Abstract 38: Bioreactor Based Approach for Valve Tissue Engineering: Novel Application of Decellularized Porcine Pericardium

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Francesco Amadeo ◽  
Gianluca Polvani ◽  
Marco Agrifoglio ◽  
Federica Boschetti ◽  
Maurizio Pesce ◽  
...  
Keyword(s):  
Smooth Muscle Actin ◽  
Sodium Dodecylsulfate ◽  
Perfusion Bioreactor ◽  
Cell Seeding ◽  
Cell Nuclei ◽  
Dapi Staining ◽  
Cellular Debris ◽  
Before And After ◽  
Homogeneous Cell ◽  
Porcine Pericardium

Background: Aldehyde-fixed pericardium is commonly used in valve implant manufacturing. Despite its wide employment, this tissue undergoes chronic rejection that limits implant performance and durability. In this work, we employed a method to engineer a leaflet-like tissue by seeding aortic valve interstitial cells (VIC) into fixative-free decellularized pericardium using a bioreactor based approach. Methods: Following treatment with hypotonic buffer (Tris-HCl) to induce cell lysis, porcine pericardium was incubated with TritonX-100, to remove adipose tissue and then treated with sodium dodecylsulfate to wash cellular debris. DNA was removed by incubation into a DNAse I solution. Pericardium permeability was measured on samples before and after decellularization (pressure from 735 Pa to 2200 Pa). A direct perfusion bioreactor was employed to seed (3days, 3ml/min) porcine VICs (6.5E+5 cell/scaffold) into decellularized pericardium patches (6mm diameters) and perform long-term culture (up to 14 days, 0.03ml/min). Cell seeding efficiency (Day 3) and cell proliferation (Day 7-14) were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) staining, histological analyses and fluorescence staining for quiescent/activated VIC markers (DAPI, vimentin, α-smooth muscle actin). Results: Permeability tests revealed a significant increase in decellularized samples (1-way ANOVA p < 0.05), thus supporting its use in a direct perfusion bioreactor system. MTT staining revealed homogeneous cell seeding distribution, supported by DAPI staining, showing efficient cellularization through the whole patch volume. Computer-based cell nuclei counting revealed a significant cell increase from day 3 to 7 and 14 ( p <0.05 1-way ANOVA). Immunofluorescence showed a marked reduction of αSMA in cells populating the inner layers. Conclusions: Our data show, for the first time, the capability to seed and culture VICs into a cell/fixative free pericardium with a direct perfusion system. Moreover, the use of pericardium treated with our decellularization procedure and recellularized under dynamic conditions supports a more physiological growth of VIC, as suggested by downregulation of αSMA in the inner pericardium layer.

Effect of white spot syndrome virus (WSSV) infection on immune enzyme activity and ultrastructure in the haemolymph tissue of Cherax quadricarinatus (Decapoda, Astacidea)

Crustaceana ◽  
2016 ◽  
Vol 89 (6-7) ◽  
pp. 669-684
Author(s):  
J. J. Ning ◽  
M. M. Zhang ◽  
Q. Q. Tong ◽  
X. Cao ◽  
D. L. Wang ◽  
...  
Keyword(s):  
Enzyme Activities ◽  
White Spot Syndrome Virus ◽  
White Spot ◽  
Immune Protection ◽  
Cell Nuclei ◽  
Cherax Quadricarinatus ◽  
Deformation And Fracture ◽  
Before And After ◽  
Protection Rate ◽  
White Spot Syndrome

