Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.

High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding.

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze-thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol-1. These polymers were found to have IRI activity down to 5 mg mL-1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL-1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications.

Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants.

Spheroids are a powerful tool for basic research and to reduce or replace in vivo (animal) studies but are not routinely banked nor shared. Here, we report the successful cryopreservation of hepatocyte spheroids using macromolecular (polyampholyte) cryoprotectants supplemented into dimethyl sulfoxide (DMSO) solutions. We demonstrate that a polyampholyte significantly increases post-thaw recovery, minimizes membrane damage related to cryo-injury, and remains in the extracellular space making it simple to remove post-thaw. In a model toxicology challenge, the thawed spheroids matched the performance of fresh spheroids. F-actin staining showed that DMSO-only cryopreserved samples had reduced actin polymerization, which the polyampholyte rescued, potentially linked to intracellular ice formation. This work may facilitate access to off-the-shelf and ready-to-use frozen spheroids, without the need for in-house culturing. Readily accessible 3-D cell models may also reduce the number of in vivo experiments.

Poly(vinyl alcohol) Molecular Bottlebrushes Nucleate Ice.

Ice binding proteins (IBP) have evolved to limit the growth of ice but also to promote ice formation by ice-nucleating proteins (INPs). IBPs, which modulate these seemingly distinct processes, often have high sequence similarities, and molecular size/assembly is hypothesized to be a crucial determinant. There are only a few synthetic materials that reproduce INP function, and rational design of ice nucleators has not been achieved due to outstanding questions about the mechanisms of ice binding. Poly(vinyl alcohol) (PVA) is a water-soluble synthetic polymer well known to effectively block ice recrystallization, by binding to ice. Here, we report the synthesis of a polymeric ice nucleator, which mimics the dense assembly of IBPs, using confined ice-binding polymers in a high-molar-mass molecular bottlebrush. Poly(vinyl alcohol)-based molecular bottlebrushes with different side-chain densities were synthesized via a combination of ring-opening metathesis polymerization (ROMP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization, using "grafting-to" and "grafting-through" approaches. The facile preparation of the PVA bottlebrushes was performed via selective hydrolysis of the acetate of the poly(vinyl acetate) (PVAc) side chains of the PVAc bottlebrush precursors. Ice-binding polymer side-chain density was shown to be crucial for nucleation activity, with less dense brushes resulting in colder nucleation than denser brushes. This bio-inspired approach provides a synthetic framework for probing heterogeneous ice nucleation and a route toward defined synthetic nucleators for biotechnological applications.

Water-organizing motif continuity is critical for potent ice nucleation protein activity.

Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section's deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Assay-ready Cryopreserved Cell Monolayers Enabled by Macromolecular Cryoprotectants.

Cell monolayers underpin the discovery and screening of new drugs and allow for fundamental studies of cell biology and disease. However, current cryopreservation technologies do not allow cells to be stored frozen while attached to tissue culture plastic. Hence, cells must be thawed from suspension, cultured for several days or weeks, and finally transferred into multiwell plates for the desired application. This inefficient process consumes significant time handling cells, rather than conducting biomedical research or other value-adding activities. Here, we demonstrate that a synthetic macromolecular cryoprotectant enables the routine, reproducible, and robust cryopreservation of biomedically important cell monolayers, within industry-standard tissue culture multiwell plates. The cells are simply thawed with media and placed in an incubator ready to use within 24 h. Post-thaw cell recovery values were >80% across three cell lines with low well-to-well variance. The cryopreserved cells retained healthy morphology, membrane integrity, proliferative capacity, and metabolic activity; showed marginal increases in apoptotic cells; and responded well to a toxicological challenge using doxorubicin. These discoveries confirm that the cells are "assay-ready" 24 h after thaw. Overall, we show that macromolecular cryoprotectants can address a long-standing cryobiological challenge and offers the potential to transform routine cell culture for biomedical discovery.

Baicalein May Act as a Caloric Restriction Mimetic Candidate to Improve the Antioxidant Profile in a Natural Rodent Model of Aging.

Caloric restriction (CR) is the most effective intervention for extending the life span of vertebrate and invertebrate aging models. Calorie restriction mimetics (CRMs), which are synthetic or natural chemicals that mimic the biochemical, hormonal, and physiological consequences of calorie restriction, are being researched for antiaging benefits. Baicalein is a plant-derived polyphenol that has the potential of antioxidant, anti-inflammatory, and autophagy inducer. The objective of this study is to evaluate the antiaging, anti-inflammatory, and antioxidant role of Baicalein in erythrocyte membrane and plasma, and evaluate the efficacy of Baicalein to act as a CRM candidate. This study evaluates the effect of Baicalein on aging biomarkers in normal and aged rats. We study various pro- and antioxidant markers, erythrocyte membrane transporters, and eryptosis. Baicalein supplementation in male Wistar rats significantly alleviated pro-oxidant markers and improved antioxidant profile. Improvement was also observed in age-induced alterations in membrane transporters, and eryptosis. Based on the aforementioned observations we conclude that Baicalein has the potential to maintain extracellular reactive oxygen species levels and redox homeostasis during the aging process, an effect that is similar to CR. Thus, Baicalein may be a potent CRM candidate for antiaging interventions.

Metformin ameliorates acetaminophen-induced sub-acute toxicity via antioxidant property.

Acetaminophen or N-acetyl-p-amino-phenol (APAP) is a drug which is available over-the-counter for fever and pain. Its overdosing causes oxidative stress and subsequent acute liver damage. In the present study, we scrutinized the protective effect of metformin co-treatment in APAP induced blood and liver sub-acute toxicity. This is a pre-clinical study in which male Wistar Rats (BW: 300 ± 20 g) were orally co-treated with APAP (1 g/kg/day) and metformin (300 mg/kg/day) for 28-days. Pro- and anti-oxidant markers viz reactive oxygen species, protein carbonyl, malondialdehyde (MDA), the ferric reducing ability of plasma (FRAP), plasma membrane redox system(PMRS) and reduced glutathione (GSH) were evaluated in blood. Additionally, in liver tissue, catalase (CAT), superoxide dismutase (SOD), MDA and GST level were also evaluated. Histological study and estimation of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) level in serum were performed. APAP induces pro-oxidant markers as well as reduces anti-oxidant markers in blood and liver. Hepatic tissues degeneration and vacuolization of hepatocytes were evident after APAP treatment. Metformin treatment reduces pro-oxidant markers as well as increases anti-oxidant markers in both tissues. It also improves liver tissue architecture after treatment. The outcome of this study suggests that metformin has protective capability against APAP-induced blood and liver toxicity. Thus, metformin co-treatment with APAP attenuates oxidative stress and its consequences.

Baicalein maintains redox balance in experimental hyperlipidemic rats.

Context: An altered lipid profile may lead to the development of CVD.Objective: We evaluated the protective role of baicalein (BAC) against lipidemic and oxidative stress in hyperlipidemic challenged Wistar rats.Materials and methods: Male Wistar rats were given a high-fat diet (HFD) (suspension (w/v) of 0.5% cholesterol, 3% coconut oil and 0.25% cholic acid for 30 days) to create a hyperlipidemic model. BAC was supplemented to experimental rats (80 mg/kg body weight). Biomarkers of oxidative stress including ROS, FRAP, GSH, PMRS, AGE, MDA, PCO, AOPP, and other parameters (Paraoxonase-1, SGOT, SGPT) including TNF-α and IL-6, were estimated in blood.Results: Oxidative stress and inflammatory markers were significantly increased in the HFD treated group. BAC treatment protected rats from HFD mediated alterations.Discussion & conclusion: Our results indicate that baicalein provides protection against hyperlipidemic stress and redox imbalance induced by HFD in rats.

Adherent cell thawing by infrared radiation.

