Ethidium Homodimer Research Articles

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

P-349 Impact of an Aurora kinase inhibitor on AML cells and mouse ovarian follicles in vitro

Abstract Study question Can an aurora kinase B/C inhibitor suppress AML cell line proliferation while not impacting on mouse follicles in vitro? Summary answer Exposure to an aurora kinase inhibitor at a predetermined cytotoxic concentration suppressed AML cell proliferation but had no impact on subsequent follicle health in vitro. What is known already There is a risk that cryopreserved ovarian tissue from young women with a leukaemia diagnosis harbours leukemic cells, which on grafting could transfer disease. Novel chemotherapy agents, specifically aurora kinase inhibitors [ e.g. GSK 1070916 (GSK) ], target proteins involved in chromosomal alignment and segregation during mitosis and meiosis. It is anticipated that aurora kinase inhibitors would have an impact on the development of all somatic cells, including granulosa cells proliferating during follicle growth, however a previous study using ovarian tissue pieces indicated no impact on follicles. Study design, size, duration Primary and secondary follicles (diameter ≤ 100 µm) were manually dissected from adult mouse ovaries and embedded in an extracellular scaffold together with &amp;gt;1,000 AML cells. Individual follicles and AML cells were cultured for 7 days followed by exposure to GSK for 24 hours. GSK was removed by washing and the culture continued for another two days at which time cells were assessed for normal morphology and survival with live/dead staining (Calcein AM/ Ethidium Homodimer-1). Participants/materials, setting, methods The leukemic cell line OC1-AML-3 (DSMZ) was cultured in RPMI-1640 +10% FCS and routinely passaged as recommended. Cell proliferation and cytotoxicity were assessed using the Alamar Blue assay. 20% DMSO was used as a positive control in the cytotoxicity assays and live/dead evaluation. Follicles were cultured in αMEM, ITS, FSH, ascorbic acid and 10% FCS under oil in 5%CO2 : 95% air. Survival and growth were evaluated in two extracellular scaffolds; UltiMatrix and Hystem™-HP. Main results and the role of chance Follicle survival and growth was better (p &amp;lt; 0.05) in UltiMatrix (84.7%; 61/72) compared to Hystem™-HP (72.0%;77/107) with an average increase of 20 µm in diameter at day 7. When OCI-AML-3 cells (0.7 x105/ml) were exposed to a range of concentrations of GSK (10nM to 10µM) for 24hr in an in vitro cytotoxicity assessment, survival was reduced to 0% at a concentration of 10µM. The 20% DMSO positive control also resulted in 0% survival. However, in the 3- dimensional scaffold culture, exposure to 10 µM GSK for 24hr resulted in a mean survival of OCI-AML-3 cells of 27% (100 cells counted/ well) which was similar to the 20% DMSO positive control (32%) and significantly different to the negative (no GSK) control (95%; p &amp;lt; 0.01). Follicles exposed to GSK appeared alive, emitting a strong green fluorescence for both oocyte and granulosa cells, although some of the surface follicular cells were dead. Live/dead staining of GSK exposed follicles was similar to follicles not exposed to GSK. In contrast, the majority of the follicular cells were dead following exposure to 20% DMSO. Limitations, reasons for caution Due to whole mount assessment of the follicles for live/dead staining, it is difficult to determine whether the oocyte is alive. In scaffold culture some of the free cells present may be of follicular origin, potentially resulting in overestimation of the proportion of AML cells alive after GSK treatment. Wider implications of the findings This 3- dimensional culture system, incorporating a follicle and a leukemic cell line within a scaffold provides a good model to assess the impact of chemotherapy on both cell types and could be applicable for human follicles. Trial registration number Not applicable

Viability and Proliferation Assessment of Gingival Fibroblasts Cultured on Silver Nanoparticle-Doped Ti-6Al-4V Surfaces.

To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces. AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.

