Limits...
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.

Show MeSH

Related in: MedlinePlus

BV2 microglia are viable in three-dimensional (3D) collagen gels. (A) BV2 microglia in monolayer, coated monolayer and 3D culture conditions were assessed for lactate dehydrogenase (LDH) activity at 48 hrs. Data are mean ± SD from three independent experiments. (B) Cells were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) and propidium iodide (PI) to assess viability at 48 hrs post-culture. LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: BV2 microglia are viable in three-dimensional (3D) collagen gels. (A) BV2 microglia in monolayer, coated monolayer and 3D culture conditions were assessed for lactate dehydrogenase (LDH) activity at 48 hrs. Data are mean ± SD from three independent experiments. (B) Cells were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) and propidium iodide (PI) to assess viability at 48 hrs post-culture. LPS, lipopolysaccharide.

Mentions: To examine the morphology of microglia in the various culture systems, we performed staining of cells with FITC-tagged lectin. Microglia cultured on uncoated monolayer surfaces mostly exhibited round cytoplasm with some bipolar projections (Figure 2A). In collagen-coated monolayer cultures, the extent of amoeboidal morphology seemed increased (Figure 2B) with cytoplasmic area appearing minimal, indicating the extent of deramification. The morphology of cells cultured in 3D collagen was distinct from monolayer, with clear multiplanar projections (Figure 2C). The microglia were suspended within the collagen matrix and evenly distributed across the width of the gel. When viewed with confocal microscopy, the extent of ramification of microglia cultured in the 3D matrix was evident, with cells displaying long and multidirectional cellular projections (Figure 2F and video in Additional file2).To determine whether microglia cultured in 3D were viable, the lactate dehydrogenase (LDH) assay and DAPI/PI staining were performed. Compared to the respective positive controls, BV2 microglia in all three culture formats showed low LDH release (Figure 3A). Even following treatment with 1 μg/ml LPS, LDH levels in the culture supernatants remained low. Additionally, BV2 microglia showed negligible PI staining at 24 (data not shown) and 48 hrs post-culture in 3D collagen (Figure 3B).


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 are viable in three-dimensional (3D) collagen gels. (A) BV2 microglia in monolayer, coated monolayer and 3D culture conditions were assessed for lactate dehydrogenase (LDH) activity at 48 hrs. Data are mean ± SD from three independent experiments. (B) Cells were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) and propidium iodide (PI) to assess viability at 48 hrs post-culture. LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: BV2 microglia are viable in three-dimensional (3D) collagen gels. (A) BV2 microglia in monolayer, coated monolayer and 3D culture conditions were assessed for lactate dehydrogenase (LDH) activity at 48 hrs. Data are mean ± SD from three independent experiments. (B) Cells were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) and propidium iodide (PI) to assess viability at 48 hrs post-culture. LPS, lipopolysaccharide.
Mentions: To examine the morphology of microglia in the various culture systems, we performed staining of cells with FITC-tagged lectin. Microglia cultured on uncoated monolayer surfaces mostly exhibited round cytoplasm with some bipolar projections (Figure 2A). In collagen-coated monolayer cultures, the extent of amoeboidal morphology seemed increased (Figure 2B) with cytoplasmic area appearing minimal, indicating the extent of deramification. The morphology of cells cultured in 3D collagen was distinct from monolayer, with clear multiplanar projections (Figure 2C). The microglia were suspended within the collagen matrix and evenly distributed across the width of the gel. When viewed with confocal microscopy, the extent of ramification of microglia cultured in the 3D matrix was evident, with cells displaying long and multidirectional cellular projections (Figure 2F and video in Additional file2).To determine whether microglia cultured in 3D were viable, the lactate dehydrogenase (LDH) assay and DAPI/PI staining were performed. Compared to the respective positive controls, BV2 microglia in all three culture formats showed low LDH release (Figure 3A). Even following treatment with 1 μg/ml LPS, LDH levels in the culture supernatants remained low. Additionally, BV2 microglia showed negligible PI staining at 24 (data not shown) and 48 hrs post-culture in 3D collagen (Figure 3B).

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