To explore the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish (Cherax quadricarinatus), we analysed activities of the three immune-related enzymes PO, SOD and LSZ in haemolymph tissue ofC. quadricarinatusbefore and after infection, and simultaneously studied the ultrastructural pathology. The results show that WSSV infection affects activities of the three enzymes. After 6-24 h of WSSV infection, the activities of PO, SOD and LSZ increased, but decreased significantly during longer infection times. The enzyme activities in WSSV-infected crayfish were significantly lower than those in controls at 72 h, except for LSZ (). Interestingly, the activities of PO, SOD and LSZ in the group treated with immune-polysaccharides before challenge with WSSV were higher than in the directly infected group, and the immune protection rate reached 51.9%, suggesting that the polysaccharides could improve enzyme activities and enhance antiviral defences of the organism. Ultrastructural pathological changes showed damaged haemolymph tissue, deformed golgiosomes, fuzzy damage in the mitochondrial structures, and nuclear membrane deformation and fracture. High levels of heterochromatin appeared in the nucleus; organoid and chromatin dissolved in dying blood cells, cytoplasm appeared oedematous and cells dissolved. WSSV particles were visible in blood cell nuclei of infected crayfish.

scholarly journals An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadja Engel ◽  
Carsten Fechner ◽  
Annika Voges ◽  
Robert Ott ◽  
Jan Stenzel ◽  
...  
Keyword(s):  
In Silico ◽  
Bone Substitute ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cell Distribution ◽  
Cell Seeding ◽  
Outlet Port ◽  
Short Period ◽  
Homogeneous Cell ◽  
Perfusion Mode

AbstractA clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

scholarly journals Microscopic Observation of Proliferative Membranes in Fibrocontractive Retinal Disorders

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Rino Frisina ◽  
Francesco Tessarolo ◽  
Ivan Marchesoni ◽  
Federico Piccoli ◽  
Emiliana Bonomi ◽  
...  
Keyword(s):  
Smooth Muscle Actin ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Fibrous Tissue ◽  
Morphological Characteristics ◽  
Repair Process ◽  
Polymorphonuclear Cells ◽  
Cell Nuclei ◽  
Alpha Smooth Muscle Actin ◽  
Retinal Disorders

Proliferative membranes of fibrocontractive retinal disorders are extensively studied from the morphological and evolutive point of view. Despite this, little is known of their cellular composition. In this study, the authors investigated the morphological characteristics and cell composition of various types of surgically excised proliferative membranes and internal limiting membranes (ILMs), in order to provide new data supporting or challenging the pathogenic theories proposed until now. Sixty-nine specimens from 64 eyes of 64 consecutive patients were collected at surgery and subjected to a multilevel analysis by means of optical and electron microscopy. Membrane samples were semiquantitatively evaluated for the amount and distribution of cell nuclei and pigment. Immunohistochemical staining was performed with antibodies to alpha smooth muscle actin and CD68. Data were analyzed after grouping according to the following tissue types: ILM (20 specimens), epiretinal membrane (ERM) (22 specimens), ILM + ERM (20 specimens), and proliferative vitreoretinopathy (PVR) (7 specimens). The cell components found in the ERM specimens, like myofibroblasts, macrophages, and polymorphonuclear cells, were recognized as the expression of cell migration and differentiation that induced an inflammatory process and a fibroproliferative repair process. The detection of pigments in specific types of ERM, like those associated with lamellar macular hole (LMH) or secondary to retinal detachment (RD), diabetes, and PVR, suggested that retinal pigment epithelium (RPE) cells may have a role in the development of these vitreoretinal disorders. The reduction of the ERM cellularity with the patient’s age supports the hypothesis that ERM evolves in time up to a fibrous tissue formation.

scholarly journals P0544INHIBITION OF 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE (15-PGDH) PROTECTS MICE AGAINST CONTRAST-INDUCED ACUTE KIDNEY INJURY

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Byeong Woo KIm ◽  
Sun hee Kim ◽  
Ki beom Bae
Keyword(s):  
Acute Kidney Injury ◽  
Blood Flow ◽  
Protective Effect ◽  
Renal Blood Flow ◽  
Smooth Muscle Actin ◽  
Kidney Injury ◽  
Plasma Creatinine ◽  
Histological Evaluation ◽  
Vehicle Group ◽  
Before And After