Abstract 4919: AI-enabled hit selection of drug screening on human pancreatic cancer organoids

Abstract Cancer remains one of the leading causes of death in the 21st century. Despite the latest advances in oncology, most cancer patients lack tailored therapeutic approaches with lasting benefit. Measuring the impact of anticancer compounds and their combinations is only possible on ex vivo assays. To this end, patient-derived organoids (PDOs) have been proposed as viable and efficient models for ex vivo testing. PDOs show long-term expansion potential while retaining tumor histopathology as well as cancer gene mutations. However, the translation of organoids in screening applications has so far been hampered by the lack of homogeneity and difficulties in handling and automation. Moreover, organoids are typically randomly distributed across the culture which complicates imaging and images analyses. To overcome these challenges, we set up a compound screening workflow with PDOs using Gri3D platform, comprising plates with micro-cavities suitable for high-throughput and reproducible organoid culture. Based on a standard 96 microtiter plate, each well contains a microwell array patterned in a cell repellent hydrogel. On Gri3D®, organoids are robustly generated in the microwells and are located in the same imaging plane. This greatly facilitates quantitative analyses in high content image-based screens. Furthermore, the pipetting port enables automation of cell seeding, media exchange and compound incubation with liquid-handlers, thus increasing assay reproducibility. In the presented work, we exposed human pancreatic cancer PDOs to a panel of anti-cancer compounds at different doses and followed their response to the drugs using viability dyes Calcein AM (live stain) and Ethidium Homodimer-1 (dead stain) with high-content confocal imaging. Using an AI-based approach, we efficiently detected each single organoid and extracted phenotypic features from all three channels (TL, live stain and dead stain) which correlate with cytotoxicity. The extracted features (more than 100) were first dimensionally reduced to 3 components using either PCA or UMAP, the treatments were then visualized in 3D scatter plots, ranked and clustered using machine learning. The data suggests that the compound treatment Palbociclib 50uM has significant cytotoxicity effects similar to the positive control, which is consistent with the traditional live/dead analysis. The AI approach demonstrates feasibility to perform drug screening in a robust and un-biased data analysis approach. Citation Format: Zhisong Tong, Angeline Lim, Marine Meyer, Maria Clapes, Oksana Sirenko, Nathalie Brandenberg. AI-enabled hit selection of drug screening on human pancreatic cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4919.

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of invitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from invitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 μm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured invitro.

Pipette-free field-deployable molecular diagnostic kit for bimodal visual detection of infectious RNA viruses.

Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO4 ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1h, and the limits of detection (LOD) were approximately 1 virus μL-1 for SFTSV and 103 copies μL-1 for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.

PROGRESSIVE MUSCLE HYPOXIA RESEARCH: COULD PHARMACOLOGICAL AGENTS BE USED TO LIMIT REPERFUSION INJURY AT THE TIME OF FASCIOTOMY TO TREAT ACUTE COMPARTMENT SYNDROME?

AbstractBackgroundProgressive muscle ischaemia results in reduced aerobic respiration and increased anaerobic respiration, as cells attempt to survive in a hypoxic environment. Acute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency resulting in the production of Lactic acid by cells through anaerobic respiration. Our previous research has shown that it is possible to measure H+ ions concentration (pH) as a measure of progressive muscle ischaemia (in vivo) and hypoxia (in vitro). Our aim was to correlate intramuscular pH readings and cell viability techniques with the intramuscular concentration of key metabolic biomarkers [adenosine triphosphate (ATP), Phosphocreatine (PCr), lactate and pyruvate], to assess overall cell health in a hypoxic tissue model.MethodsNine euthanised Wistar rats were used in a non-circulatory model. A pH catheter was used to measure real-time pH levels from one of the exposed gluteus medius muscles, while muscle biopsies were taken from the contralateral gluteus medius at the start of the experiment and subsequently at every 0.1 of a pH unit decline. The metabolic biomarkers were extracted from the snap frozen muscle biopsies and analyzed with standard fluorimetric method. Another set of biopsies were stained with Hoechst 33342, Ethidium homodimer-1 and Calcein am and imaged with a Zeiss LSM880 confocal microscope.ResultsOur study shows that the direct pH electrode readings decrease with time and took an average of 69 minutes to drop to a pH of 6.0. The concentrations of ATP, pyruvate and PCr declined over time, and the concentration of lactate increased over time. At pH 6.0, both ATP and PCr concentrations had decreased by 20% and pyruvate has decreased by 50%, whereas lactate had increased 6-fold. The majority of cells were still viable at a pH of 6.0, suggesting that skeletal muscle cells are remarkably robust to hypoxic insult, although this was a hypoxic model where reperfusion was not possible.ConclusionsOur research suggests that histologically, skeletal muscle cells are remarkably robust to hypoxic insult despite the reduction in the total adenine nucleotide pool, but this may not reflect the full extent of cell injury and quite possibly irreversible injury. The timely restoration of blood flow in theory should halt the hypoxic insult, but late reperfusion results in cellular dysfunction and cell death due to localised free radical formation. Further research investigating the effects of reperfusion in vivo are warranted, as this may identify an optimal time for using pharmacological agents to limit reperfusion injury, around the time of fasciotomy to treat acute compartment syndrome.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

