scholarly journals Protein Fibrillar Hydrogels for three-Dimensional Tissue Engineering

2009 ◽  
Vol 2009 ◽  
pp. 1-4 ◽  
Author(s):  
Hui Yan ◽  
Antti Nykanen ◽  
Janne Ruokolainen ◽  
David Farrar ◽  
Aline F. Miller
Keyword(s):  
Cell Culture ◽  
Self Assembly ◽  
Culture Media ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Disease States ◽  
Environmental Responsiveness ◽  
Transmission Electron ◽  
Biological Compatibility ◽  
Oscillatory Rheology

Protein self-assembly into highly ordered fibrillar aggregates has attracted increasing attention over recent years, due primarily to its association with disease states such as Alzheimer's. More recently, however, research has focused on understanding the generic behavior of protein self-assembly where fibrillation is typically induced under harsh conditions of low pH and/or high temperature. Moreover the inherent properties of these fibrils, including their nanoscale dimension, environmental responsiveness, and biological compatibility, are attracting substantial interest for exploiting these fibrils for the creation of new materials. Here we will show how protein fibrils can be formed under physiological conditions and their subsequent gelation driven using the ionic strength of cell culture media while simultaneously incorporating cells homogeneously throughout the gel network. The fibrillar and elastic nature of the gel have been confirmed using cryo-transmission electron microscopy and oscillatory rheology, respectively; while cell culture work shows that our hydrogels promote cell spreading, attachment, and proliferation in three dimensions.

Three-dimensional cell culture of chondrocytes on modified di-phenylalanine scaffolds

2007 ◽  
Vol 35 (3) ◽  
pp. 535-537 ◽  
Author(s):  
V. Jayawarna ◽  
A. Smith ◽  
J.E. Gough ◽  
R.V. Ulijn
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Cell Proliferation ◽  
Self Assembly ◽  
Three Dimensional ◽  
Building Blocks ◽  
Culture Conditions ◽  
The Self ◽  
Three Dimensions ◽  
Dimensional Cell

The design of self-assembled peptide-based structures for three-dimensional cell culture and tissue repair has been a key objective in biomaterials science for decades. In search of the simplest possible peptide system that can self-assemble, we discovered that combinations of di-peptides that are modified with aromatic stacking ligands could form nanometre-sized fibres when exposed to physiological conditions. For example, we demonstrated that a number of Fmoc (fluoren-9-ylmethyloxycarbonyl) modified di- and tri-peptides form highly ordered hydrogels via hydrogen-bonding and π–π interactions from the fluorenyl rings. These highly hydrated gels allowed for cell proliferation of chondrocytes in three dimensions [Jayawarna, Ali, Jowitt, Miller, Saiani, Gough and Ulijn (2006) Adv. Mater. 18, 611–614]. We demonstrated that fibrous architecture and physical properties of the resulting materials were dictated by the nature of the amino acid building blocks. Here, we report the self-assembly process of three di-phenylalanine analogues, Fmoc-Phe-Phe-OH, Nap (naphthalene)-Phe-Phe-OH and Cbz (benzyloxycarbonyl)-Phe-Phe-OH, to compare and contrast the self-assembly properties and cell culture conditions attributable to their protecting group difference. Fibre morphology analysis of the three structures using cryo-SEM (scanning electron microscopy) and TEM (transmission electron microscopy) suggested fibrous structures with dramatically varying fibril dimensions, depending on the aromatic ligand used. CD and FTIR (Fourier-transform IR) data confirmed β-sheet arrangements in all three samples in the gel state. The ability of these three new hydrogels to support cell proliferation of chondrocytes was confirmed for all three materials.

convergent-beam diffraction from surfaces

1987 ◽  
Vol 45 ◽  
pp. 30-33
Author(s):  
J. A. Eades ◽  
A. E. Smith ◽  
D. F. Lynch
Keyword(s):  
Reciprocal Lattice ◽  
Three Dimensional ◽  
Grazing Incidence ◽  
Three Dimensions ◽  
Plane Figure ◽  
Transmission Electron ◽  
Convergent Beam ◽  
Beam Patterns ◽  
Beam Diffraction

It is quite simple (in the transmission electron microscope) to obtain convergent-beam patterns from the surface of a bulk crystal. The beam is focussed onto the surface at near grazing incidence (figure 1) and if the surface is flat the appropriate pattern is obtained in the diffraction plane (figure 2). Such patterns are potentially valuable for the characterization of surfaces just as normal convergent-beam patterns are valuable for the characterization of crystals.There are, however, several important ways in which reflection diffraction from surfaces differs from the more familiar electron diffraction in transmission.GeometryIn reflection diffraction, because of the surface, it is not possible to describe the specimen as periodic in three dimensions, nor is it possible to associate diffraction with a conventional three-dimensional reciprocal lattice.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Novel low shear 3D bioreactor for high purity mesenchymal stem cell production

