A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia
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Haw et al. Journal of Neuroinflammation 2014, 11:134 http://www.jneuroinflammation.com/content/11/1/134
RESEARCH
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A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia Randy Tatt Yhew Haw1†, Chih Kong Tong1†, Andrew Yew1, Han Chung Lee2, James B Phillips3 and Sharmili Vidyadaran1*
Abstract Background: We report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma. Methods: BV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison. Results: BV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 μg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P < .05). BV2 microglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation. Conclusions: In summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation. Keywords: Microglia, Lipopolysaccharide, Collagen matrix, Three-dimensional cultures
Background Microglia are tissue-specific macrophages of the central nervous system (CNS) and derive from primitive haematopoietic progenitors of erythromyeloid origin [1,2]. These mononuclear phagocytes are the resident immune cells of the CNS, along with other subsets of mononuclear phagocytes including meningeal macrophages, choroid plexus macrophages and perivascular macrophages [3]. Microglia in the brain are disseminated * Correspondence: [email protected] † Equal contributors 1 Neuroinflammation Group, Immunology Laboratory, Department of Pathology, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Full list of author information is available at the end of the article
throughout the parenchyma and are highly motile. In the healthy mature CNS, microglia exist mainly in a ramified form, continuously traversing the CNS and using their cytoplasmic processes to sample the tissue environment [4]. Althoug
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