Cryopreservation of adherent cells is crucial for commercial cell therapy technology, including effective distribution and storage. Fast thawing has been shown to increase cell recovery in vitrified samples. Previously, radiofrequency (RF) has been investigated as a heating source on large samples, either with or without magnetic particles. Also, laser heating with the aid of dye or nanoparticles has been utilized on sub-millimeter samples successfully. For slow freezing cryopreservation methods, the influence of rate of thawing on viability is less clear. Cryopreservation of surface adhered cells result in many cases in detachment from the surface. We illustrate how intense infrared radiation from a focused halogen illuminator accelerates thawing. We show that two epithelial cell lines, retinal pigment epithelium cells and heterogeneous human epithelial colorectal adenocarcinoma cells, can be effectively cryopreserved and recovered using a combination of slow freezing and fast thawing under infrared illumination. We were able to successfully thaw samples, of 2-4 mm thick, including the media, on the order of a second, providing a heating rate of thousands of Kelvin per minute. Under optimal conditions, we observed higher post-thawing cell viability rates and higher cell adhesion with infrared thawing than with water bath thawing. We suggest that bulk warming with infrared radiation has an advantage over surface warming of surface-attached cells, as it alleviates cell stress during the process of thawing. These findings will pave the way for novel approaches to treating substrate-adhered cells and 3D scaffolds with cells and organoids. This technology may serve as a crucial component in lab-on-chip systems for medical testing and therapeutic use.

Spermidine, a caloric restriction mimetic, provides neuroprotection against normal and D-galactose-induced oxidative stress and apoptosis through activation of autophagy in male rats during aging.

Spermidine (SPD) is a natural polyamine present in all living organisms and is involved in the maintenance of cellular homeostasis by inducing autophagy in different model organisms. Its role as a caloric restriction mimetic (CRM) is still being investigated. We have undertaken this study to investigate whether SPD, acting as a CRM, can confer neuroprotection in D-galactose induced accelerated senescence model rat and naturally aged rats through modulation of autophagy and inflammation. Young male rats (4 months), D-gal induced (500 mg/kg b.w., subcutaneously) aging and naturally aged (22 months) male rats were supplemented with SPD (10 mg/kg b.w., orally) for 6 weeks. Standard protocols were employed to measure prooxidants, antioxidants, apoptotic cell death and electron transport chain complexes in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy and inflammatory marker genes. Our data demonstrate that SPD significantly (p ≤ 0.05) decreased the level of pro-oxidants and increased the level of antioxidants. SPD supplementation also augmented the activities of electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. RT-PCR data revealed that SPD up-regulated the expression of autophagy genes (ATG-3, Beclin-1, ULK-1 and LC3B) and down-regulated the expression of the inflammatory gene (IL-6) in aging brain. Our results provide first line of evidence that SPD provides neuroprotection against aging-induced oxidative stress by regulating autophagy, antioxidants level and also reduces neuroinflammation. These results suggest that SPD may be beneficial for neuroprotection during aging and age-related disorders.

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas.

Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.

Fisetin, a potential caloric restriction mimetic, attenuates senescence biomarkers in rat erythrocytes.

An imbalanced redox status is a hallmark of the aging process. Caloric restriction mimetics (CRMs) are compounds that produce caloric restriction benefits at the molecular, cellular, and physiological level, translating into health-promoting effects. Fisetin is the least explored CRM, and its role in modulating oxidative stress during aging is not clearly known. This study investigated the antioxidative and protective potential of fisetin in a rat model of d-galactose (D-gal)-induced accelerated senescence, and in naturally aged rat erythrocytes. Young rats (4 months), aged D-gal-induced rats [24 months; 500 mg/kg body mass (b.m.); subcutaneous injection] and naturally aged D-gal-induced rats [24 months; 500 mg/kg b.m.; subcutaneous injection] were supplemented with fisetin (15 mg/kg b.m.; orally) for 6 weeks. The resulting data indicated that supplementation with fisetin suppresses aging-induced increases in the levels of reactive oxygen species, eryptosis, lipid peroxidation, and protein oxidation. Our data also show that fisetin significantly increases the levels of antioxidants and activates the plasma membrane redox system. Taken together, the findings show that a fisetin-rich diet could be an anti-aging intervention strategy.

Rapamycin Confers Neuroprotection Against Aging-Induced Oxidative Stress, Mitochondrial Dysfunction, and Neurodegeneration in Old Rats Through Activation of Autophagy.

