Results for “Cell viability” — Page 8

NASA NTRS2017

Development of Storage Methods for Saccharomyces Strains to be Utilized for In Situ Nutrient Production in Long-Duration Space Missions

Long-duration space missions will benefit from closed-loop life support technologies that minimize mass, volume, and power as well as decrease reliance on Earth-based resupply. A system for In situ production of essential vitamins and nutrients can address the documented problem of degradation of stored food and supplements. Research has shown that the edible yeast Saccharomyces cerevisiae can be used as an on-demand system for the production of various compounds that are beneficial to human health. A critical objective in the development of this approach for long-duration space missions is the effective storage of the selected microorganisms. This research investigates the effects of different storage methods on survival rates of the non-sporulating probiotic S. boulardii, and S. cerevisiae spores and vegetative cells. Dehydration has been shown to increase long-term yeast viability, which also allows increased shelf-life and reduction in mass and volume. The process of dehydration causes detrimental effects on vegetative cells, including oxidative damage and membrane disruption. To maximize cell viability, various dehydration methods are tested here, including lyophilization (freeze-drying), air drying, and dehydration by vacuum. As a potential solution to damage caused by lyophilization, the efficacy of various cryoprotectants was tested. Furthermore, in an attempt to maintain higher survival rates, the effect of temperature during long-term storage was investigated. Data show spores of the wild-type strain to be more resilient to dehydration-related stressors than vegetative cells of either strain, and maintain high viability rates even after one year at room temperature. In the event that engineering the organism to produce targeted nutrient compounds interferes with effective sporulation of S. cerevisiae, a more robust method for improving vegetative cell storage is being sought. Therefore, anhydrobiotic engineering of S. cerevisiae and S. boulardii is being conducted.

life support↗

DOE OSTI2021

Immobilization of transgenic plant cells towards bioprinting for production of a recombinant biodefense agent

Abstract Transgenic rice cells ( Oryza sativa ) producing recombinant butyrylcholinesterase (BChE) as a prophylactic/therapeutic against organophosphate nerve agent poisoning, cocaine toxicity, and neurodegenerative diseases like Alzheimer's were immobilized in a polyethylene glycol‐based hydrogel. The cells were sustained for 14 days in the semi‐solid matrix, undergoing a growth phase from days 0–6, a BChE production phase in sugar‐free medium from days 6–12, and a growth/recovery phase from days 12–14. Throughout this period, the cells maintained similar viability to those in suspension cultures and displayed analogous sugar consumption trends. The rice cells in the hydrogel also produced a significant amount of active BChE, comparable to the levels produced in liquid cultures. A considerable fraction of this BChE was secreted into the media, allowing for easier product separation. To the best of our knowledge, this proof‐of‐concept is the first report of immobilization of recombinant plant cells for continuous production of high‐value heterologous proteins. This work serves as a foundation for further investigation towards plant cell bioprinting and the development of a simple, efficient, robust, modular, and potentially field‐deployable bioreactor system for the manufacture of biologics.

Varma, Anika↗

NASA NTRS1994

Inhibition of murine splenic T lymphocyte proliferation by 2-deoxy-D-glucose-induced metabolic stress

Female Swiss-Webster mice were injected with the glucose analogue 2-deoxy-D-glucose (2-DG), which when administered to rodents induces acute periods of metabolic stress. A single or multiple injections of 2-DG invoked a stress response, as evidenced by increases in serum corticosterone levels. The influence of this metabolic stressor on the blastogenic potential of splenic T lymphocytes was then examined. It was found that one, two, or three injections of 2-DG resulted in depressed T cell proliferative responses, with an attenuation of the effect occurring by the fifth injection. The 2-DG-induced inhibition of T cell proliferation was not attributable to 2-DG-induced cytolysis, as in vitro incubation of naive T cells with varying concentrations of 2-DG did not result in a reduction in cell number or viability, and flow cytometric analysis demonstrated that percentages of CD3, CD4, and CD8 splenic T cells were not altered as a result of 2-DG-induced stress. Incubating naive T cells in varying concentrations of 2-DG resulted in a dose-dependent inhibition of T cell blastogenic potential. Following in vivo exposure to 2-DG, T cell proliferation did not return to normal levels until 3 days after the cessation of 2-DG injections. Administering the beta-adrenergic receptor antagonist propranolol did not reverse the inhibited lymphoproliferation in 2-DG-treated mice. The inhibition in T cell proliferation was not observed, however, in mice that had been adrenalectomized or hypophysectomized and injected with 2-DG.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗

