PrestoBlue Assay Research Articles

Leveraging the nanotopography of filamentous fungal chitin-glucan nano/microfibrous spheres (FNS) coated with collagen (type I) for scaffolded fibroblast spheroids in regenerative medicine.

Numerous naturally occurring biological structures have inspired the development of innovative biomaterials for a wide range of applications. Notably, the nanotopographical architectures found in natural materials have been leveraged in biomaterial design to enhance cell adhesion and proliferation and improve tissue regeneration for biomedical applications. In this study, we fabricated three-dimensional (3D) chitin-glucan micro/nanofibrous fungal-based spheres coated with collagen (type I) to mimic the native extracellular matrix (ECM) microenvironment. These collagen-coated fungal nano/microfibrous spheres (C-FNS) were utilized to construct 3D scaffolded spheroids of human fibroblasts through suspension culture for tissue engineering and regenerative medicine. The particle sizes of C-FNS ranged from 1.4 to 3.25 µm (average: 2.27 ± 0.38 µm), with a porosity of 81.17 %. Field emission-scanning electron microscopy (FE-SEM) revealed that C-FNS comprised continuous chitin-glucan fibers with an average diameter of 363 ± 61 nm (range: 203-512 nm), exhibiting a highly interconnected structure. The reduced arithmetic average roughness (Ra) and root mean square roughness (Rq) values of C-FNS compared to uncoated FNS suggested that collagen coating reduced surface roughness, resulting in a smoother surface that enhanced hydrophilicity, crucial for mammalian cell adhesion and spheroid formation. Moreover, the in vitro cytocompatibility of C-FNS with fibroblasts was evaluated using a resazurin-based PrestoBlue assay, which demonstrated a time-dependent increase in the metabolic activity of C-FNS/fibroblast spheroids during suspension culture for up to 14 days. FE-SEM images of C-FNS/fibroblast spheroids further revealed enhanced adhesion and proliferation of fibroblasts on the nano/microfibrous mycelial architecture, accompanied by the secretion of ECM components and formation of multilayered cell sheets over the 14-day culture period. Similarly, an assessment of the hemocompatibility of C-FNS with erythrocytes revealed the non-hemolytic properties of the biomaterial. Overall, the interaction between collagen-coated fungal chitin-glucan nano/microfibrous structures and mammalian cells holds significant potential for the development of novel, sustainable biomaterials with tailored properties for a myriad of biomedical applications, including tissue engineering, regenerative medicine, drug screening, and wound healing.

The Effects of Photobiomodulation Therapy on Tensile-Cultured Cementoblasts Cells.

Background: Studies show that photobiomodulation therapy (PBMT) boosts cellular ATP production and cell growth and reduces inflammation. Additionally, mechanical tension affects gene expression, impacting cellular functions like proliferation and migration. Objective: We investigated the impact of PBMT on OCCM-30 cementoblast cells under tensile stress, focusing on cell survival, differentiation, and inflammatory responses, particularly relating to orthodontic tooth movement and root resorption. Methods: Cultured OCCM-30 cells under negative pressure received PBMT with a 10.6 μm wavelength in continuous mode at 1.0 W power for 3, 5, or 10 sec, corresponding to energy densities of 3, 5, or 10 J/cm2. We assessed cell viability with the Presto Blue assay and inflammatory markers Interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) through western blots at 1, 12, 24 h, and 7 days post-irradiation. Results: PBMT improved cell viability over time while maintaining levels of inflammatory markers. alkaline phosphatase levels dropped initially but increased after 7 days, suggesting enhanced cementoblast differentiation. IL-6 levels rose gradually, with 3J and 5J treatments showing significantly higher levels than the control. iNOS levels spiked within the first 24 h, then declined by day 7. COX-2 levels consistently rose, with the 5J treatment showing greater increases. Conclusions: PBMT appears to support cementoblast survival and differentiation while managing inflammation, potentially aiding root repair during orthodontic treatments and reducing inflammatory root resorption.

