Cargando…
Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy
PURPOSE: The purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing. METHODS: PVR membranes were obtained from six patients with grade C PVR. Membrane fragmen...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Association for Research in Vision and Ophthalmology
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544356/ https://www.ncbi.nlm.nih.gov/pubmed/28777835 http://dx.doi.org/10.1167/iovs.16-20912 |
_version_ | 1783255248580640768 |
---|---|
author | Amarnani, Dhanesh Machuca-Parra, Arturo Israel Wong, Lindsay L. Marko, Christina K. Stefater, James A. Stryjewski, Tomasz P. Eliott, Dean Arboleda-Velasquez, Joseph F. Kim, Leo A. |
author_facet | Amarnani, Dhanesh Machuca-Parra, Arturo Israel Wong, Lindsay L. Marko, Christina K. Stefater, James A. Stryjewski, Tomasz P. Eliott, Dean Arboleda-Velasquez, Joseph F. Kim, Leo A. |
author_sort | Amarnani, Dhanesh |
collection | PubMed |
description | PURPOSE: The purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing. METHODS: PVR membranes were obtained from six patients with grade C PVR. Membrane fragments were analyzed by gross evaluation, fixed for immunohistologic studies to establish cell identity, or digested with collagenase II to obtain single cell suspensions for culture. PVR-derived primary cultures were used to examine the effects of methotrexate (MTX) on proliferation, migration, and cell death. RESULTS: Gross analysis of PVR membranes showed presence of pigmented cells, indicative of retinal pigment epithelial cells. Immunohistochemistry identified cells expressing α-smooth muscle actin, glial fibrillary acidic protein, Bestrophin-1, and F4/80, suggesting the presence of multiple cell types in PVR. Robust PVR primary cultures (C-PVR) were successfully obtained from human membranes, and these cells retained the expression of cell identity markers in culture. C-PVR cultures formed membranes and band-like structures in culture reminiscent of the human condition. MTX significantly reduced the proliferation and band formation of C-PVR, whereas it had no significant effect on cell migration. MTX also induced regulated cell death within C-PVR as assessed by increased expression of caspase-3/7. CONCLUSIONS: PVR cells obtained from human membranes can be successfully isolated, cultured, and profiled in vitro. Using these primary cultures, we identified MTX as capable of significantly reducing growth and inducing cell death of PVR cells in vitro. |
format | Online Article Text |
id | pubmed-5544356 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | The Association for Research in Vision and Ophthalmology |
record_format | MEDLINE/PubMed |
spelling | pubmed-55443562017-08-07 Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy Amarnani, Dhanesh Machuca-Parra, Arturo Israel Wong, Lindsay L. Marko, Christina K. Stefater, James A. Stryjewski, Tomasz P. Eliott, Dean Arboleda-Velasquez, Joseph F. Kim, Leo A. Invest Ophthalmol Vis Sci Retina PURPOSE: The purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing. METHODS: PVR membranes were obtained from six patients with grade C PVR. Membrane fragments were analyzed by gross evaluation, fixed for immunohistologic studies to establish cell identity, or digested with collagenase II to obtain single cell suspensions for culture. PVR-derived primary cultures were used to examine the effects of methotrexate (MTX) on proliferation, migration, and cell death. RESULTS: Gross analysis of PVR membranes showed presence of pigmented cells, indicative of retinal pigment epithelial cells. Immunohistochemistry identified cells expressing α-smooth muscle actin, glial fibrillary acidic protein, Bestrophin-1, and F4/80, suggesting the presence of multiple cell types in PVR. Robust PVR primary cultures (C-PVR) were successfully obtained from human membranes, and these cells retained the expression of cell identity markers in culture. C-PVR cultures formed membranes and band-like structures in culture reminiscent of the human condition. MTX significantly reduced the proliferation and band formation of C-PVR, whereas it had no significant effect on cell migration. MTX also induced regulated cell death within C-PVR as assessed by increased expression of caspase-3/7. CONCLUSIONS: PVR cells obtained from human membranes can be successfully isolated, cultured, and profiled in vitro. Using these primary cultures, we identified MTX as capable of significantly reducing growth and inducing cell death of PVR cells in vitro. The Association for Research in Vision and Ophthalmology 2017-08 /pmc/articles/PMC5544356/ /pubmed/28777835 http://dx.doi.org/10.1167/iovs.16-20912 Text en Copyright 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. |
spellingShingle | Retina Amarnani, Dhanesh Machuca-Parra, Arturo Israel Wong, Lindsay L. Marko, Christina K. Stefater, James A. Stryjewski, Tomasz P. Eliott, Dean Arboleda-Velasquez, Joseph F. Kim, Leo A. Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title | Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title_full | Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title_fullStr | Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title_full_unstemmed | Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title_short | Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy |
title_sort | effect of methotrexate on an in vitro patient-derived model of proliferative vitreoretinopathy |
topic | Retina |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544356/ https://www.ncbi.nlm.nih.gov/pubmed/28777835 http://dx.doi.org/10.1167/iovs.16-20912 |
work_keys_str_mv | AT amarnanidhanesh effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT machucaparraarturoisrael effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT wonglindsayl effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT markochristinak effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT stefaterjamesa effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT stryjewskitomaszp effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT eliottdean effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT arboledavelasquezjosephf effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy AT kimleoa effectofmethotrexateonaninvitropatientderivedmodelofproliferativevitreoretinopathy |