Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “cell free”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8

Improving Satellite Compatible Microdevices to Study Biology in Space

The technology for biology in space lags far behind the gold standard for biological experiments on Earth. To remedy this disparity, the Rothschild lab works on proof of concept, prototyping, and developing of new sensors and devices to further the capabilities of biology research on satellites. One such device is the PowerCell Payload System. One goal for synthetic biology in aiding space travel and colonization is to genetically engineer living cells to produce biochemicals in space. However, such farming in space presupposes bacteria retain their functionality post-launch, bombarded by radiation, and without the 1G of Earth. Our questions is, does a co-culture of cyanobacteria and protein-synthesizing bacteria produce Earth-like yields of target proteins? Is the yield sensitive to variable gravitational forces? To answer these questions, a PowerCell Payload System will spend 1 year aboard the German Aerospace Center's Euglena and Combined Regenerative Organic-food Production In Space (Eu:CROPIS) mission satellite. The PowerCell system is a pair of two 48-well microfluidic cards, each well seeded with bacteria. The system integrates fluidic, thermal, optical, electronic, and control systems to germinate bacteria spores, then measure the protein synthesized for comparison to parallel experiments conducted on the Earth. In developing the PowerCell Payload, we gained insight into the shortcomings of biology experiments on satellites. To address these issues, we have started three new prototyping projects: 1) The development of an extremely stable and radiation resistant cell-free system, allowing for the construction of proteins utilizing only cell components instead of living cells. This can be lyophilized on a substrate, like paper. (2) Using paper as a microfluidic platform that is flexible, stable, cheap, and wicking. The capillary action eliminates the need for pumps, reducing volume, mass, and potential failing points. Electrodes can be printed on the paper to sense for biochemicals. (3) Developing a modular, semi-autonomous microfluidic device that can be easily adapted for a variety of common biological experiments. This versatility will allow for quicker and cheaper experimentation. These improvements to satellite experiment platforms have the potential to radically increase the return from NASA's biological and field studies with reduced development time, mass, and cost with increased robustness data and interpretation.

biology in spac↗

Shear and mixing effects on cells in agitated microcarrier tissue culture reactors

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from: (1) direct interaction between microcarriers and turbulent eddies; (2) collisions between microcarriers in turbulent flow; and (3) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Impeller collisions occur when beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture reactors are discussed.

Cherry, Robert S.↗

Electrode kinetics of a water vapor electrolysis cell

The anodic electrochemical behavior of the water vapor electrolysis cell was investigated. A theoretical review of various aspects of cell overvoltage is presented with special emphasis on concentration overvoltage and activation overvoltage. Other sources of overvoltage are described. The experimental apparatus controlled and measured anode potential and cell current. Potentials between 1.10 and 2.60 V (vs NHE) and currents between 0.1 and 3000 mA were investigated. Different behavior was observed between the standard cell and the free electrolyte cell. The free electrolyte cell followed typical Tafel behavior (i.e. activation overvoltage) with Tafel slopes of about 0.15, and the exchange current densities of 10 to the minus 9th power A/sq cm, both in good agreement with literature values. The standard cell exhibitied this same Tafel behavior at lower current densities but deviated toward lower than expected current densities at higher potentials. This behavior and other results were examined to determine their origin.

Jacobs, G.↗

Design, Prototyping, and Testing of a Novel Flowpath with an Array of Six 3D Matrix Vitvo Bioreactors for the NASA Bioculture System

