Influence of basement membrane proteins and endothelial cell-derived factors on the morphology of human fetal-derived astrocytes in 2D.

Astrocytes are the most prevalent type of glial cell in the brain, participating in a variety of diverse functions from regulating cerebral blood flow to controlling synapse formation. Astrocytes and astrocyte-conditioned media are widely used in models of the blood-brain barrier (BBB), however, very little is known about astrocyte culture in 2D. To test the hypothesis that surface coating and soluble factors influence astrocyte morphology in 2D, we quantitatively analyzed the morphology of human fetal derived astrocytes on glass, matrigel, fibronectin, collagen IV, and collagen I, and after the addition soluble factors including platelet-derived growth factor (PDGF), laminin, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF). Matrigel surface coatings, as well as addition of leukemia inhibitory factor (LIF) to the media, were found to have the strongest effects on 2D astrocyte morphology, and may be important in improving existing BBB models. In addition, the novel set of quantitative parameters proposed in this paper provide a test for determining the influence of compounds on astrocyte morphology, both to screen for new endothelial cell-secreted factors that influence astrocytes, and to determine in a high-throughput way which factors are important for translation to more complex, 3D BBB models

Influence of surface coatings on astrocyte morphology.

<p>(A) The cell area defined by the area of the cell body. (B) the cell diameter is overall size defined by the diameter of the smallest circle that can enclose the cell and all of its processes. (C) The protrusion length is the total length of all protrusions. (D) The degree of branching is the number of branch points divided by the number of primary protrusions. (E) The number of primary protrusions represents the number of protrusions emanating from the cell body. (F) The number of secondary protrusions represents protrusions emanating from primary protrusions. (G) The number of tertiary protrusions represents protrusions emanating from secondary protrusions. (H) The number of branch points represents the sum of secondary and other higher order protrusions (equivalent to the number of bifurcations). Data represent mean ± SE. Statistical significance was determined using a student’s t-test test. ***P≤0.01, **P≤0.05, *P≤0.1. Only cells with astrocyte-like morphology were analyzed (the total number of cells and the fraction of cells with astrocyte-like morphology are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092165#pone-0092165-g001" target="_blank">Figure 1</a>).</p

Influence of ECM coating on astrocyte morphology after 24 hours.

<p>Fluorescence images of astrocytes stained for GFAP (green) and DAPI (blue) on (A) glass, (B) collagen I, (C) collagen IV, (D) fibronectin, (E) matrigel, and (F) co-culture on a confluent monolayer of HBMECs. (G) Astrocyte (from panel (F)) seeded on a confluent monolayer of HBMECs, stained for GFAP (green), DAPI (blue), and ZO-1 (red). (H) The percentage of cells with protrusions. Total number of cells analyzed: uncoated (N = 103), collagen I (N = 85), collagen IV (N = 61), fibronectin (N = 63), matrigel (N = 54), co-culture (N = 58).</p