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252575
Author(s):  
Andrew B. Burns ◽  
Corinna Doris ◽  
Kevin Vehar ◽  
Vinit Saxena ◽  
Cameron Bardliving ◽  
...  
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Purity ◽  
Culture Media ◽  
Human Mesenchymal Stem Cells ◽  
Three Dimensions ◽  
Bioreactor Culture ◽  
Differentiation Potential ◽  
Cell Culture Systems

Bone marrow derived human Mesenchymal Stem Cells (hMSCs) are an attractive candidate for regenerative medicine. However, their harvest can be invasive, painful, and expensive, making it difficult to supply the enormous amount of pure hMSCs needed for future allogeneic therapies. Because of this, a robust method of scaled bioreactor culture must be designed to supply the need for high purity, high density hMSC yields. Here we test a scaled down model of a novel bioreactor consisting of an unsubmerged 3D printed Polylactic Acid (PLA) lattice matrix wetted by culture media. The growth matrix is uniform, replicable, and biocompatible, enabling homogenous cell culture in three dimensions. The goal of this study was to prove that hMSCs would culture well in this novel bioreactor design. The system tested resulted in comparable stem cell yields to other cell culture systems using bone marrow derived hMSCs, while maintaining viability (96.54% ±2.82), high purity (>98% expression of combined positive markers), and differentiation potential.

scholarly journals Polyhedra Self-Assembled from DNA Tripods and Characterized with 3D DNA-PAINT

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 65-69 ◽  
Author(s):  
Ryosuke Iinuma ◽  
Yonggang Ke ◽  
Ralf Jungmann ◽  
Thomas Schlichthaerle ◽  
Johannes B. Woehrstein ◽  
...  
Keyword(s):  
Self Assembly ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Super Resolution ◽  
Triangular Prism ◽  
Hexagonal Prism ◽  
General Strategy ◽  
Transmission Electron ◽  
Pentagonal Prism ◽  
Means Of Transmission

DNA self-assembly has produced diverse synthetic three-dimensional polyhedra. These structures typically have a molecular weight no greater than 5 megadaltons. We report a simple, general strategy for one-step self-assembly of wireframe DNA polyhedra that are more massive than most previous structures. A stiff three-arm-junction DNA origami tile motif with precisely controlled angles and arm lengths was used for hierarchical assembly of polyhedra. We experimentally constructed a tetrahedron (20 megadaltons), a triangular prism (30 megadaltons), a cube (40 megadaltons), a pentagonal prism (50 megadaltons), and a hexagonal prism (60 megadaltons) with edge widths of 100 nanometers. The structures were visualized by means of transmission electron microscopy and three-dimensional DNA-PAINT super-resolution fluorescent microscopy of single molecules in solution.

A Model of Force Generation in a Three-Dimensional Toroidal Cluster of Cells

2012 ◽  
Vol 79 (5) ◽  
Author(s):  
Asha Nurse ◽  
L. B. Freund ◽  
Jacquelyn Youssef
Keyword(s):  
Potential Energy ◽  
Gravitational Potential ◽  
Self Assembly ◽  
Three Dimensional ◽  
Dissipative System ◽  
The Self ◽  
Three Dimensions ◽  
Force Generation ◽  
Gravitational Potential Energy ◽  
Kinetics Of

Observation of the self-assembly of clusters of cells in three dimensions has raised questions about the forces that drive changes in the shape of the cell clusters. Cells that self-assemble into a toroidal cluster about the base of a conical pillar have been observed in the laboratory to spontaneously climb the conical pillar. Assuming that cell cluster reorganization is due solely to surface diffusion, a mathematical model based on the thermodynamics of an isothermal dissipative system is presented. The model shows that the cluster can reduce its surface area by climbing the conical pillar, however, this is at the expense of increasing its gravitational potential energy. As a result, the kinetics of the climb are affected by parameters that influence this energy competition, such as the slope of the conical pillar and a parameter of the model κ that represents the influence of the surface energy of the cluster relative to its gravitational potential energy.

