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. 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. < 0.05 was considered statistically significant. Results Clinical Demographics Six patients with grade C PVR that required surgical excision were enrolled in the study. The demographics of the patients are summarized in the Table. All patients had recurrent rhegmatogenous retinal detachment due to PVR. One patient had a recent history of a zone 3 open globe injury with rhegmatogenous retinal detachment and retinal incarceration in the scleral wound. This patient underwent vitrectomy, but returned with recurrent rhegmatogenous retinal detachment due to PVR. Table Clinical Demographics Characterization by Immunohistochemistry of PVR Membranes and C-PVR A total of six grade C PVR membranes were surgically excised from patients with retinal detachment (Fig. 1A) AV-951 and processed in the laboratory. Under light microscopy, PVR membranes grossly appeared to consist of pigmented and nonpigmented cells embedded in a fibrous matrix (Fig. 1B). We examined the cellular constituents of PVR membranes using immunohistochemistry in specimens PVR-02, PVR-03, and PVR-05. Staining of these samples revealed positive localization of SMA a marker of myofibroblasts.38 CD14, a marker expressed by most tissue macrophages,39 displayed more robust expression in PVR-02 compared with PVR-03. BEST-1, a marker for RPE cells,40 did not show expression in PVR-02 yet was localized to the pigmented cells of PVR-03, suggesting these cells were derived from the RPE. Both cytokeratin, found in epithelial cells,41 and GFAP, found in astrocytes,42 were not expressed in PVR-02, whereas PVR-03 showed low expression AV-951 for both markers. Interestingly, PVR-05 showed positive signal for all these markers (Figs. 1GC1U). Figure 1 Culture of human PVR membranes and histopathology of PVR membranes. (A) Fundus photograph of the left eye of a patient, case 6 (PVR-06) with recurrent retinal detachment; note presence of a gas bubble within AV-951 the eye from previous retinal surgery. There … Establishment of C-PVR Primary Cultures Cells from these PVR membranes, which we called C-PVR, were CD164 successfully isolated in each case and cultured as described in the Methods section (Figs. 1CC1F; Supplementary Fig. S2). Distinct populations of C-PVR cells contained AV-951 pigmented granules characteristic of RPE cells (Fig. 1B). SMA-positive cells were abundant in C-PVR derived from PVR-03, PVR-04, and PVR-05 but not from PVR-02 (Figs. 2AC2D), suggesting the presence of myofibroblasts in culture. Other cell populations, including GFAP-positive cells, were also identified in the cell culture for PVR-02, PVR-03, and PVR-04 (Figs. 2EC2G), with nonspecific staining of PVR-05 (Fig. 2H), suggesting the presence of stellate shaped glial cells. Cytokeratin was positive for all cells in culture for PVR-03 and PVR-05 but was rare in PVR-02 and PVR-04 (Figs. 2IC2L), whereas F4-80, a marker for macrophages and microglial cells, was positive in PVR-03 and PVR-05 but negative in PVR-02 and PVR-04 cultures (Figs. 2MC2P). These findings suggest that C-PVR cells retain the expression of some markers expressed in the patient-derived tissue. Figure 2 Characterization of cultured cells by immunofluorescence. Immunofluorescence of AV-951 C-PVR cells from four different cases using primary.