PROGRESSIVE MUSCLE HYPOXIA RESEARCH: A MODIFIED METHOD FOR ASSESSING CELL VIABILITY IN SKELETAL MUSCLE BIOPSIES USING VITALITY DYES AND CONFOCAL MICROSCOPY

AbstractObjectivesAcute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency and can have detrimental outcomes for patients if not treated optimally. The current problem is that there is no clear diagnostic threshold for ACS or guidance as to when fasciotomies should be performed. A new diagnostic method(s) is necessary to provide real-time information about the extent of muscle ischaemia in ACS. Given that lactic acid is produced by cells through anaerobic respiration, it may be possible to measure H+ ion concentration and to use this as a measure of ischaemia within muscle. Although we are familiar with the key biochemical metabolites involved in ischaemia; and the use of viability dyes in cell culture to distinguish between living or dead cells is well recognised; research has not been undertaken to correlate the biochemical and histological findings of ischaemia in skeletal muscle biopsies. Our primary aim was to investigate the potential for viability dyes to be used on live skeletal muscle biopsies (explants). Our secondary aim was to correlate the intramuscular pH readings with muscle biopsy viability.MethodsNine euthanised Wistar rats were used. A pH catheter was inserted into one exposed gluteus medius muscles to record real-time pH levels and muscle biopsies were taken from the contralateral gluteus medius at the start of experiment and subsequently at every 0.1 of pH unit drop. Prior to muscle biopsy, the surface of the gluteus medius was painted with a layer of 50µmol/l Brilliant blue FCF solution to facilitate biopsy orientation. A 4mm punch biopsy tool was used to take biopsies. Each muscle biopsy was placed in a base mould filled with 4% ultra-low melting point agarose. The agarose embedded tissue block was sectioned to generate 400 micron thick tissue slices with a vibratome. The tissue slices were then placed in the staining solution with Hoechst 33342, Ethidium homodimer-1 and Calcein am. The tissue slices were imaged with Zeiss LSM880 confocal microscope's Z stack function. A dead muscle control was created by adding TritonX-100 to other tissue slices. For quantitative analyses, the images were analysed in Image J using the selection tool. This permitted individual cells to be identified and the mean grey value of each channel to be defined. Using the dead control, we were able to identify the threshold value for living cells using the Calcein AM channel.ResultsViability dyes, used primarily for cell cultures, can be used with skeletal muscle explants. Our study also showed that despite a significant reduction in tissue pH concentration over time, that almost 100% of muscle cells were still viable at pH 6.0, suggesting that skeletal muscle cells are robust to hypoxic insult in the absence of reperfusion.ConclusionsViability dyes can be used on skeletal muscle biopsies. Further research investigating the likely associations between direct measured pH using a pH catheter, the concentrations of key cellular metabolic markers, and muscle tissue histology using vitality dyes in response to ischaemia, rather than hypoxia, is warranted.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

A novel chalcone derivative exerts anticancer effects by promoting apoptotic cell death of human pancreatic cancer cells

Aggressive pancreatic cancer is typically treated using chemotherapeutics to reduce the tumor pre-operatively and prevent metastasis post-operatively, as well as surgical approaches. In the present study, we synthesized a hydroxyl group-introduced chalcone derivative (1, IC50 = 32.1 μM) and investigated its potential as an anticancer drug candidate by evaluating its apoptosis-promoting effects on BXPC-3 cancer cells. The viability of BXPC-3 cells treated with 1 was measured using the water-soluble tetrazolium 1 reagent. BXPC-3 cells induced by 1 were stained with diverse probes or antibodies, such as ethidium homodimer-1, Hoechst, anti-Ki67, and MitoTracker. Protein expression was measured using an immunoblotting assay, and mRNA expression was determined using real-time polymerase chain reaction. Apoptotic molecular features, such as lipid accumulation and protein degradation, were monitored directly using stimulated Raman scattering microspectroscopy. Through incubation time- and concentration-dependent studies of 1, we found that it significantly reduced the proliferation and increased the number of apoptotic BXPC-3 cells. Compound 1 induced mitochondrial dysfunction, phosphorylation of p38, and caspase 3 cleavage. These results indicate that 1 is a potential therapeutic agent for pancreatic cancer, providing valuable insights into the development of new anticancer drug candidates.

