when comparisons among sexes and/or tissues are integral to the experimental design. There is a risk of incomplete dissolution of contaminants unless carrier solvents are used. Therefore, in practice, a solvent control along with a water control is recommended for experimentation.

Dimethyl sulfoxide (DMSO) is widely used as an adjuvant in dissolving insoluble compounds in an aqueous medium; however, it can induce significant molecular changes in cells. The possible damages may occur obeying a tissue-specific profile, and the effect on human apical papilla cells (hAPC) remains unknown. Therefore, this study aimed to evaluate DMSO effects on the viability and mineralization activity in hAPC cultures in vitro and to establish standards of maximum concentrations for its use in laboratory routines. hAPCs were cultured, plated, and maintained in media containing increasing concentrations of Dimethyl sulfoxide (0.1%, 0.5%, 1%, 5%, and 10%) for 24 h, 48 h, 72 h, and 7 days. At each time point, the cells were subjected to the MTT assay. The Alizarin red S staining assay was performed to evaluate the osteo/odontogenic mineralization potential of hAPC DMSO-exposed (0.1%, 0.5%, and 1%) in the 21-day time-point. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test (p<0.05). In general, the 5% and 10% DMSO concentrations were shown to be cytotoxic for hAPC at all analyzed time points, and the hAPC DMSO-stimulated presented higher osteo/odontogenic mineralization potential. Therefore, the 5% and 10% DMSO concentrations should be avoided, and the mineralization activity assay should be carefully designed in order to avoid biases at in vitro assays using hAPC cultures.

This study provides a comprehensive analysis of the interactions between dimethyl sulfoxide (DMSO) and two small peptides, diglycine and N-acetyl-glycine-methylamide (NAGMA), in aqueous solutions using FTIR spectroscopy and density functional theory (DFT) calculations. ATR-FTIR spectroscopy and DFT results revealed that DMSO does not form direct bonds with the peptides, suggesting that DMSO indirectly influences both peptides by modifying the surrounding water molecules. The analysis of HDO spectra allowed for the isolation of the contribution of water molecules that were simultaneously altered by the peptide and DMSO, and it also explained the changes in the hydration shells of the peptides in the presence of DMSO. In the DMSO-diglycine system, DMSO contributes to the additional strengthening of water hydrogen bonds in the reinforced hydration sphere of diglycine. In contrast, DMSO has a more moderate effect on the water molecules surrounding NAGMA due to the similarity of their hydration shells, leading to a slight weakening of the hydrogen bonds in the NAGMA hydration sphere. DFT/ONIOM calculations confirmed these observations. These findings demonstrated that DMSO influences peptide stability differentially based on their structural characteristics.

Liposomes have gained prominence as nanocarriers in drug delivery, and the number of products in the market is increasing steadily, particularly in cancer therapeutics. Remote loading of drugs in liposomes is a significant step in the translation and commercialization of the first liposomal product. Low drug loading and drug leakage from liposomes is a translational hurdle that was effectively circumvented by the remote loading process. Remote loading or active loading could load nearly 100% of the drug, which was not possible with the passive loading procedure. A major drawback of conventional remote loading is that only a very small percentage of the drugs are amenable to this method. Therefore, methods for drug loading are still a problem for several drugs. The loading of multiple drugs in liposomes to improve the efficacy and safety of nanomedicine has gained prominence recently with the introduction of a marketed formulation (Vyxeos) that improves overall survival in acute myeloid leukemia. Different strategies for modifying the remote loading process to overcome the drawbacks of the conventional method are discussed here. The review aims to discuss the latest developments in remote loading technology and its implications in liposomal drug delivery.