The NASA Bioculture System is an advanced cell culture closed-loop system containing highly automated flowpaths designed to conduct long term biology experiments on ISS with earth remote controllable medium flow, temperature, gas composition, medium exchange, cell sampling and fixation. This technology was already demonstrated with successful cardiomyocyte and osteocyte cultures experiments onboard the ISS and is now supporting NASA PI science. The Bioculture System, however, can only support 10 cassettes with disposable flowpaths, each containing a single hollow fiber bioreactor with a culture capacity of about 2ml. This constraint not only severely limits the number of investigators that can conduct experiments in space, but also subjects the experiments to limitations in the number of replicates and conditions that can be studied. To address these limitations, we sought a novel design solution to maximize the number of separate bioreactor cultures and volume that can be conducted simultaneously. To this end we designed, prototyped, and are now testing a six-Vitvo 3D Matrix 2ml bioreactor insert that replaces the conventional Bioculture System hollow fiber bioreactor. This design will allow the Bioculture System to support up to 60 different bioreactors and samples at once. Specifically, the novel gas-tight containment housing insert contains six COTS Rigenerand VITVO bioreactors stacked on each side of a heat sink powered by the existing heating element and pair of temperature sensors. Medium will be distributed into each bioreactor's cell-free chamber via its built-in Luer connector, then across the 3D matrix to the cell chamber, dissipating laminar flow and limiting fluid shear stresses that might mechanostimulate cell cultures. Gas (5% CO2 in air) will be supplied directly to the bioreactor gas-tight housing for exchange via the bioreactor flat-surface gas-permeable membranes, eliminating the need for the existing Bioculture System cassette oxygenator. If successfully implemented on ISS, this new multi-bioreactor insert for the Bioculture System has the potential to make real-time cell science experimentation in space more efficient and accessible to more investigators.

Ma, Kristin R.↗

Facile synthesis of Co(OH)2 nanoneedle arrays grown on stainless steel for industrial electrochemical oxygen evolution reaction

The electrochemical oxygen evolution reaction (OER) is a critical half-reaction in a variety of energy conversion and storage applications, however, OER suffers from sluggish kinetics due to the four proton-electron transfer processes. To remedy this, interfacial engineering proves an effective strategy to design high active OER catalysts. Herein, we report a facile synthesis of Co(OH)2 nanoneedle (Co-NN) arrays grown on stainless steel (SS) via a one-step hydrothermal reaction, and the resultant impressive OER performance. Particularly, the Co-NN/SS needs 267, 307, and 576 mV overpotentials to reach 10, 100, and 1000 mA cm−2 in 1 M KOH & room temperature, and the overpotentials drop to 199, 257, and 283 mV to achieve the same current densities in 30 wt% KOH & 80 °C. Additionally, an alkaline water electrolysis (AWE) cell coupled with Co-NN/SS anode and PtRu/NiMo cathode only requires 1.65 and 1.73 V iR-free cell voltage to reach 1.0 and 2.0 A cm−2, respectively. The sterling OER performance could be attributed to four possible reasons, the Fe in SS surface tailoring the electronic properties of Co(OH)2, the mixed metal oxides on SS surface accelerating surface reconstruction of Co(OH)2 nanoneedles into CoOOH active species, a Ni-rich surface layer formation cooperatively boosting the OER activity, and a local electric field effect concentrating reactants on the tip surface and accelerating the mass transfer. This work provides a facile approach for synthesizing highly efficient OER catalysts for industrial applications by interfacial engineering.

Lyu, Xiang [ORNL] (ORCID:0000000208673248)↗

Strategies to achieve high productivity, high conversion, and high yield in yeast fermentation of algal biomass hydrolysate

The conversion of carbohydrates in biomass via fermentation is an important component of an overall strategy to decarbonize the production of fuels and chemicals. Owing to the cost and resources required to produce biomass hydrolysates, the economic and environmental sustainability of these fermentation processes requires that they operate with high yields, sugar conversion, and productivity. Immobilized-cell technology in a continuous bioprocess can achieve significantly higher volumetric productivities than is possible from standard batch fermentation using free cells. Here, we demonstrate approaches for improvement of ethanol yield from algal hydrolysates and a mock hydrolysate medium. Saccharomyces cerevisiae was immobilized in alginate and incorporated into a two-column immobilized cell reactor system. Furthermore, the yeast quorum-sensing molecule, 2-phenylethanol, was added to improve ethanol yield by restricting growth and diverting sugar to ethanol. The bioreactor system could achieve high ethanol volumetric productivity (>20 g/ Lreactor ·h) and high glucose conversion (>99%) in mock hydrolysate, while the addition of 0.2% 2-phenylethanol resulted in 4.9% higher ethanol yield. With an algal hydrolysate of <10 g/L sugar, the ethanol volumetric productivity reached 9.8 g/ Lreactor ·h, and the addition of 0.2% 2-phenylethanol increased the ethanol yield by up to 7.4%. These results demonstrate the feasibility of novel strategies to achieve sustainability goals in biomass conversions.