DOE OSTI2023

Controlling the toxicity of biomass-derived difunctional molecules as potential pharmaceutical ingredients for specific activity toward microorganisms and mammalian cells

A biomass-derived difuran compound, denoted as HAH (HMF–acetone–HMF), synthesized by aldol-condensation of 5-hydroxyfurfural (HMF) and acetone, can be partially hydrogenated to provide an electron-rich difuran compound (PHAH) for Diels–Alder reactions with maleimide derivatives. The nitrogen (N) site in the maleimide can be substituted by imidation with amine-containing compounds to control the hydrophobicity of the maleimide moiety in adducts of furans and maleimide by Diels–Alder reaction, denoted as norcantharimides (Diels–Alder adducts). The structural effects on the toxicity of various biomass-derived small molecules synthesized in this manner to regulate biological processes, defined as low molecular weight (≤1000 g mol -1 ) organic compounds, were investigated against diverse microbial and mammalian cell types. The biological toxicity increased when hydrophobic N-substitutions and C[double bond, length as m-dash]C bonds were introduced into the molecular structure. Among the synthesized norcantharamide derivatives, some compounds demonstrated pH-dependent toxicities against specific cell types. Reaction kinetics analyses of the norcantharimides in biological conditions suggest that this pH-dependent toxicity of norcantharimides could arise from retro Diels–Alder reactions in the presence of a Brønsted acid that catalyzes the release of an N-substituted maleimide, which has higher toxicity against fungal cells than the toxicity of the Diels–Alder adduct. These synthetic approaches can be used to design biologically-active small molecules that exhibit selective toxicity against various cell types (e.g., fungal, cancer cells) and provide a sustainable platform for production of prodrugs that could actively or passively affect the viability of infectious cells.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2016

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

yeast↗

NASA NTRS2010

Isolation of Resistance-Bearing Microorganisms

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

Venkateswaran, Kasthuri, J.↗

NASA NTRS2010

Oscillating Cell Culture Bioreactor

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

Freed, Lisa E.↗

DOE OSTI2020

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

Abstract A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2‐µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single‐use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥10 4 colony‐forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 10 4 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1993

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

NASA Discipline Number 04-10↗

DOE OSTI2022

Biocompatibility of NbTaTiVZr with Surface Modifications for Osteoblasts

Here, we report a potential biomedical material, NbTaTiVZr, and the impact of surface roughness on the osteoblast culture and later behavior based on in vitro tests of preosteoblasts. Cell activities such as adhesion, viability, and typical protein activity on NbTaTiVZr showed comparable results with that of commercially pure Ti (CP-Ti). In addition, NbTaTiVZr with a smooth surface exhibits better cell adhesion, viability, and typical protein activity which shows that surface modification can improve the biocompatibility of NbTaTiVZr. This supports the biological evidence and shows that NbTaTiVZr can potentially be evaluated as a biomedical material for clinical use.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

TWIST2 inhibits EMT and induces oxidative stress in lung cancer cells by regulating the FGF21-mediated AMPK/mTOR pathway

