Development of a High-Throughput Drug Screening System Based on Human Peripheral Blood Derived Microglia-like Cells from Sickle Cell Patients with Chronic Pain

Abstract

Background: Targeting microglia activation is a therapeutic strategy and a clinically promising drug target to treat microglia involved diseases, such as chronic pain, neurodegenerative disorders. Despite the interest in studying microglia as drug targets, drug discovery has been stymied by species related differences and lack of healthy human CNS microglia in the current models, Importantly, the current model systems do not faithfully recapitulate pathological characteristics of patients or disease processes, leading to the failure of many drug candidates in clinical trials. To address these shortcomings, we developed an efficient suspension culture system to generate peripheral blood derived microglia like cells that can express molecular markers, functional characteristics like true microglia and preserved patient-specific phenotype. As a novel, cost-effective, efficient, and patient-specific microglia model, the system can be applied to screen for compounds that reduce microglia hyperactivity, thereby identifying promising pharmacologic agents to be developed to treat chronic pain. Methods: Human peripheral blood mononuclear cells obtained from patients with sickle cell disease (SCD) were cultured in a suspension culture system for 6 days to generate high-throughput assay-ready human microglia-like cells (HPB-MLC) with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL). The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with imaging flow cytometry. Microglial activation of HPB-MLC was induced with LPS (100 ng/mL) and characterized by increased expression of TNF-alpha and microglial activation marker CD68. To evaluate pharmaceutical blockade of microglial activation, HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM) for 24 hours. Commercial homogeneous cell-based assay kits based on HTRF (Homogeneous Time Resolved Fluorescence) and AlphaScreen technologies were used to develop the high-throughput screening (HTS) and miniaturizable assay. Results: Peripheral blood mononuclear cells can be efficiently differentiated into microglia-like cells in a suspension culture system. The suspension culture system enables fast and uniform seeding of cells to 384 well microplate for HTS applications. HPB-MLC express microglia specific markers, microglial phagocytic activity, and can be activated by LPS. HPB-MLC retain the patient pain phenotype: HPB-MLC generated from SCD patients with chronic pain exhibited higher phagocytic activity than that from SCD patients without chronic pain (Figure 1). We developed a cell-based, high-throughput screening assay by combination of our patient-specific microglia model and commercial homogeneous, high-throughput assay technologies HTRF and AlphaScreen (Figure 2). Suspension culture allowed 5 times higher cell concentration than adherent culture, which greatly reduced cost of cytokines in culture and produced assay-ready frozen HPB-MLC. To evaluate the possibility of using the HPB-MLC model system to screen compounds to inhibit microglia activation, we tested HPB-MLC cells with the several microglial inhibitors: minocycline, MRS2395, and clopidogrel. All the drugs significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner. Conclusions: Peripheral blood monocytes can be efficiently differentiated into MLC in a suspension culture system, using for drug screening as a patient-specific microglia model. The differentiation in suspension culture worked very well in higher cell concentration (5 times higher than adherent culture), which greatly reduced cost of cytokines in culture. Assay-ready frozen HPB-MLC as a bank of uniform aliquots can be directly used for HTS applications. HTRF and AlphaScreen technologies were used to replace traditional ELISA assays to detect inflammatory cytokines, providing a highly sensitive, rapid, homogeneous and miniaturizable method to efficiently perform high throughput drug screening. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal