On-chip phenotyping and harvesting of cells.

<p>(A) DCs were loaded localized in the middle compartment of the migration chamber in a collagen gel (1.7 mg/mL) and allowed to migrate for 2h in cell culture medium or in a soluble CCL19 gradient (5 μg/mL). Cells were stained on-chip with an anti-mouse I-A/I-E antibody. Scale bar, 100 μm. (B) Quantification of anti-I-A/I-E (MHC class II) staining of cells. Corrected Total Cell Fluorescence (CTCF) (= Integrated Density–Area of selected cell * mean fluorescence of background readings) is shown relative to Y-position in migration chamber where 0 μm indicates the chemokine source and 1700 μm the sink of the gradient system. (C) Nuclear staining of DCs with DRAQ5, cells are shown in the microfluidic chamber prior harvesting (left panel, scale bar: 100 μm) and in micro well following harvest (right panel, scale bar: 200 μm).</p

Quantification of microfluidic 2D and 3D migration.

<p>(A) Distribution of migration tracks of cells migrating on fibronectin (100 μg/mL) or in a collagen gel (1.7 mg/mL). Rose plot summarizing frequency of directions relative to the starting point. (B) Quantification of migration characteristics of cells migrating on fibronectin (100 μg/mL, FN) or in a collagen gel of various densities (col conc = collagen concentration). Chemotactic Index is a measure of chemotactic efficiency (calculated by dividing the distance from start to end point of cells in axis of the gradient by the total migration distance of every cell). Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. Not significant (ns) ρ ≥ 0.05; * ρ < 0.05; **** ρ < 0.0001. (C) Representative images of DC morphology in microfluidic 2D and 3D environments and mouse ear tissue. Scale bar, 10 μm. (D) Quantification of cell morphology of cells migrating on fibronectin (100 μg/mL), in a collagen gel (1.7 mg/mL) or in mouse ear tissue. Elongation factor = cell length divided by its perpendicular cell width. FN = fibronectin, col = collagen 1.7 mg/mL, crawl-in = mouse ear crawl-in migration assay. Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. ** ρ < 0.01; *** ρ < 0.001.</p

Geometry and functionality of collagen migration chip.

<p>(A) Photograph of the device, control channels are stained in red, flow channels are depicted in blue color. Inset shows air-filled migration chamber. (B) Schematic overview of device, inset shows migration chamber in detail. (C) Schematic overview of the functionality and workflow of the migration device. Insets show localized loading in migration chamber and tracking of dendritic cells migrating along a gradient of CCL19. Scale bar, 100 μm. (D) Comparison of gradient generation across migration chamber coated with fibronectin (100 μg/mL) and filled with cell culture medium or filled with collagen matrix (1.7 mg/mL). Gradient is visualized with a 10 kDa AF647-Dextran.</p

Reproducibility of microfluidic 3D migration assay.

<p>(A) Analysis of spontaneous migration of DCs in 4 distinct collagen preparations (collagen concentration: 1.7 mg/mL). Cells were tracked for 90 min during culture in cell culture medium only. Panel 1 exemplarily shows migration tracks plotted to a common starting point, migration characteristics are shown in panel 2–3. Chemotactic Index is a measure for chemotactic efficiency (calculated by dividing the distance from start to end point of cells in axis of the gradient by the total migration distance of every cell). (B) DCs were tracked for 90 min during diffusion-based soluble CCL19 gradient (maximal CCL19 concentrations: 5 μg/ml). Panel 1 exemplarily shows migration tracks plotted to a common starting point, migration characteristics are shown in panel 2/3. Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. Not significant (ns) ρ ≥ 0.05.</p