59 BASIC BIOLOGICAL SCIENCES↗

Cloning higher plants from aseptically cultured tissues and cells

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

Krikorian, A. D.↗

Topological Considerations in Electrolyte Additives for Passivating Silicon Anodes with Hybrid Solid–Electrolyte Interphases

Unlike most anodes used in high energy density batteries, lithiated Si does not form long-lasting passivating solid-electrolyte interphases (SEI) during formation or on charge due to SEI delamination, reconstruction, or dissolution. As a result, electrolyte degradation is continuous and results in a permanent loss of the Li inventory, shortening the useful life of the battery. Here, in this study, we show that perfluoroether electrolyte additives featuring either sulfonyl fluorides or trifluorovinyl ethers, when introduced in prescribed amounts to locally superconcentrated electrolytes, exhibit preferential reactivity at Si during formation due to their higher reduction potential than salts and solvents, creating a hybrid SEI that is simultaneously enriched with LiF and organics tethered to the reactive functionality. While both reactive motifs are effective in creating a hybrid SEI, perfluoroether additives bearing sulfonyl fluorides show more substantial integration. More important, however, is the combined influence of additive topology on anchoring efficacy and tether flexibility between anchoring sites on SEI resiliency. Top-performing Si|LFP cells featuring ditopic additive-enriched SEI improve capacity retention by as much as 45% over 100 cycles when compared to additive-free cells.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Rapid T cell engineering to counter emerging threats (Full Technical Report)

Instead of genetic engineering, we sought to determine if a rapid method of membrane protein delivery could generate functional CAR-T cells in a rapid, safer, cost-effective manner. Using cell free protein synthesis, we generated CAR proteins embedded in a nanodisc, and thus effectively solubilized the protein. We demonstrated the first functional CAR proteins outside of a cellular context. We next tested uptake into immune cells and found extremely high uptake into T cells and multiple other populations of immune cells. We found some evidence of cytotoxicity in vitro conferred by the nanodisc delivered protein.

59 BASIC BIOLOGICAL SCIENCES↗

Cells, embryos and development in space

Work continues to focus on the demonstrable totipotency of cultured somatic cells of various higher plants and has examined the conditions which regulate this propensity to be controllably released. This was done with special reference to cells obtained from cultured explants of daylily and carrot. For purposes of identifying the variables in question, work was carried out almost exclusively in liquid media. The events that intervene between the aseptic isolation of tissue explants, the culture of small derived units and free cells and the propagation in large numbers of adventive or somatic embryos to plantlets were traced and certain definitive stages at which control is exercised were identified. In daylily, morphologically competent units are now propagated with a high degree of precision in rotated liquid cultures in bulk, and under the conditions of continuous neutralized gravity, the development progresses so that embryo-plantlets are obtained.

Krikorian, A. D.↗

Revisiting the Impact of Anion Selection on Sulfur Redox Reaction Kinetics for High Sulfur Loading Lithium–Sulfur Batteries

Lithium bis(trifluoromethane)sulfonimide (LiTFSI) is widely used in lithium–sulfur (Li–S) battery electrolytes due to its stability with lithium polysulfides (LiPSs) and moderate compatibility with lithium metal anodes. However, LiTFSI presents environmental concerns due to its association with per- and polyfluoroalkyl substances (PFAS), which are environmentally persistent and potentially toxic, raises sustainability concerns. This research also reveals that LiTFSI limits sulfur redox reactions (SRRs), making it less effective than other lithium salts. Additionally, some salts previously considered incompatible with Li-S systems due to their reactivity with LiPSs are demonstrated to perform effectively. For the first time, a protective, porous cathode electrolyte interphase (CEI) formed in situ through reactions between salt anions and LiPS is reported. The cells delivered a high specific capacity of 1230.8 mAh g −1 at 0.05 C with a sulfur loading of ≈6 mg cm −2 , limited lithium anode, maintaining a capacity retention of 76.2% after 100 cycles at 0.1 C. Under harsh conditions, such as high sulfur loading, lean electrolyte conditions (3 µL mg −1 ), and in anode-free cells, the cells continued to deliver outstanding capacity. This work provides valuable guidelines for understanding and selecting lithium salts to advance electrolyte design for Li–S batteries.