Detoxification of paraoxon-ethyl by Lysiphyllum strychnifolium extracts in undifferentiated human neuroblastoma SH-SY5Y cells

Context Lysiphyllum strychnifolium (Craib) A. Schmitz. (Fabaceae) is a Thai traditional medicine used to remove food and alcohol toxins from the body. Objective This study investigates the molecular mechanism of L. strychnifolium extracts against paraoxon-ethyl-induced apoptosis in SH-SY5Y cells. Materials and methods The ethanol and water extracts of leaves and stems of L. strychnifolium were prepared at various concentrations (0–100 μg/mL) and co-treated to the cells with 0.375 mM paraoxon-ethyl for 24 and 48 h. Cell viability was performed using the PrestoBlue assay. ROS and caspase activity were detected using 2′,7′-dichlorodihydrofluorecein diacetate and caspase-Glo® 3/7, 8, and 9 assay kits. Apoptotic and ER stress-related gene expression were determined by real-time PCR, and nuclear and mitochondrial morphology were observed using Hoechst 33342 and MitoTracker® Deep Red FM staining. Results The most effective concentrations of each extract against paraoxon-ethyl-induced cell death were 25 μg/mL of leaf ethanol, 12.5 μg/mL of stem ethanol, 100 μg/mL of leaf water, and 25 μg/mL of stem water extracts. The leaf ethanol extract was the most effective at detoxifying, while stem extracts were highly toxic in high doses. The detoxifying L. strychnifolium extracts against paraoxon-ethyl-induced oxidative stress decreased p53, BiP/GRP78, and CHOP gene expression and minimized caspase 9 and caspase 3, protecting cells from apoptosis. The extracts could also restore mitochondrial membrane potential and reduce the swollen globule mitochondrial shape. Discussion and conclusion These findings could potentially protect neuron cells from neurodegenerative issues due to oxidative damage, apoptosis, and other potential consequences.

Mitochondrial dysfunction induced in human hepatic HepG2 cells exposed to the fungicide kresoxim-methyl and to a mixture kresoxim-methyl/boscalid

ABSTRACT The fungicides strobilurins and succinate dehydrogenase inhibitors (SDHIs) are blockers of the electron transport chain (ETC) in fungi. Here, we show that the exposure for 24 h to kresoxym-methyl, a fungicide from the class of strobilurins, alters the mitochondrial respiration in human HepG2 hepatocytes. In addition, we demonstrate an increase in production of mitochondrial superoxide radical anion, a reduction in ATP level, a decrease in the ratio reduced/oxidized glutathione and a decrease in cell viability (assessed by the LDH assay, Presto Blue assay, and Crystal Violet assay). As kresoxym-methyl is associated to boscalid (SDHI) in commercial formulations, we analyzed a potential exacerbation of the induced mitochondrial dysfunction for this combination. For the highest dose at which kresoxym-methyl (5 µM) and boscalid (0.5 µM) did not induce changes in mitochondrial function when used separately, in contrast, when both fungicides were used in combination at the same concentration, we observed a significant alteration of the mitochondrial function of hepatocytes: there was a decrease in oxygen consumption rate, in the ATP level. In addition, the level of mitochondrial superoxide radical anion was increased leading to a decrease in the ratio reduced/oxidized glutathione, and an increase in viability.