Abstract P1117: Personalized Care Using Patient-derived Cardiac Spheroids For Heart Failure Patients

Preclinical studies using currently available in vitro and in vivo models fail at fully recapitulating the in vivo human heart microenvironment and its pathophysiology, limiting their translation from the bench to the bedside. Our laboratory has developed cardiac spheroids (CSs) as advanced in vitro models of the human heart using cardiac myocytes, endothelial cells and fibroblasts at ratios approximating the ones found in the human heart. Thanks to patient-derived induced pluripotent stem cells (iPSCs), we have quickly moved to personalized CSs to establish advanced pathophysiological in vitro models of heart failure (HF). CSs have been used to evaluate short- and long-term effects of ischaemic/reperfusion (I/R) injury typical of myocardial infarction (MI), drug-induced toxicity, SARS-CoV-2 exposure and pregnancy-induced HF. This was achieved using our established protocols to induce I/R-mimic injury, as well by exposing CSs to doxorubicin (DOX), SARS-CoV-2 virus and pre-eclampsia blood samples, respectively. First, our analyses of cell toxicity ratios using calcein-AM and ethidium homodimer (staining live and dead cells, respectively) demonstrated treatment-specific loss in cell viability and increase in cell death compared to control (untreated, healthy) CSs. Other CSs stained with ethidium homodimer and cell-specific antibodies were imaged and further used for 3D rendering analyses using IMARIS software. This enabled us to identify cell-specific responses in each pathological condition. Then, our measurements of fractional shortening and contraction frequency in CSs showed a statistically significant loss of contractile function in treated CSs compared to controls. Finally, new potential targets to prevent myocardial injury were evaluated using transcriptomics analyses, with a focus on changes in expression levels for genes regulating sarcomere structure, calcium transport, cell cycle, cardiac remodelling and signal transduction. Current studies are evaluating novel potential therapeutic targets to prevent and treat myocardial injury in HF patients using CSs. This has the potential to pave the way to prevent and treat HF in a safer and personalised way, hopefully preventing undesired effects due to non-specific care.

Violacein improves vemurafenib response in melanoma spheroids

Chemotherapy resistance is one of the main challenges in melanoma treatment. Violacein, a natural pigment produced by Chromobacterium violaceum, induces apoptosis in a variety of tumours, including melanoma. Here, we used BRAF-mutated melanoma spheroids to test the potential of violacein as a sensitizer of cellular viability and levels of the proteins p62 and fatty acid synthase (FASN). Importantly, violacein in combination with vemurafenib (ViVe) was able to interfere with spheroid survival at subtoxic concentrations. The results demonstrated that the ViVe protocol triggered cell death assessed by calcein and ethidium homodimer dyes. Accordingly, melanoma cells in 2D systems also showed a higher apoptosis rate when treated with ViVe. In the current study, we show evidence that ViVe downregulates crucial mediators like FASN, which partially explains how it acts as a sensitizer and ultimately improves the effectiveness of vemurafenib against melanoma cells.

Mera: A scalable high throughput automated micro-physiological system.

There is an urgent need for scalable Microphysiological Systems (MPS's)1 that can better predict drug efficacy and toxicity at the preclinical screening stage. Here we present Mera, an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has multiple possible fluidics modes. Of these the primary mode is designed so that cells may be matured into a desired microtissue type and in the secondary mode the fluid flow can be re-orientated to create a recirculating circuit composed of inter-connected channels to allow drugging or staining. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM and Ethidium Homodimer (EtHD1) viability assays. We demonstrate the functionality of the automated image capture system. The prototype microtissue culture plate wells are laid out in a 3×3 or 4×10 grid format with viability and toxicity assays demonstrated in both formats. In this paper we set the groundwork for the Mera system as a viable option for scalable microtissue culture and assay development.