high loading batteries↗

Isobutanol production freed from biological limits using synthetic biochemistry

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L –1 h –1 , a titer of 275 g L –1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

3D Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent VZV Infection

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

Goodwin, Thomas J.↗

Exosomes secreted from mesenchymal stem cells mediate the regeneration of endothelial cells treated with rapamycin by delivering pro-angiogenic microRNAs

Highlights: • MSC-derived exosomes play an important role in promoting endothelial regeneration treated with rapamycin in vitro. • A distinct class of exosomal miRNAs is identified to be tightly related to endothelial regeneration. • The therapeutic effects of MSC-exosomes may be ascribed to the delivery of pro-angiogenic miRNAs to endothelial cells. • MSC-exosomes may serve as a cell-free therapy for clinical treatment of delayed endothelial healing after DES implantation. Delayed endothelial healing after drug eluting stent (DES) implantation is a critical clinical problem in treatment of coronary artery diseases. Exosomes exhibit proangiogenic potential in a variety of ischemic diseases. However, the association of exosomes with endothelial regeneration after DES implantation has been rarely reported. In this study, we aimed to investigate the therapeutic effects of mesenchymal stem cell (MSC)-derived exosomes on endothelial cells treated with rapamycin and explore the potential mechanisms of MSC-derived exosomes in promoting endothelial regeneration. Exosomes were isolated from MSCs by ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot assay. The in vitro effects of MSC-derived exosomes on the proliferation and migration of endothelial cells treated with rapamycin were evaluated by integrated experiment, cell counting kit-8, scratch, tube formation, and transwell assays. And the apoptosis of rapamycin-induced endothelial cells loaded with MSC-derived exosomes was detected using TUNEL and Annexin-V FITC and PI double-staining assays. The microRNA (miRNA) cargo of MSC-derived exosomes was identified by high-throughput RNA sequencing. Pro-angiogenic miRNAs and key pathways were further characterized. Our results indicated that MSC-derived exosomes could be ingested into umbilical vein endothelial cells (HUVECs) and significantly enhanced cell proliferation rate, migratory and tube-forming capabilities in vitro. MSC-derived exosomes also inhibited the apoptosis of HUVECs induced by rapamycin. A distinct class of exosomal miRNAs was further identified, including six miRNAs tightly related to neovasculogenesis. Silencing the expression of exosomal miRNA-21–5p and let-7c-5p attenuated the pro-proliferative and pro-migratory capacity of MSC-derived exosomes. Moreover, functional enrichment analysis indicated that metabolic pathways might contribute to reendothelialization. This study highlights a proregenerative effect of MSC-derived exosomes in vitro, which may be partly explained by the delivery of pro-angiogenic miRNAs to endothelial cells.

60 APPLIED LIFE SCIENCES↗

Diatom volatile organic compound production is driven by diel metabolism and the cell cycle

Introduction: Volatile organic compounds (VOCs) are small, low-vapor-pressure molecules emitted from the surface ocean into the atmosphere. In the atmosphere, VOCs can change OH reactivity and condense onto particles to become cloud condensation nuclei. VOCs are produced by phytoplankton, but the conditions leading to VOC accumulation in the surface ocean are poorly understood.Methods: In this study, VOC accumulation was measured in real time over a 12 h day−12 h night cycle in the model diatom Phaeodactylum tricornutum during exponential growth.Results: Sixty-three m/z signals were produced in higher concentrations than in cell-free controls. All VOCs, except methanol, were continuously produced over 24 h. All VOCs accumulated to higher concentrations during the day compared to the night, and 11 VOCs exhibited distinct accumulation patterns during the morning hours. Twenty-seven VOCs were associated with known metabolic pathways in P. tricornutum, with most VOCs involved in amino acid and fatty acid metabolism.Discussion: Patterns of VOC production were strongly associated with diel shifts in cell physiology and the cell cycle. Diel VOC production patterns give a fundamental understanding of the first steps in VOC accumulation in the surface ocean.

Biological and medical sciences↗

Stereoselectivity in Polyphenol Biosynthesis

Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.

Lewis, Norman G.↗