scholarly journals A human organoid system that self-organizes to recapitulate growth and differentiation of a benign mammary tumor

2019 ◽  
Vol 116 (23) ◽  
pp. 11444-11453 ◽  
Author(s):  
Stefan Florian ◽  
Yoshiko Iwamoto ◽  
Margaret Coughlin ◽  
Ralph Weissleder ◽  
Timothy J. Mitchison
Keyword(s):  
Cell Biology ◽  
Single Cells ◽  
3D Culture ◽  
Cell Types ◽  
Cell Polarization ◽  
Cell Aggregates ◽  
Usual Ductal Hyperplasia ◽  
Sequence Of Events ◽  
Ductal Hyperplasia

As 3D culture has become central to investigation of tissue biology, mammary epithelial organoids have emerged as powerful tools for investigation of epithelial cell polarization and carcinogenesis. However, most current protocols start from single cells suspended in Matrigel, which can also restrict cell differentiation and behavior. Here, we show that the noncancerous mammary cell line HMT-3522 S1, when allowed to spontaneously form cell aggregates (“spheroids”) in medium without Matrigel, switches to a collective growth mode that recapitulates many attributes of “usual ductal hyperplasia” (UDH), a common benign mammary lesion. Interestingly, these spheroids undergo a complex maturation process reminiscent of embryonic development: solid-cell cords form their own basement membrane, grow on the surface of initially homogeneous cell aggregates, and form asymmetric lumina lined by two distinct cell types that express basal and luminal cytokeratins. This sequence of events provides a cellular mechanism that explains how the characteristic crescent-shaped, asymmetrical lumina form in UDH. Our results suggest that HMT-3522 S1 spheroids are useful as an in vitro model system to study UDH biology, glandular lumen formation, and stem cell biology of the mammary gland.

scholarly journals Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application

2020 ◽  
Vol 21 (17) ◽  
pp. 6225 ◽  
Author(s):  
Kamila Białkowska ◽  
Piotr Komorowski ◽  
Maria Bryszewska ◽  
Katarzyna Miłowska
Keyword(s):  
Cell Cultures ◽  
Cell Biology ◽  
Disease Modeling ◽  
Three Dimensional ◽  
3D Culture ◽  
Matrix Interactions ◽  
Vitro Model ◽  
Extracellular Matrix Interactions ◽  
Natural Cell

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

scholarly journals Polarization of fluorescein fluorescence in single cells.

1979 ◽  
Vol 27 (1) ◽  
pp. 49-55 ◽  
Author(s):  
R Udkoff ◽  
A Norman
Keyword(s):  
Mammalian Cells ◽  
Single Cells ◽  
Classical Model ◽  
Cell Types ◽  
Wavelength Shift ◽  
Two Peaks ◽  
Vitro Data ◽  
P Distribution ◽  
In Vitro Data

Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 535 ◽  
Author(s):  
T. A. Pelton ◽  
M. D. Bettess ◽  
J. Lake ◽  
J. Rathjen ◽  
P. D. Rathjen
Keyword(s):  
Cell Biology ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Experimental Manipulation ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Cellular Origin

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.

Adequacy of herniated disc tissue as a cell source for nucleus pulposus regeneration

2011 ◽  
Vol 14 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Aldemar A. Hegewald ◽  
Michaela Endres ◽  
Alexander Abbushi ◽  
Mario Cabraja ◽  
Christian Woiciechowsky ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Cell Expansion ◽  
Cell Biology ◽  
3D Culture ◽  
Polyglycolic Acid ◽  
Herniated Disc ◽  
Marker Genes ◽  
Disc Tissue ◽  
Study Results