Tailoring metabolic activity assays for tumour-engineered 3D models

Monitoring cell behaviour in hydrogel-based 3D models is critical for assessing their growth and response to cytotoxic treatment. Resazurin-based PrestoBlue and AlamarBlue reagents are frequently used metabolic activity assays when determining cell responses. However, both assays are largely applied to cell monolayer cultures but yet to have a defined protocol for use in hydrogel-based 3D models. The assays' performance depends on the cell type, culture condition and measurement sensitivity. To better understand how both assays perform, we grew pancreatic cancer cells in gelatin methacryloyl and collagen hydrogels and evaluated their metabolic activity using different concentrations and incubation times of the PrestoBlue and AlamarBlue reagents. We tested reagent concentrations of 4 % and 10 % and incubation times of 45 min, 2 h and 4 h. In addition, we co-cultured cancer cells together with cancer-associated fibroblasts and peripheral blood mononuclear cells in gelatin methacryloyl hydrogels and subjected them to gemcitabine and nab-paclitaxel to evaluate how both assays perform when characterising cell responses upon drug treatment. CyQuant assays were conducted on the same samples and compared to data from the metabolic activity assays. In cancer monocultures, higher reagent concentration and incubation time increased fluorescent intensity. We found a reagent concentration of 10 % and an incubation time of 2 h suitable for all cell lines and both hydrogels. In multicellular 3D cultures, PrestoBlue and AlamarBlue assays detected similar cell responses upon drug treatment but overestimated cell growth. We recommend to assess cell viability and growth in conjunction with CyQuant assays that directly measure cell functions.

Cytotoxic Effects of Hibiscus surattensis L. Leaves Extracts on BSLT, MCF-7 and Hela Cells

Hibiscus surattensis L. leaves have been known as a folk medicine. Several previous reports have shown several potential therapeutic effects, including antidiabetic, antibacterial, anti-diarrheal, analgesic, hepatoprotective, and antioxidant, but no toxicity study is similar to the current one. Therefore, it is necessary to investigate the cytotoxic effect of these commonly consumed medicinal leaves. This study aimed to investigate the cytotoxicity of Hibiscus surattensis L. leaves extract (HSL). The cytotoxic effect was determined through brine shrimp lethality bioassay (BSLT) and in vitro Presto Blue assay of MCF-7 and Hela cell. HSL extract was obtained by maceration using 96% ethanol. The BSLT results showed that the HSL has LC50 value of 224.814 μg/mL. MTT assay results showed the IC50 value of the HSL extract on MCF-7 cells was 1419.83 μg/mL, and Hela cells were 2050.50 μg/mL. Based on the LC50 value that has been analyzed against HSL extract shows an LC50 value of less than 1000 μg/mL which can be concluded while having potential cytotoxic activity against experimental animal larvae of A. salina Leach. The results of the cytotoxic test with Presto Blue assay on two cancer cells used showed that the HSL extract was not toxic.

Biocompatibility of 3D-printed vs. thermoformed and heat-cured intraoral appliances.

The development of additive manufacturing has the potential to revolutionize the fabrication of medical devices. This technology, also known as 3D printing, offers precise, cost-effective, and personalized approaches, which could be particularly beneficial in the production of intraoral appliances. Despite its promise, research on the biocompatibility of 3D-printed intraoral devices is still limited. Our study aims to address this gap. We examined the cytotoxicity of materials processed via three techniques commonly used for the fabrication of different intraoral appliances: 3D printing (Dental LT Clear), thermoforming (Duran adjusted with Durasplint LC), and conventional heat-curing (Villacryl H Plus). We also investigated the impact of chemical or UVC disinfection on the biocompatibility of these materials. We assessed the biological effects induced in human gingival fibroblasts (HGFs) through both direct contact tests (MTT and LDH assays) and extract tests (PrestoBlue, DCF, and cell death type assays). Additionally, we observed changes in cellular morphology and migration rate under an inverted light microscope. The surface roughness of materials was evaluated using contact profilometry. Statistical analysis was conducted using two-way analysis of variance. Our findings suggest that all three fabrication techniques induced a slight cytotoxic effect in HGFs, as evidenced by both direct contact and extract tests. However, these materials could be considered nontoxic according to the ISO 10993-5:2009 norm, as the decrease in metabolic activity observed was always less than 30% compared to the untreated control. This novel study confirms that 3D printing may be a safe alternative to conventional methods for fabricating intraoral appliances. However, further tests assessing the long-term intraoral usage are still needed.

Photoprotective Effects of Yeast Pulcherrimin.