Abstract Background and Aims Although the mechanism of contrast-induced acute kidney injury (CI-AKI) is not fully known, the imbalance of vasoconstrictive and vasodilative mediators plays a major role. Prostaglandin E2 (PGE2) is one of the vasodilators involved in this process. Inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) causes elevation of PGE2 level in tissue by delaying the rapid degradation of PGE2 by the enzyme. We tested the hypothesis that the 15-PGE2 inhibitor would protect against CI-AKI in a mouse model and attempted to elucidate the mechanism involved. Method 10-week aged male C57/BL6 Mice were injected with 10gI/kg of iodixanol by tail vein. Renal blood flow measurement, right nephrectomy, and blood sampling were taken at 48 hours after iodixanol injection. The 15-PGDH inhibitor was injected before and after iodixanol administration. Plasma creatinine, NGAL, KIM-1 were measured as biomarkers for renal function. Histological evaluation was analyzed by the necrosis scoring system and TUNEL assay. Arteriolar area of outer medulla was analyzed by α-smooth muscle actin stain. Renal blood flow was measured by the non-invasive laser doppler. Results Plasma creatinine (1.94±0.75 vs 1.11±0.44 mg/dL, p=0.005), NGAL (299.7±115.87 vs 140.4±76.56 ng/mL, p=0.004), and KIM-1 (2.09±2.34 vs 0.43±0.89 ng/mL, p=0.024) levels were significantly lower when the 15-PGDH inhibitor was injected before and after iodixanol administration than the vehicle group. But no significant renal protective effect was shown when the 15-PGDH inhibitor was injected before or after iodixanol administration. The 15-PGDH inhibitor administration before and after iodixanol injection showed a significantly wider renal arteriolar area (683.63±248.46 vs 1132.97±357.46 μm2, p=0.039) and larger renal blood flow (360.0±49.72 vs 635.1±27.20, p=0.011) than vehicle administration. Conclusion The 15-PGDH inhibitor has a renal protective effect against CI-AKI in mice by increasing renal blood flow when injected intravenously before and after iodine contrast media administration.

scholarly journals Peroxide Electrochemical Sensor and Biosensor Based on Nanocomposite of TiO2 Nanoparticle/Multi-Walled Carbon Nanotube Modified Glassy Carbon Electrode

Nanomaterials ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 64 ◽  
Author(s):  
L. Andrés Guerrero ◽  
Lenys Fernández ◽  
Gema González ◽  
Marjorie Montero-Jiménez ◽  
Rafael Uribe ◽  
...  
Keyword(s):  
Modified Electrode ◽  
Glassy Carbon ◽  
Sodium Dodecylsulfate ◽  
Modified Electrodes ◽  
Dimethyl Formamide ◽  
X Ray Diffraction ◽  
Transmission Electron ◽  
Before And After ◽  
Multi Walled Carbon Nanotubes ◽  
Walled Carbon Nanotubes

A hydrogen peroxide (H2O2) sensor and biosensor based on modified multi-walled carbon nanotubes (CNTs) with titanium dioxide (TiO2) nanostructures was designed and evaluated. The construction of the sensor was performed using a glassy carbon (GC) modified electrode with a TiO2–CNT film and Prussian blue (PB) as an electrocalatyzer. The same sensor was also employed as the basis for H2O2 biosensor construction through further modification with horseradish peroxidase (HRP) immobilized at the TiO2–fCNT film. Functionalized CNTs (fCNTs) and modified TiO2–fCNTs were characterized by Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), and X-Ray DifFraction (XRD), confirming the presence of anatase over the fCNTs. Depending on the surface charge, a solvent which optimizes the CNT dispersion was selected: dimethyl formamide (DMF) for fCNTs and sodium dodecylsulfate (SDS) for TiO2–fCNTs. Calculated values for the electron transfer rate constant (ks) were 0.027 s−1 at the PB–fCNT/GC modified electrode and 4.7 × 10−4 s−1 at the PB–TiO2/fCNT/GC electrode, suggesting that, at the PB–TiO2/fCNT/GC modified electrode, the electronic transfer was improved. According to these results, the PB–fCNT/GC electrode exhibited better Detection Limit (LD) and Quantification Limit (LQ) than the PB–TiO2/fCNT/GC electrode for H2O2. However, the PB film was very unstable at the potentials used. Therefore, the PB–TiO2/fCNT/GC modified electrode was considered the best for H2O2 detection in terms of operability. Cyclic Voltammetry (CV) behaviors of the HRP–TiO2/fCNT/GC modified electrodes before and after the chronoamperometric test for H2O2, suggest the high stability of the enzymatic electrode. In comparison with other HRP/fCNT-based electrochemical biosensors previously described in the literature, the HRP–fCNTs/GC modified electrode did not show an electroanalytical response toward H2O2.