Brain aging is an intricate and natural phenomenon exclusively characterized by oxidative stress, accumulation of oxidatively damaged macromolecules, and alterations in structure and function of neurons that further increase the risk factor for most of the neurodegenerative diseases. In addition, age-dependent defective autophagy has also been implicated to favor the pathogenesis and prevalence of the neurological diseases. Therefore, the development of strategies that delay aging and the concomitant neurological disorders remain elusive. Thus, the present study was undertaken to investigate the effect of rapamycin-induced activation of autophagy on aging-related oxidative stress, cell death, neuroinflammation, and neurodegeneration in rat brain. Our data demonstrated the significant age-related oxidative stress, apoptotic cell death, elevated inflammatory response, and reduced level of markers associated with rejuvenation and neural integrity, including the activities of ion channel transporters (Na+/K+-ATPase and Ca2+-ATPase) and acetylcholinesterase in the brain of old aged rats. Furthermore, rapamycin (0.5 mg/kg b.w. for 28 days) induced activation of autophagy provided significant protection to aging rat brain by reducing the aging-induced oxidative stress, apoptotic cell death, and markers of neurodegeneration. Thus, our data confirmed that autophagy plays a pivotal role in delaying brain aging plausibly by maintaining the cellular homeostasis, and structural and functional integrity of cells in the brain.

Preparation and characterization of gelatin-chitosan-nanoß-TCP based scaffold for orthopaedic application.

The primary aim of this study was to fabricate gelatin/chitosan/ß-TCP (GCT) composite scaffold to improve its compressive mechanical behaviour and in-vivo biocompatibility with predictable degradation rate. Beta tricalcium phosphate (ß-TCP) powder was synthesized in size range between 70-100 nm using aqueous precipitation route at a fixed Ca/P molar ratio of 1.5:1 at pH 10 and after subsequent heat treatment of as precipitated powder at 800 °C for 4 hours. The composite scaffolds were fabricated using solid-liquid phase separation of the slurry containing gelatin, chitosan, ß-tricalcium phosphate in varying proportion and subsequent lyophilisation of the phase separated mixture. The prepared scaffolds exhibited high porosity (>80%) with pore sizes ranging between 78-382 µm as determined using Hg-porosimetry. SEM result revealed that incorporation of ß-TCP to the extent of 30 wt% resulted in well-shaped and uniformly distributed interconnected pores of average pore size of 120 ±â€¯18.6 µm in it. Compressive strength of the scaffolds was increased from 0.8 MPa to 2.45 MPa on increase in ß-TCP content from 10 wt%-30 wt% in the prepared scaffold. Human Umbilical Cord derived mesenchymal stem cells (MSCs) exhibited higher degree of lamellopodia and fillopodia extensions and better spreading behaviour onto GCT30 scaffold. MTT assay and immunocytochemistry studies with cultured MSCs revealed that GCT30 scaffolds were more conducive to MSC's proliferation and differentiation into osteoblast lineage. In vivo implantation of GCT30 scaffold subcutaneously into mice did not indicate any significant inflammatory reaction, but ongoing vascularization.

Enhanced chondrogenesis of mesenchymal stem cells over silk fibroin/chitosan-chondroitin sulfate three dimensional scaffold in dynamic culture condition.

Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46.2-50.3°, showed controlled swelling and biodegradation. The biocompatibility of scaffold was confirmed by subcutaneous implantation in mouse. Human mesenchymal stem cells (hMSCs) seeded scaffolds cultured under spinner flask bioreactor promoted cell attachment, proliferation, distribution, and metabolic activity in vitro. The histology and immunofluorescence studies revealed that combined effect of Ch and dynamic condition resulted in higher glycosaminoglycan secretion and native cartilage type matrix synthesis in comparison to SF/CS scaffolds used as control. Higher expression of collagen-II, Sox9, aggrecan and decrease in collagen-I expression represented by quantitative polymerase chain reaction study confirmed the progression of chondrogenic differentiation. This study successfully demonstrates the potentiality of SF/CS-Ch scaffold for hMSCs recruitment and redirecting cartilage tissue regeneration with enhanced chondrogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2576-2587, 2018.