Twist related protein 2 (TWIST2) plays an important role in bone development, tumorigenesis, tumour progression and epithelial mesenchymal transition (EMT). At present, there are few reports about the role of TWIST2 in lung cancer, which need to be further explored. Therefore, the purpose of this study is to explore the role and molecular mechanism of TWIST2 in the occurrence and development of lung cancer. The expression of TWIST2 in tissues of patients and cell lines was measured using RT-qPCR and western blotting. MTT and CCK8 assays were used to detect cell proliferation and viability. Western blotting was used to measure the expression of EMT-related proteins, including E-cadherin, N-cadherin, Vimentin and Slug. The results revealed that TWIST2 is lowly expressed in the tissues of lung cancer patients and cell lines. Further studies found that overexpression of TWIST2 significantly induced apoptosis and promoted the expression of E-cadherin, as well as inhibiting the expression of N-cadherin, Vimentin and Slug. More importantly, TWIST2 induced oxidative stress in lung cancer cells. In addition, TWIST2 regulated the FGF21 and AMPK/mTOR signalling pathway, which is involved in the molecular mechanism of the gene in lung cancer cells. We suggest that the mechanism of TWIST2 inhibition of the progression of lung cancer is by regulating the FGF21-mediated AMPK/mTOR signalling pathway.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1999

Induction of Three-Dimensional Growth of Human Liver Cells in Simulated Microgravity

We previously reported that a NASA-developed bioreactor that simulates microgravity environment and creates the unique environment of low shear force and high-mass transfer is conducive for maintaining long term 3-D cell cultures of functional hepatocytes (60 days). However, significant further expansion of liver mass, or the remodeling of liver in vitro was jeopardized by the appearance of apoptotic zones in the center of large cell aggregates. To optimize oxygenation and nutritional uptake within growing cellular aggregates we cultured primary human liver cells (HLC) in a bioreactor in the presence or absence of microcarriers and biodegradable scaffolds. Also, to promote angiogenesis, HLC were cultured with or without microvascular endothelial cells. HLC were harvested from human livers by collagenase perfusion. While microcarriers did not affect cell growth, HLC cultured with biodegradable scaffolds made from polyglycolic acid (PGA) formed aggregates up to 3 cm in length. Culturing cells with PGA scaffolds increased the efficiency of cell self-assembly and the formation of larger cell aggregates. Based on histological evaluation it appears that the degree of apoptotic cells was diminished as compared to cultures without scaffolds. Histology of HLC with PGA-scaffolds revealed cell distribution between the fibers of the scaffolds, and cell-cell and cell-fiber interactions. Analyses of HLC spheroids revealed tissue-like structures comprised of hepatocytes, biliary epithelial cells and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes and bile canaliculi with multiple microvilli and tight cellular junctions. Hepatocytes were further organized into tight clusters surrounded by complex stromal structures and reticulin fibers. Also, we observed higher levels of albumin mRNA expression when hepatocytes were co-cultured with endothelial cells. To evaluate viability and microsomal deethylation activity of hepatocytes we assessed the metabolism of midazolam by gas chromatography. Samples that were collected from HLC cultured in the bioreactor contained higher levels of midazolam metabolites (1-OH-midazolam and 4-OH-midazolam) than samples collected from conventional cultures. In summary, we have shown that co-culture of HLC with endothelial cells and/or culturing in the presence of PGA scaffolds provides additional advantages in maintaining functional activity of 3-D hepatocyte cultures that resemble liver tissue. This cell culture system may facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo and suggest the feasibility of using this approach for pre-clinical metabolic screening of novel drug candidates.