Object The object of this study was to characterize the regenerative potential of cells isolated from herniated disc tissue obtained during microdiscectomy. The acquired data could help to evaluate the feasibility of these cells for autologous disc cell transplantation. Methods From each of 5 patients (mean age 45 years), tissue from the nucleus pulposus compartment as well as from herniated disc was obtained separately during microdiscectomy of symptomatic herniated lumbar discs. Cells were isolated, and in vitro cell expansion for cells from herniated disc tissue was accomplished using human serum and fibroblast growth factor-2. For 3D culture, expanded cells were loaded in a fibrin-hyaluronan solution on polyglycolic acid scaffolds for 2 weeks. The formation of disc tissue was documented by histological staining of the extracellular matrix as well as by gene expression analysis of typical disc marker genes. Results Cells isolated from herniated disc tissue showed significant signs of dedifferentiation and degeneration in comparison with cells from tissue of the nucleus compartment. With in vitro cell expansion, further dedifferentiation with distinct suppression of major matrix molecules, such as aggrecan and Type II collagen, was observed. Unlike in previous reports of cells from the nucleus compartment, the cells from herniated disc tissue showed only a weak redifferentiation process in 3D culture. However, propidium iodide/fluorescein diacetate staining documented that 3D assembly of these cells in polyglycolic acid scaffolds allows prolonged culture and high viability. Conclusions Study results suggested a very limited regenerative potential for cells harvested from herniated disc tissue. Further research on 2 major aspects in patient selection is suggested before conducting reasonable clinical trials in this matter: 1) diagnostic strategies to predict the regenerative potential of harvested cells at a radiological or cell biology level, and 2) clinical assessment strategies to elucidate the metabolic state of the targeted disc.

scholarly journals Non-proliferative neurogenesis in human periodontal ligament stem cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Bueno ◽  
Marta Martínez-Morga ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Periodontal Ligament ◽  
Cell Biology ◽  
Basic Knowledge ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Periodontal Ligament Stem Cells ◽  
Sequence Of Events

AbstractUnderstanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to in vitro neurogenesis. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.

ColorCells: a database of expression, classification and functions of lncRNAs in single cells

2020 ◽  
Author(s):  
Ling-Ling Zheng ◽  
Jing-Hua Xiong ◽  
Wu-Jian Zheng ◽  
Jun-Hao Wang ◽  
Zi-Liang Huang ◽  
...  
Keyword(s):  
Single Cell ◽  
Tissue Specificity ◽  
Single Cells ◽  
Noncoding Rnas ◽  
Cell Types ◽  
Marker Genes ◽  
Rna Seq ◽  
Expression Variability ◽  
Expression Classification

Abstract Although long noncoding RNAs (lncRNAs) have significant tissue specificity, their expression and variability in single cells remain unclear. Here, we developed ColorCells (http://rna.sysu.edu.cn/colorcells/), a resource for comparative analysis of lncRNAs expression, classification and functions in single-cell RNA-Seq data. ColorCells was applied to 167 913 publicly available scRNA-Seq datasets from six species, and identified a batch of cell-specific lncRNAs. These lncRNAs show surprising levels of expression variability between different cell clusters, and has the comparable cell classification ability as known marker genes. Cell-specific lncRNAs have been identified and further validated by in vitro experiments. We found that lncRNAs are typically co-expressed with the mRNAs in the same cell cluster, which can be used to uncover lncRNAs’ functions. Our study emphasizes the need to uncover lncRNAs in all cell types and shows the power of lncRNAs as novel marker genes at single cell resolution.

scholarly journals Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture.

1980 ◽  
Vol 87 (2) ◽  
pp. 370-378 ◽  
Author(s):  
U Rutishauser ◽  
G M Edelman
Keyword(s):  
Neurite Outgrowth ◽  
Cell Adhesion Molecule ◽  
Capillary Tube ◽  
Single Cells ◽  
Spinal Ganglia ◽  
Cell Aggregates ◽  
Preferential Growth ◽  
Nerve Growth ◽  
High Concentrations

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.

scholarly journals Current Advances in 3D Tissue and Organ Reconstruction

2021 ◽  
Vol 22 (2) ◽  
pp. 830
Author(s):  
Georgia Pennarossa ◽  
Sharon Arcuri ◽  
Teresina De Iorio ◽  
Fulvio Gandolfi ◽  
Tiziana A. L. Brevini
Keyword(s):  
Cell Biology ◽  
Drug Testing ◽  
Three Dimensional ◽  
3D Culture ◽  
In Vitro Models ◽  
The Body ◽  
Cellular Functions ◽  
Culture Systems

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.

scholarly journals Quantification of nematic cell polarity in three-dimensional tissues

2020 ◽  
Vol 16 (12) ◽  
pp. e1008412
Author(s):  
André Scholich ◽  
Simon Syga ◽  
Hernán Morales-Navarrete ◽  
Fabián Segovia-Miranda ◽  
Hidenori Nonaka ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Liver Tissue ◽  
Cell Biology ◽  
Single Cells ◽  
Orientational Order ◽  
Three Dimensional ◽  
Order Parameters ◽  
Imaging Data ◽  
Unique Type

How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarrete et al., eLife 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues and in-vitro organoids.

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