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Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy
Dhanesh Amarnani1, Arturo Israel Machuca-Parra1, Lindsay L Wong1
1Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States.
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.
Insights
Researchers developed a method to culture cells from proliferative vitreoretinopathy (PVR) membranes for drug testing. Methotrexate (MTX) was found to reduce PVR cell growth and induce cell death in these cultures.
Area of Science:
- Ophthalmology and cell biology.
- Investigating cellular mechanisms in retinal diseases.
Background:
- Proliferative vitreoretinopathy (PVR) is a severe complication of retinal detachment.
- Current drug testing methods for PVR lack patient-specific cellular models.
Purpose of the Study:
- To establish a reliable method for isolating and culturing cells from patient-derived PVR membranes.
- To create an in vitro model for drug screening and testing against PVR.
Main Methods:
- PVR membranes from Grade C patients were obtained and processed.
- Cells were isolated, cultured, and characterized using immunohistochemistry.
- The effects of methotrexate (MTX) on cell proliferation, migration, and death were evaluated.
Main Results:
- Successful isolation and culture of primary PVR cells (C-PVR) exhibiting characteristic markers.
- C-PVR cultures formed membrane and band-like structures in vitro.
- Methotrexate significantly inhibited C-PVR proliferation and band formation while inducing cell death.
Conclusions:
- Patient-derived PVR cells can be effectively isolated, cultured, and profiled in vitro.
- This novel cell culture system provides a valuable platform for PVR drug discovery.
- Methotrexate demonstrates potential as a therapeutic agent by reducing PVR cell growth and inducing apoptosis.