Sunscreen products can protect the skin against the harmful effects of UV radiation, including reddening, aging, and cancer. The aim of this research was to evaluate the photoprotective effects of yeast pulcherrimin, an iron-chelating dipeptide. We first investigated the cytotoxicity of pulcherrimin produced by Metschnikowia pulcherrima yeast on the human keratinocyte HaCaT cell line, using the PrestoBlue assay. We assessed the ability of pulcherrimin to induce DNA repair after the exposure of HaCaT cells to oxidative stress. We also evaluated its protective activity against UVC radiation. The sun protective factor (SPF) was calculated using the Mansur equation. The UVA/UVB ratio values for pure pulcherrimin were evaluated using the Boots Star Rating system. The critical wavelength was determined by calculating the integrated optical density curve area. Based on the results, pulcherrimin shows strong cytoprotective effects through antioxidant and photoprotective activities on the HaCaT cell line. The calculated SPFs were 20 and 15 at pH = 7 and pH = 10, respectively. The critical wavelength above 370 nm and the UVA/UVB ratio R > 1 suggest that yeast pulcherrimin-a cyclic dipeptide containing iron-may be considered a promising photoprotective agent.

Exploring Senolytic and Senomorphic Properties of Medicinal Plants for Anti-Aging Therapies.

Senolytic and senomorphic therapies have gained more and more attention in the last decade. This kind of therapy is based on the killing of cellular senescent cells without harming the "normal" cells. Aging is not a disease. Clinical studies on healthy people will be difficult to conduct. Therefore, one possibility is to draw on the large repertoire of medicinal plants and use their senolytic properties to provide mild anti-aging therapies. Chamomile, goldenrod, reishi, and green tea were tested for their ability to trigger senolysis. Quercetin was used as control substance. Cellular senescence was induced with 25 µM etoposide in human dermal fibroblasts and established for at least 14 days. The plant extracts were tested for their antioxidant potential (DPPH assay) and their polyphenol content. Senolysis was determined by presto blue assay of young and etoposide-induced senescent cells, and SA-β-Gal assays were also performed. The senomorphic properties of the plants were investigated using IL-6 ELISA and qPCR. It turned out that chamomile triggers a kind of cytokine storm and causes the cytokine values in the ELISA and in the qPCR to rise extremely, and other senescence-associated phenotype (SASP) markers were also elevated. Goldenrod and quercetin tend to have a senolytic and senomorphic effect, respectively. Regarding the senolytic and senomorphic properties of herbs, we found that all tested herbs can have a senolytic effect, and a senomorphic effect of quercetin has also been discovered. With regard to the effect of chamomile, however, we can say that seemingly harmless tea products may have harmful effects, especially in combination with chemotherapy, at least in cell culture experiments. Nevertheless, inflammation is a double-bladed mechanism with positive effects, for example, in healing, but also known negative effects.

Encapsulation of photosensitizer worsen cell responses after photodynamic therapy protocol and polymer micelles act as biomodulators on their own

Photodynamic therapy (PDT) is a photochemical therapeutic modality used clinically for dermatological, ophthalmological and oncological applications. Pheo a was used as a model photosensitizer, either in its free form or encapsulated within poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) polymer micelles. Block copolymer micelles are water-soluble biocompatible nanocontainers with great potential for delivering hydrophobic drugs. Empty PEO-PCL micelles were also tested throughout the experiments. The goal was to conduct an in vitro investigation into human colorectal tumor HCT-116 cellular responses induced by free and encapsulated Pheo a in terms of cell architecture, plasma membrane exchanges, mitochondrial function, and metabolic disturbances. In a calibrated PDT protocol, encapsulation enhanced Pheo a penetration (flow cytometry, confocal microscopy) and cell death (Prestoblue assay), causing massive changes to cell morphology (SEM) and cytoskeleton organization (confocal), mitochondrial dysfunction and loss of integrity (TEM), rapid and massive ion fluxes across the plasma membrane (ICP-OES, ion chromatography), and metabolic alterations, including increased levels of amino acids and choline derivatives (1H NMR). The detailed investigation provides insights into the multifaceted effects of encapsulated Pheo-PDT, emphasizing the importance of considering both the photosensitizer and its delivery system in understanding therapeutic outcomes. The study also raises questions as to the broader impact of empty nanovectors per se, and encourages a more comprehensive exploration of their biological effects.

