by Keyword: injection

Yeste, J, Azagra, M, Ortega, MA, Portela, A, Matajsz, G, Herrero-Gómez, A, Kim, Y, Sriram, R, Kurhanewicz, J, Vigneron, DB, Marco-Rius, I, (2023). Parallel detection of chemical reactions in a microfluidic platform using hyperpolarized nuclear magnetic resonance Lab On A Chip 23, 4950-4958

The sensitivity of NMR may be enhanced by more than four orders of magnitude via dissolution dynamic nuclear polarization (dDNP), potentially allowing real-time, in situ analysis of chemical reactions. However, there has been no widespread use of the technique for this application and the major limitation has been the low experimental throughput caused by the time-consuming polarization build-up process at cryogenic temperatures and fast decay of the hyper-intense signal post dissolution. To overcome this limitation, we have developed a microfluidic device compatible with dDNP-MR spectroscopic imaging methods for detection of reactants and products in chemical reactions in which up to 8 reactions can be measured simultaneously using a single dDNP sample. Multiple MR spectroscopic data sets can be generated under the same exact conditions of hyperpolarized solute polarization, concentration, pH, and temperature. A proof-of-concept for the technology is demonstrated by identifying the reactants in the decarboxylation of pyruvate via hydrogen peroxide (e.g. 2-hydroperoxy-2-hydroxypropanoate, peroxymonocarbonate and CO2). dDNP-MR allows tracing of fast chemical reactions that would be barely detectable at thermal equilibrium by MR. We envisage that dDNP-MR spectroscopic imaging combined with microfluidics will provide a new high-throughput method for dDNP enhanced MR analysis of multiple components in chemical reactions and for non-destructive in situ metabolic analysis of hyperpolarized substrates in biological samples for laboratory and preclinical research.

JTD Keywords: injections, nmr, pyruvate, Polarization

Picazo-Frutos, R, Stern, Q, Blanchard, JW, Cala, O, Ceillier, M, Cousin, SF, Eills, J, Elliott, SJ, Jannin, S, Budker, D, (2023). Zero- to Ultralow-Field Nuclear Magnetic Resonance Enhanced with Dissolution Dynamic Nuclear Polarization Analytical Chemistry 95, 720-729

Zero- to ultralow-field nuclear magnetic resonance is a modality of magnetic resonance experiment which does not require strong superconducting magnets. Contrary to conventional high-field nuclear magnetic resonance, it has the advantage of allowing high-resolution detection of nuclear magnetism through metal as well as within heterogeneous media. To achieve high sensitivity, it is common to couple zero-field nuclear magnetic resonance with hyperpolarization techniques. To date, the most common technique is parahydrogen-induced polarization, which is only compatible with a small number of compounds. In this article, we establish dissolution dynamic nuclear polarization as a versatile method to enhance signals in zero-field nuclear magnetic resonance experiments on sample mixtures of [13C]sodium formate, [1-13C]glycine, and [2-13C]sodium acetate, and our technique is immediately extendable to a broad range of molecules with >1 s relaxation times. We find signal enhancements of up to 11,000 compared with thermal prepolarization in a 2 T permanent magnet. To increase the signal in future experiments, we investigate the relaxation effects of the TEMPOL radicals used for the hyperpolarization process at zero- and ultralow-fields.

JTD Keywords: injection, liquids, relaxation, times, Spectroscopy

Clua-Ferre, L, De Chiara, F, Rodriguez-Comas, J, Comelles, J, Martinez, E, Godeau, AL, Garcia-Alaman, A, Gasa, R, Ramon-Azcon, J, (2022). Collagen-Tannic Acid Spheroids for beta-Cell Encapsulation Fabricated Using a 3D Bioprinter Advanced Materials Technologies 7, 2101696

Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.© 2022 The Authors. Advanced Materials Technologies published by Wiley‐VCH GmbH.

JTD Keywords: 3d bioprinter, beta-cell, biomaterial, collagen, encapsulation, mechanics, microspheres, survival, 3d bioprinter, ?-cell, Advanced material technologies, Biocompatibility, Cell encapsulations, Cells, Collagen, Cross-linking, Cytology, Drug delivery, Encapsulation, Fabrication, Flavonoids, Gelation, In-vitro, Insulin injections, Insulin release, Microspheres, Tannic acid, Tannins, Throughput, Tissue grafts, Type 1 diabetes, Β‐cell

Bonilla-Pons, SA, Nakagawa, S, Bahima, EG, Fernández-Blanco, A, Pesaresi, M, D'Antin, JC, Sebastian-Perez, R, Greco, D, Domínguez-Sala, E, Gómez-Riera, R, Compte, RIB, Dierssen, M, Pulido, NM, Cosma, MP, (2022). Müller glia fused with adult stem cells undergo neural differentiation in human retinal models Ebiomedicine 77, 103914

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

Lozano-Hernández, N, Llanos, GP, Comet, CS, del Valle, LJ, Puiggali, J, Fontdecaba, E, (2022). Micro- and Nanotexturization of Liquid Silicone Rubber Surfaces by Injection Molding Using Hybrid Polymer Inlays Macromolecular Materials And Engineering 307, 2100741

Micro- and nanotexturization of surfaces can give to the parts different advanced functionalities, such as superhydrophobicity, self-cleaning, or antibacterial capabilities. These advanced properties in combination with the biocompatibility of Liquid Silicone Rubber are an interesting approach for obtaining high-performance medical devices. The industrial production of surface textures in polymeric materials is through the replication technique, and the best option to attain a high production rate is injection molding. Moreover, its low viscosity during processing can provide an accurate replication capacity by the easy filling by capillarity of the microtextures. An innovative replicating technique for Liquid Silicone Rubber is presented by studying the replication of different shaped textures within a diameter range of between 2 and 50 mu m. The copying process consists in the overmolding of a textured polymeric inlay obtained by nanoimprint lithography. At the end of the process, a textured part is obtained, while the imprinted film remains in the mold. The injection molding parameters are optimized to increase the replication accuracy, and their effect on texture replicability is analyzed and discussed. Finally, it is shown that the textured surfaces improve their wettability behavior, which is a necessary and important characteristic in the development of biomedical devices.

JTD Keywords: Cross-linking density, Injection molding, Microtextures, Nanoimprint lithography, Polymeric inlays, Silicone rubber, Stamp, Wettability

Georgiev, VN, Avalos-Padilla, Y, Fernàndez-Busquets, X, Dimova, R, (2022). Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles Bio Protoc 12, e4328

The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle. Copyright: © 2022 The Authors.

JTD Keywords: adhesion, budding, electroformation, escrt-iii, exosomes, extracellular vesicles, giant unilamellar vesicle (guv), light, microinjection, microparticles, plasma, Adhesion, Budding, Escrt-iii, Extracellular vesicles, Giant unilamellar vesicle (guv), Membrane, Microinjection

Chausse, V, Schieber, R, Raymond, Y, Ségry, B, Sabaté, R, Kolandaivelu, K, Ginebra, MP, Pegueroles, M, (2021). Solvent-cast direct-writing as a fabrication strategy for radiopaque stents Additive Manufacturing 48, 102392