scholarly journals Niche-inspired synthetic matrices for epithelial organoid culture

2019 ◽  
Author(s):  
Victor Hernandez-Gordillo ◽  
Timothy Kassis ◽  
Arinola Lampejo ◽  
GiHun Choi ◽  
Mario E. Gamboa ◽  
...  
Keyword(s):  
Single Cells ◽  
Biophysical Properties ◽  
Organoid Culture ◽  
Apicobasal Polarity ◽  
Induction Of Differentiation ◽  
Synthetic Matrix ◽  
Synthetic Matrices ◽  
Α2β1 Integrin ◽  
Dominant Parameter ◽  
Human Implantation

AbstractEpithelial organoids are now an important tool in fields ranging from regenerative medicine to drug discovery. Organoid culture requires Matrigel, a complex, tumor-derived, extracellular matrix. An alternative completely synthetic matrix could improve culture reproducibility, clarify mechanistic phenomena, and enable applications involving human implantation. Here, we designed synthetic matrices with tunable biomolecular and biophysical properties that allowed us to identify critical gel parameters in organoid formation. Inspired by known epithelial integrin expression in the proliferative niche of the human intestine, we identified an α2β1 integrin-binding peptide as a critical component of the synthetic matrix that supports human duodenal colon and endometrial organoid propagation. We show that organoids emerge from single cells, retain their proliferative capacity, are functionally responsive to basolateral stimulation and have correct apicobasal polarity upon induction of differentiation. The local biophysical presentation of the cues, rather than bulk mechanical properties, appears to be the dominant parameter governing epithelial cell proliferation and organoid formation in the synthetic matrix.

On the Role of Biophysical Properties of Cortical Neurons in Binding and Segmentation of Visual Scenes

1999 ◽  
Vol 11 (5) ◽  
pp. 1113-1138 ◽  
Author(s):  
Paul F. M. J. Verschure ◽  
Peter König
Keyword(s):  
Leakage Current ◽  
Cortical Neurons ◽  
Single Cells ◽  
Spatial Dimension ◽  
Differential Regulation ◽  
System Level ◽  
Biophysical Properties ◽  
Moving Stimulus ◽  
Context Dependent ◽  
Different Levels

Neuroscience is progressing vigorously, and knowledge at different levels of description is rapidly accumulating. To establish relationships between results found at these different levels is one of the central challenges. In this simulation study, we demonstrate how microscopic cellular properties, taking the example of the action of modulatory substances onto the membrane leakage current, can provide the basis for the perceptual functions reflected in the macroscopic behavior of a cortical network. In the first part, the action of the modulatory system on cortical dynamics is investigated. First, it is demonstrated that the inclusion of these biophysical properties in a model of the primary visual cortex leads to the dynamic formation of synchronously active neuronal assemblies reflecting a context-dependent binding and segmentation of image components. Second, it is shown that the differential regulation of the leakage current can be used to bias the interactions of multiple cortical modules. This allows the flexible use of different feature domains for scene segmentation. Third, we demonstrate how, within the proposed architecture, the mapping of a moving stimulus onto the spatial dimension of the network results in an increased speed of synchronization. In the second part, we demonstrate how the differential regulation of neuromodulatory activity can be achieved in a self-consistent system. Three different mechanisms are described and investigated. This study thus demonstrates how a modulatory system, affecting the biophysical properties of single cells, can be used to achieve context-dependent processing at the system level.

scholarly journals Mouse intestinal organoid time-course experiments from single cells

2019 ◽  
Author(s):  
Denise Serra ◽  
Urs Mayr ◽  
Andrea Boni ◽  
Ilya Lukonin ◽  
Markus Rempfler ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Time Course ◽  
Single Cells ◽  
Rna Extraction ◽  
Cell Types ◽  
The Self ◽  
Organoid Culture ◽  
Tissue Organization ◽  
Grown Structure

Abstract Organoids recapitulate the self-organizing capacity of stem cells and the tissue organization of the original organ in a controlled and trackable environment. Intestinal organoids, in particular, can develop from a single cell into a fully-grown structure that contains most of the cell types, patterns, and morphogenetic properties of the adult intestine. Here we present a protocol for high-throughput organoid culture in multi-well plate format combined with high content immunofluorescence imaging and RNA extraction. Our protocol allows recording and analysis of thousands of organoids during several days of development.

scholarly journals Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 804-815 ◽  
Author(s):  
Donny Hanjaya-Putra ◽  
Vivek Bose ◽  
Yu-I Shen ◽  
Jane Yee ◽  
Sudhir Khetan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
In Vitro Models ◽  
Cellular Interactions ◽  
Vascular Networks ◽  
3 Dimensional ◽  
Endothelial Colony Forming Cells ◽  
Vascular Morphogenesis ◽  
Synthetic Matrix ◽  
Synthetic Matrices