Hybrid three-dimensional (3D) bioprinting of retina equivalent for ocular research.

In this article, a hybrid retina construct was created via three-dimensional (3D) bioprinting technology. The construct was composed of a PCL ultrathin membrane, ARPE-19 cell monolayer and Y79 cell-laden alginate/pluronic bioink. 3D bioprinting technology was applied herein to deliver the ARPE-19 cells and Y79 cell-laden bioink to ensure homogeneous ARPE-19 cell seeding; subsequently, two distinctive Y79 cell-seeding patterns were bioprinted on top of the ARPE-19 cell monolayer. The bioprinted ARPE-19 cells were evaluated by prestoblue assay, F-actin, and hematoxylin/eosin (HE) staining, and then the cells were observed under laser scanning and invert microscopy for 14 days. The Y79 cells in alginate/pluronic bioink after bioprinting had been closely monitored for 7 days. Live/dead assay and scanning electrical microscopy (SEM) were employed to investigate Y79 cell viability and morphology. Both the ARPE-19 and Y79 cells were in excellent condition, and the successfully bioprinted retina model could be utilized in drug delivery, disease mechanism and treatment method discoveries.

Sesquiterpenoids from The Stem Bark of Aglaia cucullata (Meliaceae) and Their Cytotoxic Activity Against A549 Lung Cancer Cell Lines

ABSTRACT. Sesquiterpenoids are a class of terpenoid compounds with a remarkable diversity of structures and biological activities. Sesquiterpenoids are primarily found in higher plants, such as the Meliaceae family's Aglaia genus. Aglaia cucullata is a species of Aglaia that is still rarely explored and can potentially contain sesquiterpenoid compounds with cytotoxic activity. Hence, the research intended to isolate sesquiterpenoids from the n-hexane extract of A. cucullata stem bark and evaluate their cytotoxic effect against A549 lung cancer cells. Compounds 1 and 2 were isolated and purified from n-hexane extracts utilizing various chromatographic techniques. The structure of compounds 1 and 2 was determined by analyzing various spectroscopic methods (IR, MS, and NMR) and comparing them to previously reported spectral data. Compound 1 was identified as an isodaucane-type sesquiterpenoid, 10-hydroxy-6,10-epoxy-7(14)-isodaucane, and was first reported in Meliaceae family. Compound 2 was confirmed as an eudesmane-type sesquiterpenoid, eudesm-4(15)-ene-1β,6α-dihydroxy, and was first reported in Aglaia cucullata. Cytotoxic activity of 1 and 2 were investigated in vitro against A549 lung cancer cells using the PrestoBlue assay and resulted in inactive and low cytotoxicity with IC50 values of 292.77 and 90.55 μM, respectively. Key words: Aglaia cucullata, , A549 cell lines, cytotoxic activity, Meliaceae, sesquiterpenoids.

Effects of hydroxyapatite content on cytotoxicity, bioactivity and strength of metakaolin/hydroxyapatite composites

The development of new materials is a critical challenge in advancing bone repair technology. Nowadays, the use of high strength geopolymers is extended to various fields, including biomedical research. This study investigates the influence of hydroxyapatite (HAp) content on the in vitro properties and characteristics of the metakaolin/HAp (MK/HAp) composites for potential application as bone substitutes. The MK/HAp composites with varying HAp contents were fabricated and subjected to cytotoxicity, bioactivity, physical and mechanical testing. PrestoBlue assay results confirmed acceptable osteoblast cell compatibility for all composites, with cell viability increases with increasing HAp content. Simulated body fluid (SBF) immersion demonstrated enhanced bioactivity and bone-like apatite formation on the composite surfaces with increasing HAp content. The compressive strength of the composite decreased mainly due to the reduced amount of silica and alumina contents and the increased porosity with higher HAp. Based on the present results, the relationship between bioactivity and strength with variations in HAp content indicates a promising composite. The HAp content of the MK/HAp composites for a bone substitute material should be 50% or higher to match the apatite formation and strength of specific human bones.