scholarly journals Deflection of a vibrissa leads to a gradient of strain across mechanoreceptors in a mystacial follicle

2015 ◽  
Vol 114 (1) ◽  
pp. 138-145 ◽  
Author(s):  
Samuel J. Whiteley ◽  
Per M. Knutsen ◽  
David W. Matthews ◽  
David Kleinfeld
Keyword(s):  
Laser Scanning ◽  
Ex Vivo ◽  
Volumetric Strain ◽  
Laser Scanning Microscopy ◽  
Cell Nuclei ◽  
Two Photon ◽  
Before And After ◽  
Mechanical Transduction ◽  
Lower Magnitude ◽  
Active Exploration

Rodents use their vibrissae to detect and discriminate tactile features during active exploration. The site of mechanical transduction in the vibrissa sensorimotor system is the follicle sinus complex and its associated vibrissa. We study the mechanics within the ring sinus (RS) of the follicle in an ex vivo preparation of the mouse mystacial pad. The sinus region has a relatively dense representation of Merkel mechanoreceptors and longitudinal lanceolate endings. Two-photon laser-scanning microscopy was used to visualize labeled cell nuclei in an ∼100-nl vol before and after passive deflection of a vibrissa, which results in localized displacements of the mechanoreceptor cells, primarily in the radial and polar directions about the vibrissa. These displacements are used to compute the strain field across the follicle in response to the deflection. We observe compression in the lower region of the RS, whereas dilation, with lower magnitude, occurs in the upper region, with volumetric strain ΔV/V ∼ 0.01 for a 10° deflection. The extrapolated strain for a 0.1° deflection, the minimum angle that is reported to initiate a spike by primary neurons, corresponds to the minimum strain that activates Piezo2 mechanoreceptor channels.

scholarly journals A Perfusion Bioreactor System for Cell Seeding and Oxygen-Controlled Cultivation of Three-Dimensional Cell Cultures

2018 ◽  
Vol 24 (10) ◽  
pp. 585-595 ◽  
Author(s):  
Jakob Schmid ◽  
Sascha Schwarz ◽  
Robert Meier-Staude ◽  
Stefanie Sudhop ◽  
Hauke Clausen-Schaumann ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Three Dimensional ◽  
Perfusion Bioreactor ◽  
Cell Seeding ◽  
Bioreactor System ◽  
Dimensional Cell

scholarly journals Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses

2021 ◽  
Author(s):  
Miguel Angel Pena-Ortiz ◽  
Sarfraz Shafiq ◽  
Megan E Rowland ◽  
Nathalie G Berube
Keyword(s):  
Sample Preparation ◽  
Glial Cell ◽  
Mouse Brain ◽  
Cellular Damage ◽  
Rna Seq ◽  
Cell Type ◽  
Cell Nuclei ◽  
Dapi Staining ◽  
High Quality ◽  
Cell Type Specific

Background: Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. New Method: Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. Results: We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. Comparison with existing methods: Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. Conclusions : We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.

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