Electrochemical Lipolysis Induces Adipocyte Death and Fat Necrosis: In Vivo Pilot Study in Pigs.

Current minimally invasive fat reduction modalities use equipment that can cost thousands of U.S. dollars. Electrochemical lipolysis (ECLL), using low-cost battery and electrodes (approximately $10), creates acid/base within fat (width, approximately 3 mm), damaging adipocytes. Longitudinal effects of ECLL have not been studied. In this pilot study, the authors hypothesize that in vivo ECLL induces fat necrosis, decreases adipocyte number/viability, and forms lipid droplets. Two female Yorkshire pigs (50 to 60 kg) received ECLL. In pig 1, 10 sites received ECLL, and 10 sites were untreated. In pig 2, 12 sites received ECLL and 12 sites were untreated. For ECLL, two electrodes were inserted into dorsal subcutaneous fat and direct current was applied for 5 minutes. Adverse effects of excessive pain, bleeding, infection, and agitation were monitored. Histology, live-dead (calcein, Hoechst, ethidium homodimer-1), and morphology (Bodipy and Hoechst) assays were performed on day 0 and postprocedure days 1, 2, 7, 14 (pig 1 and pig 2), and 28 (pig 2). Average particle area, fluorescence signal areas, and adipocytes and lipid droplet numbers were compared. No adverse effects occurred. Live-dead assays showed adipocyte death on the anode on days 0 to 7 and the cathode on days 1 to 2 (not significant). Bodipy showed significant adipocyte loss at all sites ( P < 0.001) and lipid droplet formation at the cathode site on day 2 ( P = 0.0046). Histology revealed fat necrosis with significant increases in average particle area at the anode and cathode sites by day 14 (+277.3% change compared with untreated, P < 0.0001; +143.4%, P < 0.0001) and day 28 (+498.6%, P < 0.0001; +354.5%, P < 0.0001). In vivo ECLL induces fat necrosis in pigs. Further studies are needed to evaluate volumetric fat reduction. In vivo ECLL induces adipocyte death and fat necrosis. ECLL has the potential to be utilized in body fat contouring.

EVALUATION OF CELL VIABILITY IN LOW CELL DENSITY INTERVERTEBRAL DISC (IVD) TISSUE: TIPS AND TECHNICAL NOTES

Little information exists when using cell viability assays to evaluate cells within whole tissue, particularly specific types such as the intervertebral disc (IVD). When comparing the reported methodologies and the protocols issued by manufacturers, the processing, working times, and dye concentrations vary significantly, making the assay's reproducibility a costly and time-consuming trial and error process. This study aims to develop a detailed step-by-step cell viability assay protocol for evaluating IVD tissue.IVDs were harvested from bovine tails (n=8) and processed at day 0 and after 7 days of culture. Nucleus pulposus (NP) and the annulus fibrosus (AF) 3 mm cuts were incubated at room temperature (26˚C) with a Viability/Cytotoxicity Kit containing Calcein AM and Ethidium Ethidium homodimer-1 for 2 hr, followed by flash freezing in liquid nitrogen. Thirty µm sections were placed in glass slides and sealed with nail varnish or Antifade Mounting Medium. The IVD tissue was imaged within the next 4h after freezing using an inverted confocal laser-scanning microscope equipped with 488 and 543 nm laser lines.Cell viability at day 0 (NP: 92±9.6 % and AF:80±14.0%) and day 7 (NP: 91±7.9% and AF:76±20%) was successfully maintained and evaluated. The incubation time required is dependent on the working temperatures and tissue thickness. The calcein-AM dye will not be retained in the cells for more than four hours.The specimen preparation and culturing protocol have demonstrated good cell viability at day 0 and after seven days of culture. Processing times and sample preparation play an essential role as the cell viability components in most kits hydrolyse or photobleach quickly. A step-by-step replicable protocol for evaluating the cell viability in IVD will facilitate the evaluation of cell and toxicity-related outcomes of biomechanical testing protocols and IVD regenerative therapies.