Anticancer Activities of Bromelain Hydrolysate of Soy Protein Against Breast Cancer Cells MCF-7

Soybeans contain proteins that have the potential to produce anticancer bioactive peptides. This study aims to determine the anticancer activity of soy protein hydrolysate against MCF-7 breast cancer cells. Soybean protein hydrolyzed by bromelain enzyme 0.5% (w/v) at the optimum temperature and pH for protein hydrolysis using the Bergmeyer and Grassl method. The degree of hydrolysis value of protein hydrolysate was determined by the Alder-Niesen method and the protein profile was analyzed by SDS-PAGE. The hydrolysate with the best degree of hydrolysis value was analyzed for anticancer activity against MCF-7 breast cancer cells by the Presto Blue assay method, and fractionation of protein hydrolysates by gel filtration chromatography (Sephadex G-15). The molecular weight of the peptide was characterized by LCMS/MS. Soy protein hydrolysis using 0.5% (w/v) bromelain enzyme was optimum at 65 ºC and pH 7.0 for 4 hours, with a hydrolysis degree value of 20.57%. The SDS-PAGE analysis showed that the protein hydrolysates had quite thick protein bands in the range of <35 kDa with an IC50 value of 70.37 mg/mL. Based on the LCMS/MS results, the peptide from fractionation has a molecular weight of 5.133 kDa.

Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.

Anti-inflammatory effect of curcuminoids and their analogs in hyperosmotic human corneal limbus epithelial cells

BackgroundTo assess the efficacy of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin [BDC]) and their analogs (tetrahydrocurcumin [THC], tetrahydrodemethoxycurcumin [THDC], tetrahydrobisdemethoxycurcumin) in reducing inflammatory cytokines and their toxicity to primary human corneal limbal epithelial cells, these cells were cultured and exposed to these compounds.MethodsThe PrestoBlue assay assessed cell viability after treatment. Anti-inflammatory effects on hyperosmotic cells were determined using real-time polymerase chain reaction and significance was gauged using one-way analysis of variance and Tukey’s tests, considering p-values < 0.05 as significant.ResultsCurcuminoids and their analogs, at 1, 10, and 100 µM, exhibited no effect on cell viability compared to controls. However, cyclosporin A 1:500 significantly reduced cell viability more than most curcuminoid treatments, except 100 µM curcumin and BDC. All tested curcuminoids and analogs at these concentrations significantly decreased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-17 A, matrix metallopeptidase-9, and intercellular adhesion molecule-1 after 90 mM NaCl stimulation compared to untreated cells. Furthermore, proinflammatory cytokine levels from hyperosmotic cells treated with 1, 10, and 100 µM curcumin, 100 µM BDC, 100 µM THC, 1 and 100 µM THDC mirrored those treated with cyclosporin A 1:500.ConclusionThe anti-inflammatory efficiency of 1 and 10 µM curcumin, 100 µM THC, 1 and 100 µM THDC was comparable to that of cyclosporin A 1:500 while maintaining cell viability.

Bioprinting of a Liposomal Oxygen-Releasing Scaffold for Ovary Tissue Engineering.