Pellis, Neal R.↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗

NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗

DOE OSTI2024

Confinement performance predictions for a high field axisymmetric tandem mirror

This paper presents Hammir tandem mirror confinement performance analysis based on Realta Fusion's first-of-a-kind model for axisymmetric magnetic mirror fusion performance. This model uses an integrated end plug simulation model including, heating, equilibrium, and transport combined with a new formulation of the plasma operation contours (POPCONs) technique for the tandem mirror central cell. Using this model in concert with machine learning optimization techniques, it is shown that an end plug utilizing high temperature superconducting magnets and modern neutral beams enables a classical tandem mirror pilot plant producing a fusion gain Q > 5. The approach here represents an important advance in tandem mirror design. The high fidelity end plug model enables calculations of heating and transport in the highly non-Maxwellian end plug to be made more accurately. The detailed end plug modelling performed in this work has highlighted the importance of classical radial transport and neutral beam absorption efficiency on end plug viability. The central cell POPCON technique allows consideration of a wide range of parameters in the relatively simple near-Maxwellian central cell, facilitating the selection of more optimal central cell plasmas. These advances make it possible to find more conservative classical tandem mirror fusion pilot plant operating points with lower temperatures, neutral beam energies, and end plug performance requirements than designs in the literature. Despite being more conservative, it is shown that these operating points have sufficient confinement performance to serve as the basis of a viable fusion pilot plant provided that they can be stabilized against MHD and trapped particle modes.

FOS: Physical sciences↗

DOE OSTI2025

Confinement performance predictions for a high field axisymmetric tandem mirror

This paper presents a Hammir tandem mirror confinement performance analysis based on Realta Fusion’s first-of-a-kind model for axisymmetric magnetic mirror fusion performance. This model uses an integrated end plug simulation model including, heating, equilibrium and transport combined with a new formulation of the plasma operation contours (POPCONs) technique for the tandem mirror central cell. Using this model in concert with machine learning optimization techniques, it is shown that an end plug utilizing high temperature superconducting magnets and modern neutral beams enables a classical tandem mirror pilot plant producing a fusion gain Q > 5. The approach here represents an important advance in tandem mirror design. The high-fidelity end plug model enables calculations of heating and transport in the highly non-Maxwellian end plug to be made more accurately. The detailed end plug modelling performed in this work has highlighted the importance of classical radial transport and neutral beam absorption efficiency on end plug viability. The central cell POPCON technique allows consideration of a wide range of parameters in the relatively simple near-Maxwellian central cell, facilitating the selection of more optimal central cell plasmas. These advances make it possible to find more conservative classical tandem mirror fusion pilot plant operating points with lower temperatures, neutral beam energies and end plug performance requirements than designs in the literature. Despite being more conservative, it is shown that these operating points have sufficient confinement performance to serve as the basis of a viable fusion pilot plant provided that they can be stabilized against magnetohydrodynamic and trapped particle modes.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

DOE OSTI2021

Extracellular vesicles promote epithelial-to-mesenchymal transition of lens epithelial cells under oxidative stress

Posterior capsule opacification (PCO), resulting from residual lens epithelial cell (LEC) epithelial–mesenchymal transition (EMT), abnormal proliferation, and migration, is the most common complication of cataract surgery. A recent study determined that extracellular vesicles (EVs) and reactive oxygen species (ROS) regulate the EMT process during cutaneous wound healing and tumour metastasis. However, their underlying mechanism in PCO is unclear. In this study, we examined the secreted EVs from a scratch model in vitro. We found that the production of ROS was increased after mechanical injury, especially at the wound edge, and there was an increased viability of LECs, which can be blocked by diphenyleneiodonium, an NADPH oxidase inhibitor. Cell viability and migration were increased upon treatment with 1 μM H{sub 2}O{sub 2}, but significantly reduced when the concentration of H{sub 2}O{sub 2} increased to 100 μM. Transwell assay showed that both post-surgery LECs and LECs treated with 1 μM H{sub 2}O{sub 2} significantly induced the migration of normal LECs by EV secretion. Extraction and quantification of EVs derived from injured and H{sub 2}O{sub 2}-treated LECs showed a similar increase in production. Co-incubation of EVs from both injured and H{sub 2}O{sub 2}-treated LECs with normal LECs and organ-cultured mouse lenses activated EMT, which was attenuated by a ROS inhibitor. These results suggest that EVs participate in ROS-induced lens EMT, making EVs a potential target for treating PCO.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1997

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

NASA Center ARC↗