Abstract Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells (ECFCs) to form efficient vascular networks. Entrapped ECFCs underwent tubulogenesis dependent on the cellular interactions with the HA hydrogel during each stage of vascular morphogenesis. Vacuole and lumen formed through integrins α5β1 and αVβ3, while branching and sprouting were enabled by HA hydrogel degradation. Vascular networks formed within HA hydrogels containing ECFCs anastomosed with the host's circulation and supported blood flow in the hydrogel after transplantation. Collectively, we show that the signaling pathways of vascular morphogenesis of ECFCs can be precisely regulated in a synthetic matrix, resulting in a functional microvasculature useful for the study of 3-dimensional vascular biology and toward a range of vascular disorders and approaches in tissue regeneration.

scholarly journals New tools to study biophysical properties of single molecules and single cells

2007 ◽  
Vol 79 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Márcio S. Rocha ◽  
Oscar N. Mesquita
Keyword(s):  
Optical Tweezers ◽  
Osmotic Shock ◽  
Single Cells ◽  
Single Molecules ◽  
Numerical Aperture ◽  
Single Individual ◽  
Biophysical Properties ◽  
Individual Object ◽  
Transparent Objects ◽  
Volume Measurements

We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.

scholarly journals Synthetic Matrices for Intestinal Organoid Culture: Implications for Better Performance

ACS Omega ◽  
2021 ◽  
Author(s):  
Humendra Poudel ◽  
Karie Sanford ◽  
Peter K. Szwedo ◽  
Rupak Pathak ◽  
Anindya Ghosh
Keyword(s):  
Organoid Culture ◽  
Synthetic Matrices

scholarly journals Induction of differentiation of intrahepatic cholangiocarcinoma cells to functional hepatocytes using an organoid culture system

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Yoshimasa Saito ◽  
Toshiaki Nakaoka ◽  
Toshihide Muramatsu ◽  
Hidenori Ojima ◽  
Aoi Sukeda ◽  
...  
Keyword(s):  
Intrahepatic Cholangiocarcinoma ◽  
Culture System ◽  
Organoid Culture ◽  
Induction Of Differentiation

scholarly journals Dissecting single-cell genomes through the clonal organoid technique

2021 ◽  
Author(s):  
Jeonghwan Youk ◽  
Hyun Woo Kwon ◽  
Ryul Kim ◽  
Young Seok Ju
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
De Novo ◽  
Single Cells ◽  
Somatic Mosaicism ◽  
Adult Stem Cell ◽  
Clonal Expansion ◽  
De Novo Mutations ◽  
Organoid Culture

AbstractThe revolution in genome sequencing technologies has enabled the comprehensive detection of genomic variations in human cells, including inherited germline polymorphisms, de novo mutations, and postzygotic mutations. When these technologies are combined with techniques for isolating and expanding single-cell DNA, the landscape of somatic mosaicism in an individual body can be systematically revealed at a single-cell resolution. Here, we summarize three strategies (whole-genome amplification, microdissection of clonal patches in the tissue, and in vitro clonal expansion of single cells) that are currently applied for single-cell mutational analyses. Among these approaches, in vitro clonal expansion, particularly via adult stem cell-derived organoid culture technologies, yields the most sensitive and precise catalog of somatic mutations in single cells. Moreover, because it produces living mutant cells, downstream validation experiments and multiomics profiling are possible. Through the synergistic combination of organoid culture and genome sequencing, researchers can track genome changes at a single-cell resolution, which will lead to new discoveries that were previously impossible.

Effects of carbamylcholine on chick embryo aggregate retina cell cultures

1988 ◽  
Vol 46 ◽  
pp. 350-351
Author(s):  
Glenn M. Cohen ◽  
Radharaman Ray
Keyword(s):  
Cell Cultures ◽  
Single Cells ◽  
Retinal Cell ◽  
Cell Aggregates ◽  
High Concentration ◽  
In Ovo ◽  
Sterile Culture ◽  
Retinal Tissue ◽  
Synaptic Junctions ◽  
And Control

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.

Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

1968 ◽  
Vol 26 ◽  
pp. 38-39
Author(s):  
J. H. Luft
Keyword(s):  
Electron Microscope ◽  
Light Microscopy ◽  
Light Microscope ◽  
Osmium Tetroxide ◽  
Single Cells ◽  
Ruthenium Red ◽  
Collagen Fibers ◽  
Selective Staining ◽  
Pectic Substances ◽  
Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

Fluorescent potentiometric dyes for membrane-potential imaging

1994 ◽  
Vol 52 ◽  
pp. 166-167
Author(s):  
Leslie M. Loew
Keyword(s):  
Membrane Potential ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Optical Recording ◽  
Biological Processes ◽  
Major Focus ◽  
The Past ◽  
Excitable Systems ◽  
Major Application ◽  
The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

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