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A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia.

Haw RT, Tong CK, Yew A, Lee HC, Phillips JB, Vidyadaran S - J Neuroinflammation (2014)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroinflammation Group, Immunology Laboratory, Department of Pathology, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. sharmili@upm.edu.my.

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.

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BV2 microglia cultured in three-dimensional (3D) collagen gels express nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. BV2 microglia in monolayer, coated monolayer and 3D culture conditions were activated with 1 μg/ml LPS and assessed for NO2- expression at 36 and 48 hrs with the Griess assay. Data are mean ± SD from three independent experiments.
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Figure 5: BV2 microglia cultured in three-dimensional (3D) collagen gels express nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. BV2 microglia in monolayer, coated monolayer and 3D culture conditions were activated with 1 μg/ml LPS and assessed for NO2- expression at 36 and 48 hrs with the Griess assay. Data are mean ± SD from three independent experiments.

Mentions: To assess NO production by BV2 microglia in monolayer and 3D cultures, cells were seeded in the different culture formats with equal seeding number per well. Microglia in monolayer and 3D cultures had negligible NO production (<3 μM at both 36 and 48 hrs; Figure 5), indicating that the culture conditions alone do not induce NO expression. Following stimulation with LPS, BV2 microglia in all culture formats showed an induction of NO expression, approximately 4 to 8 times higher at 36 hrs and 6 to 10 times higher at 48 hrs compared to untreated BV2 (P < .05). Negligible NO induction was detected at 24 hrs post-LPS (data not shown). At 36 hrs, BV2 grown as uncoated monolayer cultures produced the highest amount of NO (20.03 ± 0.76 μM), followed by collagen-coated monolayer cultures (6.59 ± 2.39 μM) and 3D cultures (10.54 ± 1.46 μM). These levels increased for uncoated monolayer, collagen-coated monolayer and 3D culture at the 48-hr time point to 26.96 ± 1.76 μM, 17.87 ± 2.27 μM and 24.47 ± 1.75 μM NO, respectively. The NO expression by LPS-treated BV2 cells in 3D cultures was not significantly different compared to microglia cultured in uncoated or collagen-coated monolayer cultures at either time points (P > .05; Kruskal-Wallis with Dunn’s multiple comparison test).


A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia.

Haw RT, Tong CK, Yew A, Lee HC, Phillips JB, Vidyadaran S - J Neuroinflammation (2014)

BV2 microglia cultured in three-dimensional (3D) collagen gels express nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. BV2 microglia in monolayer, coated monolayer and 3D culture conditions were activated with 1 μg/ml LPS and assessed for NO2- expression at 36 and 48 hrs with the Griess assay. Data are mean ± SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128540&req=5

Figure 5: BV2 microglia cultured in three-dimensional (3D) collagen gels express nitric oxide (NO) in response to lipopolysaccharide (LPS) stimulation. BV2 microglia in monolayer, coated monolayer and 3D culture conditions were activated with 1 μg/ml LPS and assessed for NO2- expression at 36 and 48 hrs with the Griess assay. Data are mean ± SD from three independent experiments.
Mentions: To assess NO production by BV2 microglia in monolayer and 3D cultures, cells were seeded in the different culture formats with equal seeding number per well. Microglia in monolayer and 3D cultures had negligible NO production (<3 μM at both 36 and 48 hrs; Figure 5), indicating that the culture conditions alone do not induce NO expression. Following stimulation with LPS, BV2 microglia in all culture formats showed an induction of NO expression, approximately 4 to 8 times higher at 36 hrs and 6 to 10 times higher at 48 hrs compared to untreated BV2 (P < .05). Negligible NO induction was detected at 24 hrs post-LPS (data not shown). At 36 hrs, BV2 grown as uncoated monolayer cultures produced the highest amount of NO (20.03 ± 0.76 μM), followed by collagen-coated monolayer cultures (6.59 ± 2.39 μM) and 3D cultures (10.54 ± 1.46 μM). These levels increased for uncoated monolayer, collagen-coated monolayer and 3D culture at the 48-hr time point to 26.96 ± 1.76 μM, 17.87 ± 2.27 μM and 24.47 ± 1.75 μM NO, respectively. The NO expression by LPS-treated BV2 cells in 3D cultures was not significantly different compared to microglia cultured in uncoated or collagen-coated monolayer cultures at either time points (P > .05; Kruskal-Wallis with Dunn’s multiple comparison test).

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroinflammation Group, Immunology Laboratory, Department of Pathology, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. sharmili@upm.edu.my.

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.

Show MeSH
Related in: MedlinePlus