SEARCH · Search bioRxiv

Results for “Bioengineering”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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

At least 235 records · Page 13Linked to original sources

bioRxiv2017

A synthetic stroma-free germinal center niche using material-surface driven polyvalent signaling efficiently induces antigen-specific humoral immunity ex vivo

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial \"follicular T helper cells (TFH)\" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40; and by mimicking artificial \"follicular dendritic cells (FDC)\" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

bioengineering↗

bioRxiv2017

Fundamentals of injection molding for microfluidic cell-based assays

Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. Rapid injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

bioengineering↗

bioRxiv2017

Zwitterionic hydrogels modulate the foreign body response in a modulus-dependent manner

Reducing the foreign body response (FBR) to implanted biomaterials will enhance their in vivo performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this application due to their low cost and ease of use. PEG hydrogels can elicit chronic inflammation upon implantation, but recent evidence has suggested that extremely hydrophilic, zwitterionic hydrogels can reduce the FBR to particles and gels. To expand on this approach, we synthesized hydrogels of co-monomers PEG and the zwitterion phosphorylcholine (PC) to quantify the combinatorial effects of modulus and hydrophilicity on the FBR. Surprisingly, hydrogels with the highest amount of zwitterionic co-monomer elicited the highest FBR we observed. Lowering the hydrogel modulus (165 kPa to 3 kPa), or PC content (20 wt% to 0 wt%), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment to these hydrogels. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design.\n\nHighlightsO_LIModulus and zwitterion content independently modulate the foreign body response to soft hydrogels\nC_LIO_LISoft PEG hydrogels synthesized with the zwitterionic PC co-monomer are pro-inflammatory as modulus is increased\nC_LIO_LIThe chemical and physical properties of hydrogels influence the foreign body response via macrophage recruitment and protein adsorption\nC_LI

bioengineering↗

bioRxiv2017

High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

Construction of genetic variant libraries with phenotypic measurement is central to advancing todays functional genomics, and remains a grand challenge. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions and insertions), along with methods for tracking their fitness en masse. We demonstrate the utility of our approach in performing focused analysis of hundreds of mutants of a single protein and in investigating the biological function of an entire family of poorly characterized genetic elements. Our platform allows fundamental biology questions to be investigated in a quick, easy and affordable manner.

bioengineering↗

bioRxiv2017

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs - BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

bioengineering↗

bioRxiv2017

A radial pillar device (RAPID) for continuous and high-throughput separation of multi-sized particles

Pillar-based microfluidic sorting devices are preferred for isolation of rare cells due to their simple designs and passive operation. Dead-end pillar filters can efficiently capture large rare cells, such as, circulating tumor cells (CTCs), nucleated red blood cells (NRBCs), etc., but they get clogged easily. Cross flow filters are preferred for smaller rare particles (e.g. separating bacteria from blood), but they need additional buffer inlets and a large device footprint for efficient operation. We have designed a new microparticle separation device i.e. Radial Pillar Device (RAPID) that combines the advantages of dead-end and cross flow filters. RAPID can simultaneously isolate both large and small rare particles from a mixed population, while functioning for several hours without clogging. We have achieved simultaneous separation of 10{micro}m and 2{micro}m polystyrene particles from a mixture of 2 {micro}m, 7 {micro}m and 10 {micro}m beads. RAPID achieved average separation purity and recovery in excess of 90%. The throughput of our device ( 3ml/min) is 10 and 100 times higher compared to cross flow and dead-end filters respectively, thereby justifying the name RAPID.

bioengineering↗

bioRxiv2017

Control of Thiol-Maleimide Reaction Kinetics in PEG Hydrogel Networks

Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous, because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buffers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications.

bioengineering↗

bioRxiv2017

Enzymatic synthesis of gene-length single-stranded DNA

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxic ity, yet ssDNA synthesis strategies are limited by the maximum length attainable, as well control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kb using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and imaging applications.

bioengineering↗

bioRxiv2017

Aqueous ethanol permeation across Sterlitech flat sheet aquaporin membranes

Aquaporin (Aqp) embedded membranes have become a topic of recent research due to the superior selectivity of Aqp to water and its acceptable flux. Most researchers have utilized Aqp membranes for water purification purposes such as reverse and forward osmosis desalination of sea and brackish water and wastewater treatment. This paper investigated the permeation of aqueous ethanol across commercial aquaporin membranes for possible applications in ethanol dehydration in the process and biofuel refining industries. The study revealed that rather than being selectively rejected, ethanol was able to diffuse through the membrane with minimal changes in composition from feed to permeate, even at low feed concentrations of ethanol. The total flux across the membranes were shown to average 3.86 liters/m2h (LMH). The presence of ethanol increased the relative rate of water transport across the membrane. When comparing total flux of pure water to aqueous ethanol solutions, the flux was higher for solutions containing any ethanol.

