Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Epithelial cell guidance by self-generated EGF gradients †

doi: 10.1039/c2ib00106c

Figure Lengend Snippet: Epithelial cells can spontaneously migrate through mazes in the absence of pre-existent chemical gradients. (a) Migration of one PC9 cell through a maze. The maze combines one short path, one long path, and numerous dead end channels. Fluorescent images of GFP–tubulin PC9 cells at 1, 3, 4.5, 6, 7, 8.5 and 10 hours are overlapped on a phase contrast image of the microfluidic maze. Scale bar is 50 µm. (b) Percentages of HMEC (human mammary epithelial cells), HPC (human pancreatic cells), PC9 and MDA MB231 cell lines moving through the maze along the short (red) or the long (green) paths or getting trapped (yellow) inside the maze. N represents the number of cells observed moving through the mazes. Only the first cell entering each maze was analyzed. (c) Schematics of the self-ordination strategy of epithelial cells through mazes. Immediately after seeding the cells in the cell compartment, the concentration of EGF is uniform throughout the device (t = 0). As cells consume EGF a gradient is established between the cell and outer compartments (t = 1 h). The process depends on the consumption of EGF (1) and its diffusion through the channels (2) from the outer compartment. The gradient that is subsequently formed can guide the directional migration of cells (3) from the cell compartment to the outer compartment. (d) Simulation of normalized concentration gradient in front of a moving cell in a straight channel at t = 1 h, plotted vs. diffusion constant D and normalized consumption rate rc. The gradient was normalized so that a value of 1 indicates a linear decrease in concentration along the entire length of the channel. Thick solid lines indicate iso-gradient contours. Thin solid lines indicate similar contours when consumption by the moving cell is neglected; the similarity of these lines to the thick ones indicates that the contribution of moving cells to the overall gradient was minimal for most conditions. Dashed lines indicate contours when consumption by cells in the central well is neglected. Ovals indicate regions of D and rc corresponding to several biologically relevant molecules for the cell types used in this study. The gradient for oxygen is quite small; for glucose and glutamine, moderate; for EGF, very high.

Article Snippet: For experiments targeting the role of EGF in epithelial cell migration, assay media were prepared by adding different concentrations of EGF (from 0 to 10 ng mL −1 ) to the epithelial growth media (MEGM, Lonza) supplemented with bovine pituitary extract, hydrocortisone, insulin and gentamicin/ amphotericin-B (Lonza).

Techniques: Migration, Concentration Assay, Diffusion-based Assay

Journal: Integrative biology : quantitative biosciences from nano to macro

Article Title: Epithelial cell guidance by self-generated EGF gradients †

doi: 10.1039/c2ib00106c

Figure Lengend Snippet: Epithelial cell EGF-uptake changes the orientation bias at bifurcations. (a) Microscopy image of one MDA cell migrating past the channel junction (orange dot), from the cell compartment (blue dot) toward the outer compartment (green dot). The ratio between the lengths of the channels toward the outer compartment and the side chamber (yellow dot) was 3 : 1 for the device to the left and 1 : 1 for the device to the right. (b) Inversion of gradient slope toward the outer compartment and the side chamber due to cellular EGF uptake, for 3 : 1 length channel ratio and 1/2× EGF concentration in the side chamber. In the absence of cellular EGF uptake, a large positive gradient toward the outer compartment (green dot) and a negative gradient toward the side chamber (yellow dot) were predicted. When cellular EGF uptake is taken into consideration, the EGF concentration in the cell compartment decreases (blue dot) and a stronger gradient toward the side chamber compared to the one toward the outer compartment develops by the time a cell arrives at the junction (orange dot). (c and d) Bias of HMEC, HPC, and PC9 cells toward side chambers with various concentrations of EGF (1× in the outer compartment vs. 5×, 1×, 1/2×, 1/10× in the side chamber) and 1 : 1 ratio between the length of the channels after bifurcation, toward the outer compartment and the side chamber (c), or 3 : 1 ratio (d). Epithelial cells consistently migrate toward the higher concentration of EGF when the ratio of the two channels is 1 : 1. Cells can migrate toward a lower concentration of EGF (1/2×) when the side chamber is positioned closer to the bifurcation (3 : 1 length ratio), suggestive of significant EGF concentration decrease at the bifurcation. N represents the number of cells observed moving through the bifurcating channels, only the first cell reaching the bifurcation was analyzed.

Article Snippet: For experiments targeting the role of EGF in epithelial cell migration, assay media were prepared by adding different concentrations of EGF (from 0 to 10 ng mL −1 ) to the epithelial growth media (MEGM, Lonza) supplemented with bovine pituitary extract, hydrocortisone, insulin and gentamicin/ amphotericin-B (Lonza).

Techniques: Microscopy, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Prostaglandin D 2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine

doi: 10.1084/jem.20022218

Figure Lengend Snippet: Effects of PGD 2 on expression of MDC mRNA in vitro. MDC mRNA was expressed in primary human bronchial epithelial cells in a dose-dependent manner to 10 −9 –10 −5 M PGD 2 after 48-h incubation, whereas TARC mRNA was constitutively expressed and not influenced by PGD 2 . These results are representative of three independent experiments.

Article Snippet: Cells were maintained in bronchial epithelial growth media (SABM; Sanko Junyaku) and supplemented with 7.5 g/liter of bovine pituitary extract, 0.5 g/liter hydrocortisone, 0.5 mg/l of human recombinant epidermal growth factor, 0.5 g/liter epinephrine, 10 g/liter transferrin, 5 g/liter insulin, 0.1 mg/l retinoic acid, 6.5 mg/l 3,3′,5-triiodo- l -thryonine, 50 g/liter bovine serum albumin, and 0.1% GA-1000 (Sanko Junyaku) in 25-cm 2 tissue plates at 37°C in 5% CO 2 .

Techniques: Expressing, In Vitro, Incubation