Fungal specialized metabolites are known for their potent biological activities, among which tropolone sesquiterpenoids (TS) stand out for their diverse bioactivities. Here, we report cytotoxic and proliferation inhibitory effects of the recently discovered TS compounds 4-hydroxyxenovulene B and 4-dihydroxy norpycnidione, and the structurally related 4-hydroxy norxenovulene B and xenovulene B. Inhibition of metabolic activity after TS treatment was observed in Jurkat, PC-3 and FAIK3-5 cells, whereas MDA-MB-231 cells were unresponsive to treatment. Structurally similar epolones were shown to induce erythropoietin (EPO). Therefore, FAIK3-5 cells, which can naturally produce EPO, were applied to test the compounds in this regard. While no effect on EPO production in FAIK3-5 cells could be demonstrated, effects on their proliferation, viability, and morphology were observed depending on the presence of tropolone moieties in the molecules. Our study underlines the importance of relevant cell models for bioactivity testing of compounds with unknown mechanisms of action.

Pesticides are diluted in products called solvents for spraying fields and for cell viability studies. This study aimed to determine whether pesticide solvents can alter the toxicity of endosulfan and Vero cell viability. Thus, the cytotoxicity of some diluents commonly used in pesticide solutions was evaluated by the neutral red incorporation technique and cell growth. Vero cells were exposed to endosulfan dissolved in sunflower (Helianthus annus, Linnaeus) oil, corn (Zea mays, Linnaeus) oil, dimethylsulfoxide (DMSO), and Tween 80, at a concentration of 1 µg L-1 for a period of 96 h. The results showed that both DMSO and Tween 80 induced a significant increase in cytotoxicity compared to sunflower oil and corn oil. Moreover, Tween 80 had a significant cytotoxic effect (P < 0.05) when compared to DMSO. The solvent can alter the toxicity of endosulfan, decreasing Vero cell viability, as was the case with DMSO and Tween 80.

Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze-thaw as well as a controlled rate freezer to study bulk freeze-thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of -6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (-10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.

Cryopreservation is a promising technique for the long-term storage of skin. However, the formation of ice crystals during cryopreservation unavoidably damages skin structure and functionality. Currently, the lack of thorough and systematic investigation into the internal mechanisms of skin cryoinjury obstructs the advancement of cryopreservation technology. In this study, we identified 3 primary contributors to skin cryoinjury: interfacial ice nucleation, stress accumulation, and thermal stress escalation. We emphasized the paramount role of interfacial ice nucleation in provoking ice growth within the skin during the cooling process. This progress subsequently leads to stress accumulation within the skin. During the rewarming process, the brittleness of skin, previously subjected to freezing, experienced a marked increase in thermal stress due to ice recrystallization. Based on these insights, we developed a novel zwitterionic betaine-based solution formulation designed for cryopreservation skin. This cryoprotective agent formulation exhibited superior capability in lowering ice nucleation temperatures and inhibiting ice formation at interfaces, while also facilitating the growth of smooth and rounded ice crystals compared to sharp-edged and cornered crystals formed in aqueous solutions. As a result, we successfully achieved prolonged cryopreservation of the skin for at least 6 months, while preserving 98.7% of structural integrity and 94.7% of Young's modulus. This work provides valuable insights into the mechanisms of ice crystal damage during organ cryopreservation and profoundly impacts the field of organ transplantation and regenerative medicine.

Drug-induced liver injury (DILI) refers to drug-mediated damage to the structure and function of the liver, ranging from mild elevation of liver enzymes to severe hepatic insufficiency, and in some cases, progressing to liver failure. The mechanisms and clinical symptoms of DILI are diverse due to the varying combination of drugs, making clinical treatment and prevention complex. DILI has significant public health implications and is the primary reason for post-marketing drug withdrawals. The search for reliable preclinical models and validated biomarkers to predict and investigate DILI can contribute to a more comprehensive understanding of adverse effects and drug safety. In this review, we examine the progress of research on DILI, enumerate in vitro models with potential benefits, and highlight cellular molecular perturbations that may serve as biomarkers. Additionally, we discuss omics approaches frequently used to gather comprehensive datasets on molecular events in response to drug exposure. Finally, three commonly used gene modulation techniques are described, highlighting their application in identifying causal relationships in DILI. Altogether, this review provides a thorough overview of ongoing work and approaches in the field of DILI.

