Dataset Information

Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation.

ABSTRACT: The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin histologic evaluations. There are few studies that have reported detailed processes for preparing 3-D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining and light microscopic evaluations.

SUBMITTER: Clayton NP

PROVIDER: S-EPMC6117202 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Publications

Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation.

Clayton Natasha P NP Burwell Alanna A Jensen Heather H Williams Barbara F BF Brown Quashana D QD Ovwigho Pamela P Ramaiahgari Sreenivasa S Hermon Tonia T Dixon Darlene D

Toxicologic pathology 20180808 6

The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed ce ...[more]

PMID: 30089414

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Project description:Hepatic trematodiasis is a common condition in a number of free-ranging cetacean species, which occasionally result in severe hepatic and/or pancreatic lesions. However, even the basic pathological information of this disease is unknown for the majority of affected species. The current study describes and compares the histomorphology and immune reaction induced by hepatic trematodes of the family Brachycladiidae in the liver of the harbor porpoise (Phocoena phocoena, n = 8), Dall's porpoise (Phocoenoides dalli, n = 8), and Hubbs' beaked whale (Mesoplodon carlhubbsi, n = 2). Immunohistochemistry for eight antibodies (CK19, CD3, Foxp3, CD20, Iba1, CD68, CD163, and CD204) was conducted to analyze the pathology of these parasitic infections. In all three odontocete species, the changes observed in the trematode-affected biliary epithelium were comparable with marked hyperplasia and goblet cell metaplasia, as well as lymphoplasmacytic and eosinophilic inflammation. Additionally, regions of the Glisson's sheath were diffusely and severely fibrotic in all examined species, regardless of the physical presence of trematodes. Differences among the three species included the presence of characteristic lymphoid follicles formed in the fibrotic bile duct walls of only the two porpoise species. In the Hubbs' beaked whale, the degree of lymphoplasmacytic cholangitis was more severe, and ductular reaction was generally more prominent. In terms of the overall macrophage population among the three species, CD163- and CD204-positive cells (M2 macrophages) outnumbered Iba1- and CD68-positive cells (M1 macrophages), indicating a chronic infection stage in all analyzed individuals. Species-specific differences among the infiltrating macrophages included numbers of CD68-positive cells being significantly more abundant in the harbor porpoises, whereas CD163-positive cells were significantly more numerous in the Dall's porpoises. The numbers of CD204-positive macrophages were higher in the Hubbs' beaked whales compared to those in the porpoises. Trematode species of the harbor and Dall's porpoises were Campula oblonga, while they were Oschmarinella macrorchis in the Hubbs' beaked whales. This study concludes that interspecies differences in the tissue reactions to hepatic trematode infections are present among odontocete species and that the immune reaction varies depending on the species. This information aids in furthering our understanding of the pathogenesis of hepatic trematodiasis in cetaceans.

Dataset Information

Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation.

Publications

Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation.

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