bioengineering↗

bioRxiv2017

Quantitative in vivo evaluation of the reverse β-oxidation pathway for fatty acid production in Saccharomyces cerevisiae

Production of fatty acids using engineered Saccharomyces cerevisiae cells is a challenging task in part due to low efficiency of the native fatty acid biosynthesis pathway. One option for improving production efficiency relies on exploring alternative fatty acid production pathways with either improved kinetics, thermodynamics or yield properties.\n\nIn this work, we explored the reverse {beta}-oxidation pathway as an alternative pathway for free fatty acid production. Different gene combinations and analysis methods were tested for assessing pathway efficiency when expressed in the yeast Saccharomyces cerevisiae. Even though different alternatives were tested, quantitative analysis showed no improvement or major change in fatty acid production of the tested strains in our conditions. This lack of improvement suggests that the tested pathway designs and constructs are either nonfunctional in the tested conditions or the resulting strains lack a metabolic driving force that is needed for a functional pathway.\n\nWe conclude that expression of the reverse {beta}-oxidation pathway in S. cerevisiae poses many challenges when compared to expression in bacterial systems. These factors gravely hinder development efforts and success rate for producing fatty acids through this pathway.

bioengineering↗

bioRxiv2017

Restoration longevity among geriatric and adult special needs patients

This study aimed to describe the survival trajectory of dental restorations placed in an outpatient population of geriatric and adult special needs patients over a 15-year span, with particular interest in longevity of subsequent restorations in teeth that received multiple restorations over time. Dental restorations of different types and sizes in patients age [≥]65 years treated between 2000-14 at the University of Iowa, College of Dentistry were followed until they incurred an event (i.e., restoration replacement, extraction of the tooth, or endodontic treatment of the tooth). Survival analysis and extended Cox regression models were used to generate hazards ratios for selected predictor variables. A total of 9184 restorations were followed in 1551 unique patients. During the follow-up period, 28.7% of these restorations incurred an event; and overall the restorations had a median lifespan of 6.25 years. In multivariable regression models, after controlling for gender and age, composite restorations and greater number of restoration surfaces were associated with higher risks of failure; and the initial restoration recorded in the database for each subject tended to have lower risk of failure than restorations placed later that included any of those same surfaces. This information potentially could be helpful to elderly patients considering various restorative treatment options during the dental treatment planning and informed consent process.

bioengineering↗

bioRxiv2017

Automated Reactive Accelerated Aging for Rapid In Vitro Evaluation of Neural Implants Performance

ObjectiveNovel therapeutic applications for neural implants require miniaturized devices. Pilot clinical studies suggest that rapid failure of the miniaturized neural implants in the body presents a major challenge for this type of technology. Miniaturization imposes stricter requirements for reliability of materials and designs. Evaluation of neural implant performance over clinically relevant timescales presents time-and cost-prohibitive challenges for animal models.\n\nApproachIn vitro reactive accelerated aging (RAA) was developed to expedite durability testing of these devices. RAA simulates an aggressive physiological environment associated with an immune response and implicated in device failure. It uses hydrogen peroxide, which mimics reactive oxygen species (ROS), and high temperature to accelerate chemical reactions that lead to device degradation. RAA accurately simulates the degradation pattern of neural implants observed in vivo, but requires daily maintenance and is prone to variability in performance.\n\nMain resultsThis work introduces automated reactive accelerated aging (aRAA) that is compatible with multiplexing. The core of aRAA is electrochemical detection for feedback control of hydrogen peroxide concentration, implemented with simple off-the shelf components.\n\nSignificanceaRAA allows multiple parallel experiments for a high-throughput optimization of reactive aging conditions to more quickly and more rigorously simulate the in vivo environment. aRAA is a cost-effective tool for rapid in vitro evaluation of durability of neural implants, ultimately expediting the development of a new generation of miniaturized devices with long functional lifespans.

bioengineering↗

bioRxiv2017

Inter-slice motion correction using spatiotemporal interpolation for functional magnetic resonance imaging of the moving fetus

