scholarly journals Cell dissociation of fresh human lung tissue for single-cell RNA-seq v1 (protocols.io.zp2f5qe)

protocols.io ◽  
2019 ◽  
Author(s):  
Ilias Angelidis ◽  
Maximilian Strunz ◽  
Herbert Schiller
Keyword(s):  
Single Cell ◽  
Lung Tissue ◽  
Human Lung ◽  
Rna Seq ◽  
Human Lung Tissue ◽  
Cell Dissociation

Preparation of Single Cell Suspension from Human Lung Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Basak Ural ◽  
Maya M.L. Poon
Keyword(s):  
Epithelial Cells ◽  
Cell Suspension ◽  
Single Cell ◽  
Lung Tissue ◽  
Immune Cells ◽  
Human Lung ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Human Lung Tissue ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

scholarly journals Cryobanking of human distal lung epithelial cells for preservation of their phenotypic and functional characteristics.

2021 ◽  
Author(s):  
Bindu Konda ◽  
Apoorva Mulay ◽  
Changfu Yao ◽  
Edo Israely ◽  
Stephen Beil ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Tissue ◽  
Human Lung ◽  
Alveolar Type ◽  
Basal Cells ◽  
Human Lung Tissue ◽  
Downstream Analysis ◽  
Long Term Preservation

The epithelium lining airspaces of the human lung is maintained by regional stem cells including basal cells of pseudostratified airways and alveolar type 2 pneumocytes (AT2) of the alveolar gas-exchange region. Despite effective methods for long-term preservation of airway basal cells, methods for efficient preservation of functional epithelial cell types of the distal gas-exchange region are lacking. Here we detail a method for cryobanking of epithelial cells from either mouse or human lung tissue for preservation of their phenotypic and functional characteristics. Flow cytometric profiling, epithelial organoid-forming efficiency, and single cell transcriptomic analysis, were used to compare cells recovered from cryopreserved tissue with those of freshly dissociated tissue. Alveolar type 2 cells within single cell suspensions of enzymatically digested cryobanked distal lung tissue retained expression of the pan-epithelial marker CD326 and the AT2 cell surface antigen recognized by monoclonal antibody HTII-280, allowing antibody-mediated enrichment and downstream analysis. Isolated AT2 cells from cryobanked tissue were comparable with those of freshly dissociated tissue both in their single cell transcriptome and their capacity for in vitro organoid formation in 3D cultures. We conclude that the cryobanking method described herein allows long-term preservation of distal human lung tissue for downstream analysis of lung cell function and molecular phenotype, and is ideally suited for creation of an easily accessible tissue resource for the research community.

scholarly journals CellDART: Cell type inference by domain adaptation of single-cell and spatial transcriptomic data

2021 ◽  
Author(s):  
Sungwoo Bae ◽  
Kwon Joong Na ◽  
Jaemoon Koh ◽  
Dong Soo Lee ◽  
Hongyoon Choi ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Single Cell ◽  
Lung Tissue ◽  
Human Lung ◽  
Domain Adaptation ◽  
Cell Types ◽  
Cell Type ◽  
Human Lung Tissue ◽  
Suggested Approach ◽  
Transcriptomic Data

AbstractDeciphering the cellular composition in genome-wide spatially resolved transcriptomic data is a critical task to clarify the spatial context of cells in a tissue. In this study, we developed a method, CellDART, which estimates the spatial distribution of cells defined by single-cell level data using domain adaptation of neural networks and applied it to the spatial mapping of human lung tissue. The neural network that predicts the cell proportion in a pseudospot, a virtual mixture of cells from single-cell data, is translated to decompose the cell types in each spatial barcoded region. First, CellDART was applied to mouse brain and human dorsolateral prefrontal cortex tissue to identify cell types with a layer-specific spatial distribution. Overall, the suggested approach was superior to the other computational methods in predicting the spatial localization of excitatory neurons. Furthermore, CellDART elucidated the cell type predominance defined by the human lung cell atlas across the lung tissue compartments and it corresponded to the known prevalent cell types. CellDART is expected to help to elucidate the spatial heterogeneity of cells and their close interactions in various tissues.