Abstract 5824: Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins

Abstract Drug effects may vary depending on cancer cell density. We investigated discrepancies in phenotypes for high density and low-density cultured cancer cells and elucidated relevant variables that influence them. We assessed chemosensitivity as a function of tumor cell density by plating TOV-21G and OVMANA ovarian cancer cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin. CellTiterGlo assays were performed at 72 hours of treatment to determine cisplatin dose response curves. We used ethidium homodimer fluorescence staining and imaging to quantify extent of cell death at differing cell densities of TOV-21G and OVMANA cells treated with cisplatin for 48 and 72hours in 48-well plates. We performed western blot analyses of apoptotic and autophagy proteins from TOV-21G and OVMANA cells that were seeded at high and low cell densities in 24-wellplates. TOV-21G and OVMANA cells plated at higher cell seeding densities displayed less cisplatin sensitivity than TOV-21G and OVMANA cells plated at lower seeding densities, as shown by cisplatin dose response curves. Ethidium homodimer staining of TOV-21G and OVMANA cells treated with cisplatin revealed that cells seeded at lower densities experience a greater amount of cell death that cells seeded at higher densities. Western blot analyses showed that high density TOV-21G cells may express greater amounts of BCL2 and c-FLIP and lower amounts of p62 and NOXA compared to low density cells when normalized for protein expression. Similarly, high density OVMANA cells express greater amounts of c-FLIP andBCL2 and lower amounts of NOXA compared to low density cells when normalized for protein expression. Our experiments reveal that human ovarian cancer cells display different phenotypic changes depending on cell density, causing higher density cells to require increased cisplatin to achieve the same level of effect seen in lower density cells. Ovarian cancer cell resistance to cisplatin is associated with alterations in autophagy and anti-apoptotic protein expression at high cell seeding density. These alterations may also affect chemosensitivity in vivo in settings of differential tumor density, such as single circulating tumor cells compared to clumps of tumor cells, and other settings, such as metastasis. Citation Format: Thomas W. Brown, Ujwal Punyamurtula, Wafik S. El-Deiry. Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5824.

Abstract 4438: Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1

Abstract Integrin receptors have long posed a potentially attractive target for disrupting cancer hallmarks. Promising preliminary findings with integrin inhibition as an adjuvant to chemotherapy have not translated to clinical success. However, the effect of integrin inhibition on tumor-immune cell interactions remains largely unexplored. Further investigation could shed light on a connection between integrin signaling and immune checkpoint expression, opening the path for using integrin inhibitors to sensitize otherwise resistant tumors to immunotherapy. Fluorescently labeled wild-type HCT-116 colorectal cancer cells and TALL-104 T-cells were co-cultured and treated with GLPG0187, a small molecule integrin inhibitor, at various doses. Co-cultures were observed at different time points using fluorescence microscopy and cell counts were quantified using ImageJ software. The co-culture included the cell viability marker ethidium homodimer so the number of viable cells of each type could be determined using colocalization. This assay revealed synergistic cancer cell killing, indicating that integrin inhibition may be sensitizing cancer cells to immune cells. The hypothesized mechanism involves TGF-beta-mediated PD-L1 expression in cancer cells. GLPG0187 has been shown to inhibit the ɑvβ6 integrin receptor, which is responsible for the activation of latent-TGFβ into the active form. By blocking this receptor, there would potentially be a reduction of TGFβ signaling and therefore a reduced expression of PD-L1 leading to an enhanced immune response. To investigate this mechanism, both WT and p53-/- HCT-116 cells were pre-treated with GLPG0187 and subsequently with latent-TGFβ. Western blot analysis demonstrated that the addition of latent-TGFβ increased the expression of PD-L1 in cancer cells. Additionally, a low dose of integrin inhibitor rescued these effects, returning PD-L1 expression back to control levels. However, the higher dose of the drug did not reduce PD-L1 expression. This could potentially be due to off-target effects conflicting with the proposed pathway, however, these findings are still under active investigation. Ongoing proteomic experiments include a larger range of both drug and latent-TGFβ doses. Probing for additional downstream markers of TGFβ and up-stream markers of PD-L1 will help to further elucidate the mechanism. Further co-culture experiments include anti-PD-L1 and anti-PD-1 therapy to investigate the viability of integrin inhibition as an adjuvant to immune checkpoint blockade. Citation Format: William J. MacDonald, Brooke Verschleiser, Lindsey Carlsen, Kelsey E. Huntington, Wafik S. El-Deiry. Bypassing immune evasion in colorectal cancer by integrin inhibition-mediated downregulation of PD-L1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4438.