Fetal motion continues to be one of the major artifacts in in-utero functional MRI; interestingly few methods have been developed to address fetal motion correction. In this study, we propose a robust method for motion correction in fetal fMRI by which both inter-slice and inter-volume motion artifacts are jointly corrected. To accomplish this, an original volume is temporally split into odd and even slices, and then voxel intensities are spatially and temporally interpolated in the process of image registration. Our experimental data demonstrate that our method was more effective in correcting fetal motion artifact compared to traditional motion correction methods.

bioengineering↗

bioRxiv2017

Light-Activated Cell Identification and Sorting (LACIS): A New Method to Identify and Select Edited Clones on a Microfluidic Device

CRISPR-Cas9 gene editing has revolutionized cell engineering and promises to open new doors in gene and cell therapies. Despite improvements in the CRISPR-editing molecular toolbox in cell lines and primary cells, identifying and purifying properly edited clones remains slow, laborious and low-yield. Here, we establish a new method that uses cell manipulation on a chip with Opto-Electronic Positioning (OEP) technology to enable clonal isolation and selection of edited cells. We focused on editing CXCR4 in primary human T cells, a gene that encodes a co-receptor for HIV entry. T cells hold significant potential for cell-based therapy, but the gene-editing efficiency and expansion potential of these cells is limited. We describe here a method to obviate these limitations. Briefly, after electroporation of cells with CXCR4-targeting Cas9 ribonucleoproteins (RNPs), single T cells were isolated on a chip, where they proliferated over time into well-resolved colonies. Phenotypic consequences of genome editing could be rapidly assessed on-chip with cell-surface staining for CXCR4. Furthermore, independent of phenotype, individual colonies could be identified based on their specific genotype at the 5-10 cell stage. Each colony was split and sequentially exported for immediate on-target sequencing and validation, and further off-chip clonal expansion of the validated clones. We were able to assess single-clone editing efficiencies, including the rate of monoallelic and biallelic indels or precise nucleotide replacements. This new method will enable identification and selection of perfectly edited clones within 10 days from Cas9-RNP introduction in cells based on the phenotype and/or genotype.

bioengineering↗

bioRxiv2017

Minimization of muscle activation costs demanded from mechanical work and power accounts for selection of duty factor in human gaits.

Duty factor DF - the proportion of a stride a foot is in contact with the ground - is of fundamental mechanical importance, and is often viewed as a defining kinematic parameter distinguishing walking (DF>0.5) from running (DF<0.5). However, the mechanical and/or physiological considerations that determine duty factor are not well understood. Here, a model is proposed that focuses on the interaction between mechanical and muscle costs to account for duty factor in human gaits. It minimizes the activation costs associated with mechanical work or power demand during muscle contraction (whichever is the more demanding). Empirical observations match model predictions using initial muscle parameters over a range of speeds within gaits. However, a better match is achieved - and a better account for the walk-run transition - with tuned muscle parameters. The tuned model is validated with responses in duty factor to walking at a range of imposed, unnatural step frequencies.

bioengineering↗

bioRxiv2017

Optical excitation and detection of neuronal activity

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength changes with nanometer scale sensitivity. We found that optical pathlength fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Quantitative phase imaging enables label-free detection of enhanced intracellular activities induced by optogenetic stimulation.\n\nC_FIG_DISPLAY

bioengineering↗

bioRxiv2017

Automated microuidic cell culture of stem cell derived dopaminergic neurons in Parkinson’s disease

Parkinsons disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons leading mainly to motor dysfunction, but also other non-motor symptoms. The development of human in vitro cellular models with similar phenotypic characteristics to selectively vulnerable neurons is a major challenge in Parkinsons disease research. We constructed a fully automated cell culture platform optimised for long-term maintenance and monitoring of induced pluripotent stem cell derived neurons in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess neuronal activity. Using this system, we continuously monitored the differentiation of Parkinsons disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of a fully automated Organ-on-a-Chip culture and enables a versatile array of in vitro experiments for patient-specific disease modelling.

bioengineering↗

bioRxiv2017

Combinatorial Drug Discovery in Nanoliter Droplets

Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here we report a high-throughput system for nanoliter-scale phenotypic screening that stabilizes a chemical library in nanoliter droplet emulsions and automates the formulation of chemical combinations en mass using parallel droplet processing. We apply this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against E. coli, a model Gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against Gram-positive bacteria but are not effective by themselves to resolve Gram-negative infections.

bioengineering↗