This study addresses a critical challenge in bioprinting for regenerative medicine, specifically the issue of hypoxia compromising cell viability in engineered tissues. To overcome this hurdle, a novel approach using a microfluidic bioprinter is used to create a two-layer structure resembling the human ovary. This structure incorporates a liposomal oxygen-releasing system to enhance cell viability. The bioprinting technique enables the simultaneous extrusion of two distinct bioinks, namely, bioink A (comprising alginate 1% and 5 mg/mL PEGylated fibrinogen in a 20:1 molar ratio) and bioink B (containing alginate 0.5%). In addition, liposomal catalase and hydrogen peroxide (H2O2) are synthesized and incorporated into bioinks A and B, respectively. The liposomes are prepared using thin film hydration with a monodisperse size (140-160 nm) and high encapsulation efficiency. To assess construct functionality, isolated human ovarian cells are added to bioink A. The bioprinted constructs, with or without liposomal oxygen-releasing systems, are cultured under hypoxic and normoxic conditions for 3 days. Live/Dead assay results demonstrate that liposomal oxygen-releasing systems effectively preserve cell viability in hypoxic conditions, resembling viability under normoxic conditions without liposomes. PrestoBlue assay reveals significantly higher mitochondrial activity in constructs with liposomal oxygen delivery systems under both hypoxic and normoxic conditions. The evaluation of apoptosis status through annexin V immunostaining shows that liposomal oxygen-releasing scaffolds successfully protect cells from hypoxic stress, exhibiting a proportion of apoptotic cells similar to normoxic conditions. In contrast, constructs lacking liposomes in hypoxic conditions exhibit a higher incidence of cells in early-stage apoptosis. In conclusion, the study demonstrates the promising potential of bioprinted oxygen-releasing liposomal scaffolds to protect ovarian stromal cells in hypoxic environments. These innovative scaffolds not only offer protection but also recapitulate the mechanical differences between the medulla and the cortex in the normal ovary structure. This opens new avenues for advanced ovary tissue engineering and transplantation strategies.

Direct inkjet writing of polylactic acid/β-tricalcium phosphate composites for bone tissue regeneration: A proof-of-concept study.

There is an ever-evolving need of customized, anatomic-specific grafting materials for bone regeneration. More specifically, biocompatible and osteoconductive materials, that may be configured dynamically to fit and fill defects, through the application of an external stimulus. The objective of this study was to establish a basis for the development of direct inkjet writing (DIW)-based shape memory polymer-ceramic composites for bone tissue regeneration applications and to establish material behavior under thermomechanical loading. Polymer-ceramic (polylactic acid [PLA]/β-tricalcium phosphate [β-TCP]) colloidal gels were prepared of different w/w ratios (90/10, 80/20, 70/30, 60/40, and 50/50) through polymer dissolution in acetone (15% w/v). Cytocompatibility was analyzed through Presto Blue assays. Rheological properties of the colloidal gels were measured to determine shear-thinning capabilities. Gels were then extruded through a custom-built DIW printer. Space filling constructs of the gels were printed and subjected to thermomechanical characterization to measure shape fixity (Rf) and shape recovery (Rr) ratios through five successive shape memory cycles. The polymer-ceramic composite gels exhibited shear-thinning capabilities for extrusion through a nozzle for DIW. A significant increase in cellular viability was observed with the addition of β-TCP particles within the polymer matrix relative to pure PLA. Shape memory effect in the printed constructs was repeatable up to 4 cycles followed by permanent deformation. While further research on scaffold macro-/micro-geometries, and engineered porosities are warranted, this proof-of-concept study suggested suitability of this polymer-ceramic material and the DIW 3D printing workflow for the production of customized, patient specific constructs for bone tissue engineering.