Human adipose-derived stem cells (hASCs) are cryopreserved traditionally using dimethyl sulfoxide (DMSO) as the cryoprotectant agent. DMSO penetrates cell membranes and prevents cellular damage during cryopreservation. However, DMSO is not inert to cells, inducing cytotoxic effects by causing mitochondrial dysfunction, reduced cell proliferation, and impaired hASCs transplantation. Additionally, large-scale production of DMSO and contamination can adversely impact the environment. A sustainable, green alternative to DMSO is trehalose, a natural disaccharide cryoprotectant agent that does not pose any risk of cytotoxicity. However, the cellular permeability of trehalose is less compared to DMSO. Here, a microfluidic chip is developed for the intracellular delivery of trehalose in hASCs. The chip is designed for mechanoporation, which creates transient pores in cell membranes by mechanical deformation. Mechanoporation allows the sparingly permeable trehalose to be internalized within the cell cytosol. The amount of trehalose delivered intracellularly is quantified and optimized based on cellular compatibility and functionality. Furthermore, whole-transcriptome sequencing confirms that less than 1% of all target genes display at least a twofold change in expression when cells are passed through the chip compared to untreated cells. Overall, the results confirm the feasibility and effectiveness of using this microfluidic chip for DMSO-free cryopreservation of hASCs.

The cryopreservation of gametes and embryos has increased notably over the past 20 years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservation process is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming-crucial for the fate of the embryo-occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health.

Dimethyl sulfoxide (DMSO) is a common solvent in biological and medical research for dissolving water-insoluble compounds and drugs. However, the impact of DMSO goes beyond its primary function. High-throughput and in vitro assays have uncovered various effects of DMSO in mammalian cells. The present article highlights the biological effects of DMSO on normal and cancerous mammalian cells.

The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the "immortal" nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model.

Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus.

Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus.

Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain.

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications, but has long-term cytotoxic effects. Trehalose, a natural disaccharide, has been proposed as a non-toxic cryoprotectant. However, the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs, which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally, the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery, offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods.

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

In regenerative medicine, ongoing advancements in cell culture techniques, including isolation, expansion, banking, and transport, are crucial for clinical success. Cryopreservation ensures off-the-freezer availability of living cells, enabling long-term storage and transport. Customizing cryopreservation techniques and cryoprotective agents (CPAs) for specific cell types is crucial for cell source quality, sustainability, safety, and therapeutic intervention efficiency. As regenerative medicine progresses, it becomes imperative that the scientific community and industry provide a comprehensive, cell-specific landscape of available and effective cryopreservation techniques, preventing trial-and-error approaches and unlocking the full potential of cell-based therapies. Open-sharing data could lead to safer, more efficient cell therapies and treatments. Two decades of dermal progenitor cell use for burn wound treatment and Good Manufacturing Practice-compliant technology transfers have highlighted the need for further cryopreservation optimization in manufacturing workflows. In this paper, we present experimental data assessing 5 different cryopreservation formulae for long-term storage of clinical-grade FE002 primary progenitor fibroblasts, emphasizing the crucial difference between DMSO-based and DMSO-free CPAs. Our findings suggest that CryoOx, a DMSO-free CPA, is a promising alternative yielding cell viability similar to that of established commercial CPAs. This research highlights the importance of secure, robust, and efficient cryopreservation techniques in cell banking for maximizing quality, ensuring patient safety, and advancing regenerative medicine.

Using endodontic sealers with long-term antimicrobial properties can increase the success of endodontic treatment. This study aimed to assess the antimicrobial activity over time of two calcium silicate (CS)-based sealers, AH Plus Bioceramic and BioRoot RCS, and to compare them with an epoxy resin-based sealer, AH Plus Jet, against mature polymicrobial biofilms grown on human radicular dentin.

The antimicrobial activity of the sealers was tested using a direct contact test after 1 and 6 weeks of contact with the biofilms. Cell viability was determined by the adenosine triphosphate (ATP) method and flow cytometry (FC). The results of the ATP test were analyzed using an ANOVA with Welch's correction, followed by the Games-Howell test. The number of cells with damaged membranes obtained by FC in each period was compared by means of an ANOVA and Duncan's test. For the comparison between times, a Student's t-test was used.

Globally, after a week of contact, the epoxy resin-based sealer obtained the best results. However, at 6 weeks, the two CSs showed the highest antimicrobial efficacy, with a significant increase in this activity over time.

Calcium silicate-based sealers exert long-term antimicrobial activity against endodontic biofilms.

Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs.

MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3 M/mL), 1:1 dilution (from 6 M/mL), or 1:2 dilution (from 9 M/mL) with PLA/5% HA, to achieve uniform concentration at 3 M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6 h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6 h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3 M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis.

MSCs can be cryopreserved up to 9 M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

What impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells?

Cellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications.

Native and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC.

These findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Cryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing-Dulbecco's modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO.

What can't freezing hold still? This article surveys the history of substances used to protect cells and organisms from freezing damage, known as cryoprotectants. Dimethyl sulfoxide (DMSO) has since 1959 been the most widely used of these agents in cryopreservation. Here, its evolution from pulp and paper waste byproduct to wonder drug to all-but-invisible routine element of freezing protocols is used to trace the direct arc from protection to toxicity in theories of how and why cryoprotectants work, from the 1960s to today. The power of these agents to simultaneously protect and degrade is shown to reside in manipulation of chemical time via hydrogen bonding and electron exchange, thereby reframing freezing as a highly active and transformational process. Countering long-held assumptions about cryopreservation as an operation of stasis after which the thawed entity is the same as it was before, this article details recent demonstrations of effects of cryoprotectant exposure that are nonlethal but nonetheless profoundly impactful within scientific and therapeutic practices that depend on freezing infrastructures. Understanding the operationalization of chemical time in the case of cryoprotectants is broadly relevant to other modern technologies dedicated to shifting how material things exist and persist in human historical time.

Proteins are acknowledged as the phenotypical manifestation of the genotype, because protein-coding genes carry the information for the strings of amino acids that constitute the proteins. It is widely accepted that protein function depends on the corresponding "native" structure or folding achieved within the cell, and that native protein folding corresponds to the lowest free energy minimum for a given protein. However, protein folding within the cell is a non-deterministic dissipative process that from the same input may produce different outcomes, thus conformational heterogeneity of folded proteins is the rule and not the exception. Local changes in the intracellular environment promote variation in protein folding. Hence protein folding requires "supervision" by a host of chaperones and co-chaperones that help their client proteins to achieve the folding that is most stable according to the local environment. Such environmental influence on protein folding is continuously transduced with the help of the cellular stress responses (CSRs) and this may lead to changes in the rules of engagement between proteins, so that the corresponding protein interactome could be modified by the environment leading to an alternative cellular phenotype. This allows for a phenotypic plasticity useful for adapting to sudden and/or transient environmental changes at the cellular level. Starting from this perspective, hereunder we develop the argument that the presence of sustained cellular stress coupled to efficient CSRs may lead to the selection of an aberrant phenotype as the resulting adaptation of the cellular proteome (and the corresponding interactome) to such stressful conditions, and this can be a common epigenetic pathway to cancer.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is a benchmark hole-transporting (p-type) polymer that finds applications in diverse electronic devices. Most of its success is due to its facile synthesis in water, exceptional processability from aqueous solutions, and outstanding electrical performance in ambient. Applications in fields like (opto-)electronics, bioelectronics, and energy harvesting/storage devices often necessitate the complementary use of both p-type and n-type (electron-transporting) materials. However, the availability of n-type materials amenable to water-based polymerization and processing remains limited. Herein, we present a novel synthesis method enabling direct polymerization in water, yielding a highly conductive, water-processable n-type conjugated polymer, namely, poly[(2,2'-(2,5-dihydroxy-1,4-phenylene)diacetic acid)-stat-3,7-dihydrobenzo[1,2-b:4,5-b']difuran-2,6-dione] (PDADF), with remarkable electrical conductivity as high as 66 S cm-1, ranking among the highest for n-type polymers processed using green solvents. The new n-type polymer PDADF also exhibits outstanding stability, maintaining 90% of its initial conductivity after 146 days of storage in air. Our synthetic approach, along with the novel polymer it yields, promises significant advancements for the sustainable development of organic electronic materials and devices.