scholarly journals Electron Tomography

2002 ◽  
Vol 10 (2) ◽  
pp. 3-5
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Two Dimensions ◽  
Three Dimensions ◽  
Specimen Preparation ◽  
Two Dimensional Image ◽  
Transmission Electron ◽  
Different Depths ◽  
Depth Dimension ◽  
Pass Through

The transmission electron microscope (TEM) was invented in the 1930's, and developments in specimen preparation in the 1950's led to its widespread use as a tool to study structure in biologic systems. Similar in principle to the light microscope, but utilizing a much shorter wavelength for better resolution, the TEM has the image-forming beam pass through the specimen. This results in a two-dimensional image which can be difficult to interpret because features from different depths of the three dimensional specimen are superimposed. Traditionally this was dealt with by cutting sections of plastic-embedded specimens so thin (in the 40 to SO nanometer range) that they effectively had only two dimensions. To allow biologists to examine structures in three dimensions, serial sections are stacked and structures reconstructed. Even though computers have made reconstruction easier, the reality is that resolution in the depth dimension is limited by the section thickness. The technique of electron tomography is emerging as a way to overcome this limitation.

scholarly journals A stimuli-responsive, pentapeptide, nanofiber hydrogel for tissue engineering

2019 ◽  
Author(s):  
James D. Tang ◽  
Cameron Mura ◽  
Kyle J. Lampe
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Culture Media ◽  
Solvent Accessibility ◽  
Three Dimensional ◽  
Building Blocks ◽  
Weight Percent ◽  
Oligodendrocyte Progenitor Cells ◽  
Self Healing

ABSTRACTShort peptides are uniquely versatile building blocks for self-assembly. Supramolecular peptide assemblies can be used to construct functional hydrogel biomaterials—an attractive approach for neural tissue engineering. Here, we report a new class of short, five-residue peptides that form hydrogels with nanofiber structures. Using rheology and spectroscopy, we describe how sequence variations, pH, and peptide concentration alter the mechanical properties of our pentapeptide hydrogels. We find that this class of seven unmodified peptides forms robust hydrogels from 0.2–20 kPa at low weight percent (less than 3 wt. %) in cell culture media, and undergoes shear-thinning and rapid self-healing. The peptides self-assemble into long fibrils with sequence-dependent fibrillar morphologies. These fibrils exhibit a unique twisted ribbon shape, as visualized by TEM and Cryo-EM imaging, with diameters in the low tens of nanometers and periodicities similar to amyloid fibrils. Experimental gelation behavior corroborates our molecular dynamics simulations, which demonstrate peptide assembly behavior, an increase in β-sheet content, and patterns of variation in solvent accessibility. Our Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are syringe-injectable and support cytocompatible encapsulation of oligodendrocyte progenitor cells (OPCs), as well as their proliferation and three-dimensional process extension. Furthermore, RAPID gels protect OPCs from mechanical membrane disruption and acute loss of viability when ejected from a syringe needle, highlighting the protective capability of the hydrogel as potential cell carriers for trans-plantation therapies. The tunable mechanical and structural properties of these supramolecular assemblies are shown to be permissive to cell expansion and remodeling, making this hydrogel system suitable as an injectable material for cell delivery and tissue engineering applications.

scholarly journals Self-assembly, buckling and density-invariant growth of three-dimensional vascular networks

2019 ◽  
Vol 16 (159) ◽  
pp. 20190517 ◽  
Author(s):  
Julius B. Kirkegaard ◽  
Bjarke F. Nielsen ◽  
Ala Trusina ◽  
Kim Sneppen
Keyword(s):  
Cell Polarity ◽  
Self Assembly ◽  
Planar Cell Polarity ◽  
Vascular System ◽  
Initial Density ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Emergent Properties ◽  
Vascular Density ◽  
Convergent Extension

The experimental actualization of organoids modelling organs from brains to pancreases has revealed that much of the diverse morphologies of organs are emergent properties of simple intercellular ‘rules’ and not the result of top-down orchestration. In contrast to other organs, the initial plexus of the vascular system is formed by aggregation of cells in the process known as vasculogenesis. Here we study this self-assembling process of blood vessels in three dimensions through a set of simple rules that align intercellular apical–basal and planar cell polarity. We demonstrate that a fully connected network of tubes emerges above a critical initial density of cells. Through planar cell polarity, our model demonstrates convergent extension, and this polarity furthermore allows for both morphology-maintaining growth and growth-induced buckling. We compare this buckling with the special vasculature of the islets of Langerhans in the pancreas and suggest that the mechanism behind the vascular density-maintaining growth of these islets could be the result of growth-induced buckling.

scholarly journals Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

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