Abstract A074: Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway

Abstract Background: The Wnt/β-catenin pathway controls the growth of pancreatic carcinoma by regulating processes such as cell cycle progression, apoptosis, and the tumor immune microenvironment. Treating pancreatic cancer with Wnt pathway inhibitors was found to be toxic in clinical studies. Namodenoson, an A3 adenosine receptor (A3AR) agonist, is under clinical development as a treatment of advanced liver cancer, where namodenoson was shown to have an excellent safety profile in phase II studies and is a phase III study is ongoing. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. The objectives of the currents study were to examine the anti-growth effect of namodenoson on pancreatic carcinoma cells both in vitro and in vivo and to investigate the molecular mechanisms involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson, or in the presence of a combination of namodenoson (0.1 nM) and gemcitabine (0.2 µM). Cell growth was monitored via 3[H]-thymidine proliferation, MTT, and PrestoBlue assays. Western blot analyses were conducted to identify the regulatory cell growth proteins impacted by the treatment. A xenograft model with human BxPC-3 cells in nude mice was established to investigate the in-vivo effect of namodenoson. Results: A significant dose-dependent growth inhibition of BxPC-3 cells was observed with namodenoson in the3[H]-thymidine proliferation assay (1 nM: 67.4% ± 1.7%, p &amp;lt; 0.001; 0.1 nM: 53.7% ± 6.3%, p&amp;lt; 0.05; 0.01 nM: 27.9 % ± 2.3%, p&amp;lt; 0.005). The combination of namodenoson and gemcitabine showed an additive inhibitory effect in the MTT assay (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p&amp;lt;0.001 for all). Western blot analyses demonstrated that namodenoson treatment was associated with de-regulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, β-catenin and up-regulation of the BAD and BAX apoptotic proteins. In the xenograft model, daily oral treatment with 10 µg/kg of namodenoson led to a significant progressive inhibition of tumor growth. Conclusions: Nanomolar concentrations of namodenoson inhibited the growth of pancreatic carcinoma in cell culture and in a xenograft model. This effect involved A3AR activation and de-regulation of the Wnt/β-catenin pathway. Our results support a potential clinical utility for namodenoson in pancreatic cancer, a disease where an unmet clinical need exists, and novel therapeutic opportunities are required. Citation Format: Pnina Fishman, Avital Bareket-Samish, Inbal Izhak. Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A074.

10199-SPDR-2 PERSONALIZED SYNTHETIC LETHALITY INDUCED BY TARGETING ATR-CHK1 IN MEDULLOBLASTOMAS IDENTIFIED BY IMPLEMENTATION OF SLFN11 IMMUNOHISTOCHEMISTRY

Abstract Survival in medulloblastoma varies widely between molecular subgroups, and risk-adapted treatment stratification is necessary. Schlafen family member 11 (SLFN11) defines sensitivity to DNA-damaging therapies in a variety of cancers and may be useful in stratifying medulloblastomas. We previously showed that SLFN11 immunohistochemistry identifies poor-prognosis groups that lack expression; however, the additional treatment for these groups has not been fully elucidated. The serine/threonine kinase ATR (ataxia telangiectasia and Rad3-related) binds to single-stranded DNA upon DNA damage competing with SLFN11 and leads to cell cycle arrest and DNA repair process via Chk1 phosphorylation. Although therapeutic effects of ATR/Chk1 inhibitors on SLFN11-deficient cell lines have been reported in other carcinomas, little is known on brain tumors. In the present study, we investigated the effect of ATR/Chk1 inhibition on SLFN11-negative medulloblastomas. ATR knockdown was introduced to the medulloblastoma cell line DAOY and the isogenic SLFN11-knockout line DYB9, and only DYB9 showed increased sensitivity to cisplatin (Prestoblue assay, p &amp;lt; 0.05, t-test). The ATR inhibitor ceralasertib and the Chk1 inhibitor prexasertib also increased cisplatin and camptothecin sensitivity in DYB9 and SLFN11-low Group 3 medulloblastoma D425. Furthermore, combination therapy with cisplatin and prexasertib inhibited the growth of D425 cerebellar tumors (Akaluc IVIS imaging, p &amp;lt; 0.05, t-test) and prolonged survival of the mice (p &amp;lt; 0.05, Log-rank). Combination therapy with irradiation is currently under investigation. Evaluation of SLFN11 expression in medulloblastoma may lead to new treatment strategies, such as reduced radiation dose for the SLFN11-high groups and the combination therapy with ATR/Chk1 inhibitors for the low-expressing groups.