Molecules that inhibit the growth of ice crystals are highly desirable for applications in building materials, foods, and agriculture. Antifreezes are particularly essential in biomedicine for tissue banking, yet molecules currently in use have known toxic effects. Antifreeze glycoproteins have evolved naturally in polar fish species living in subzero climates, but practical issues with collection and purification have limited their commercial use. Here, we present a synthetic strategy using polymerization of amino acid N-carboxyanhydrides to produce polypeptide mimics of these potent natural antifreeze proteins. We investigated a set of mimics with varied structural properties and identified a glycopolypeptide with potent ice recrystallization inhibition properties. We optimized for molecular weight, characterized their conformations, and verified their cytocompatibility in a human cell line. Overall, we present a material that will have broad applications as a biocompatible antifreeze.

Cell culture is a critical platform for numerous research and industrial processes. However, methods for transporting cells are largely limited to cryopreservation, which is logistically challenging, requires the use of potentially cytotoxic cryopreservatives, and can result in poor cell recovery. Development of a transport media that can be used at ambient temperatures would alleviate these issues. In this study, we describe a novel transportation medium for mammalian cells. Five commonly used cell lines, (HEK293, CHO, HepG2, K562, and Jurkat) were successfully shipped and stored for a minimum of 72 hours and up to 96 hours at ambient temperature, after which, cells were recovered into standard culture conditions. Viability (%) and cell numbers, were examined, before, following the transport/storage period and following the recovery period. In all experiments, cell numbers returned to pretransport/storage concentration within 24-48 hours recovery. Imaging data indicated that HepG2 cells were fully adherent and had established typical growth morphology following 48 hours recovery, which was not seen in cells recovered from cryopreservation. Following recovery, Jurkat cells that had been subjected to a 96 hours transport/storage period, demonstrated a 1.93-fold increase compared with the starting cell number with >95% cell viability. We conclude that CellShip® may represent a viable method for the transportation of mammalian cells for multiple downstream applications in the Life Sciences research sector.

Mesenchymal stromal cells (MSCs) have become one of the most investigated and applied cells for cellular therapy and regenerative medicine. In this update of our review published in 2015, we show that studies continue to abound regarding the characterization of MSCs to distinguish them from other similar cell types, the discovery of new tissue sources of MSCs, and the confirmation of their properties and functions that render them suitable as a therapeutic. Because cryopreservation is widely recognized as the only technology that would enable the on-demand availability of MSCs, here we show that although the traditional method of cryopreserving cells by slow cooling in the presence of 10% dimethyl sulfoxide (Me2SO) continues to be used by many, several novel MSC cryopreservation approaches have emerged. As in our previous review, we conclude from these recent reports that viable and functional MSCs from diverse tissues can be recovered after cryopreservation using a variety of cryoprotectants, freezing protocols, storage temperatures, and periods of storage. We also show that for logistical reasons there are now more studies devoted to the cryopreservation of tissues from which MSCs are derived. A new topic included in this review covers the application in COVID-19 of MSCs arising from their immunomodulatory and antiviral properties. Due to the inherent heterogeneity in MSC populations from different sources there is still no standardized procedure for their isolation, identification, functional characterization, cryopreservation, and route of administration, and not likely to be a "one-size-fits-all" approach in their applications in cell-based therapy and regenerative medicine.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

Sensitivity enhanced dynamic nuclear polarization solid-state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP-ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution. We also explore possible solutions ranging from utilization of new pulse sequences, design of better performing polarizing agents, and application of additional biochemical/ cell biological methodologies.