Mast Cell Heterogeneity in Human Lung Tissue

1989 ◽  
Vol 77 (3) ◽  
pp. 297-304 ◽  
Author(s):  
F. J. Van Overveld ◽  
L. A. M. J. Houben ◽  
F. E. M. Schmitz du Moulin ◽  
P. L. B. Bruijnzeel ◽  
J. A. M. Raaijmakers ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Alcian Blue ◽  
Lung Tissue ◽  
Human Lung ◽  
Immunoglobulin E ◽  
Prostaglandin D2 ◽  
Leukotriene C4 ◽  
Human Lung Tissue ◽  
No Release

1. In this study mast cells were found to comprise 2.1% of total cells recovered by enzymatic digestion of human lung tissue. 2. This mast cell population consisted of 79% formalin-sensitive, Alcian Blue-positive mast cells and 21% formalin-insensitive, Alcian Blue-positive mast cells. 3. By the use of centrifugal elutriation and subsequent Percoll gradient centrifugation, separate mixed cell populations could be obtained in which the mast cell constituents were either of the formalin-sensitive or -insensitive type. 4. Cell suspensions in which formalin-sensitive cells comprised 97% of mast cells contained approximately 1.34 pg of histamine per mast cell, whereas in preparations in which mast cells were 84% formalin-resistant the histamine content was approximately 4.17 pg of histamine per mast cell. 5. The histamine release upon anti-immunoglobulin E challenge of formalin-sensitive mast cells was greater than the release by formalin-insensitive mast cells. 6. After challenge with opsonized zymosan, only formalin-sensitive mast cells were able to release histamine. 7. Leukotriene C4 release was observed when formalin-sensitive mast cells were challenged with antiimmunoglobulin E. Formalin-insensitive mast cells showed no release of leukotriene C4. 8. Prostaglandin D2 release was observed when formalin-insensitive mast cells were challenged with antiimmunoglobulin E. Formalin-sensitive mast cells showed no release of prostaglandin D2.

The Glucocorticosteroid, Budesonide, Partially Blocks Histamine Release from Human Lung Tissue in Vitro

Allergy ◽  
1986 ◽  
Vol 41 (5) ◽  
pp. 319-326 ◽  
Author(s):  
H. Bergstrand ◽  
B. Lundquist ◽  
B.-Å. Petersson
Keyword(s):  
Histamine Release ◽  
Lung Tissue ◽  
Human Lung ◽  
Human Lung Tissue

scholarly journals Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

2013 ◽  
Vol 82 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Jens Jäger ◽  
Sebastian Marwitz ◽  
Jana Tiefenau ◽  
Janine Rasch ◽  
Olga Shevchuk ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Legionella Pneumophila ◽  
Human Lung ◽  
Transcriptional Response ◽  
Human Infection ◽  
Bacterial Invasion ◽  
Human Lung Tissue ◽  
Content Type ◽  
Tissue Explants ◽  
Legionnaires Disease

ABSTRACTHistological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion ofL. pneumophilato the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA−strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context ofL. pneumophilainfections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.

Inhibition of ferrous-induced lipid peroxidation by dipyridamole, RA-642 and mopidamol in human lung tissue

1996 ◽  
Vol 27 (5) ◽  
pp. 855-859 ◽  
Author(s):  
J.P. De la Cruz ◽  
C. Olveira ◽  
J.A. Gonzalez-Correa ◽  
A. Benítez ◽  
F. Sánchez de la Cuesta
Keyword(s):  
Lipid Peroxidation ◽  
Lung Tissue ◽  
Human Lung ◽  
Human Lung Tissue

scholarly journals Immunofluorescence-guided segmentation of three-dimensional features in micro-computed tomography datasets of human lung tissue

2021 ◽  
Vol 8 (11) ◽  
Author(s):  
Matthew J. Lawson ◽  
Orestis L. Katsamenis ◽  
David Chatelet ◽  
Aiman Alzetani ◽  
Oliver Larkin ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Lung Tissue ◽  
Human Lung ◽  
Three Dimensional ◽  
Micro Computed Tomography ◽  
3D Segmentation ◽  
Human Lung Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Micro-computed tomography (µCT) provides non-destructive three-dimensional (3D) imaging of soft tissue microstructures. Specific features in µCT images can be identified using correlated two-dimensional (2D) histology images allowing manual segmentation. However, this is very time-consuming and requires specialist knowledge of the tissue and imaging modalities involved. Using a custom-designed µCT system optimized for imaging unstained formalin-fixed paraffin-embedded soft tissues, we imaged human lung tissue at isotropic voxel sizes less than 10 µm. Tissue sections were stained with haematoxylin and eosin or cytokeratin 18 in columnar airway epithelial cells using immunofluorescence (IF), as an exemplar of this workflow. Novel utilization of tissue autofluorescence allowed automatic alignment of 2D microscopy images to the 3D µCT data using scripted co-registration and automated image warping algorithms. Warped IF images, which were accurately aligned with the µCT datasets, allowed 3D segmentation of immunoreactive tissue microstructures in the human lung. Blood vessels were segmented semi-automatically using the co-registered µCT datasets. Correlating 2D IF and 3D µCT data enables accurate identification, localization and segmentation of features in fixed soft lung tissue. Our novel correlative imaging workflow provides faster and more automated 3D segmentation of µCT datasets. This is applicable to the huge range of formalin-fixed paraffin-embedded tissues held in biobanks and archives.

Sign in / Sign up

Export Citation Format

Share Document