Use of a Cellulase from Trichoderma reesei as an Adjuvant for Enterococcus faecalis Biofilm Disruption in Combination with Antibiotics as an Alternative Treatment in Secondary Endodontic Infection.

Apical periodontitis is an inflammation leading to the injury and destruction of periradicular tissues. It is a sequence of events that starts from root canal infection, endodontic treatment, caries, or other dental interventions. Enterococcus faecalis is a ubiquitous oral pathogen that is challenging to eradicate because of biofilm formation during tooth infection. This study evaluated a hydrolase (CEL) from the fungus Trichoderma reesei combined with amoxicillin/clavulanic acid as a treatment against a clinical E. faecalis strain. Electron microscopy was used to visualize the structure modification of the extracellular polymeric substances. Biofilms were developed on human dental apices using standardized bioreactors to evaluate the antibiofilm activity of the treatment. Calcein and ethidium homodimer assays were used to evaluate the cytotoxic activity in human fibroblasts. In contrast, the human-derived monocytic cell line (THP-1) was used to evaluate the immunological response of CEL. In addition, the secretion of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 were measured by ELISA. The results demonstrated that CEL did not induce the secretion of IL-6 and TNF-α when compared with lipopolysaccharide used as a positive control. Furthermore, the treatment combining CEL with amoxicillin/clavulanic acid showed excellent antibiofilm activity, with a 91.4% reduction in CFU on apical biofilms and a 97.6% reduction in the microcolonies. The results of this study could be used to develop a treatment to help eradicate persistent E. faecalis in apical periodontitis.

Effects of N-acetylcysteine on growth, viability and reactive oxygen species levels in small antral follicles cultured in vitro

Objective: To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology, as well as on viability, levels of reactive oxygen species (ROS) and meiotic progression of oocytes from in vitro cultured bovine early antral follicles. Methods: Isolated early antral follicles (about 500 μm) were cultured in TCM-199+ alone or supplemented with 1.0, 5.0 or 25.0 mM N-acetylcysteine at 38.5 °C with 5% CO2 for 8 days. Follicle diameters were evaluated at day 0, 4 and 8 of culture. At the end of culture, the levels of ROS, chromatin configuration and viability (calcein-AM and ethidium homodimer-1 staining) were investigated in the cumulus-oocyte complexes. Comparisons of follicle diameters between treatments were performed. Data on percentages of morphologically normal follicles, growth rates and chromatin configuration in different treatments were compared. Results: An increase in follicular diameters after culture in all treatments was observed, except for follicles cultured with 25.0 mM N-acetylcysteine. Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein-AM, and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1. Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments. The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage. Conclusions: N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles, respectively, while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.

Protective Effect of Cimicifuga racemosa (L.) Nutt Extract on Oocyte and Follicle Toxicity Induced by Doxorubicin during In Vitro Culture of Mice Ovaries

This study evaluated the potential of Cimicifuga racemosa (L.) Nutt extract (CIMI) to reduce the deleterious effects of doxorubicin (DOXO) in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. In experiment 1, mice ovaries were cultured in DMEM+ alone or supplemented with 5, 50 or 500 ng/mL CIMI, while in experiment 2, mice ovaries were cultured in DMEM+ alone or supplemented with 5 ng/mL CIMI (better concentration), 0.3 μg/mL DOXO or both. Thereafter, the ovaries were processed for histological (morphology, growth, activation, extracellular matrix configuration and stromal cell density), immunohistochemical (caspase-3) analyses. Follicle viability was evaluated by fluorescence microscopy (ethidium homodimer-1 and calcein) while real-time PCR was performed to analyses the levels of (mRNA for SOD, CAT and nuclear factor erythroid 2-related factor 2 (NRF2) analyses. The results showed that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. The DOXO reduced the percentage of primordial follicles, while the presence of CIMI alone did not influence percentage of primordial follicles. A higher staining for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. In addition, follicles from ovaries cultured with both CIMI and DOXO were stained by calcein, while those follicles cultured with only DOXO were stained with ethidium homodimer-1. Furthermore, ovaries cultured with CIMI or both CIMI and DOXO had higher levels of mRNA for SOD and CAT, respectively, than those cultured with only DOXO. In conclusion, the extract of CIMI protects the ovaries against deleterious effects of DOXO on follicular survival and ovarian stromal cells.