All of the experiments shown were performed in low throughput 15-well Angiogenesis slides (Ibidi GmbH, Munich, Germany), and growth factor-reduced Matrigel (BD Invitrogen) as the ECM of choice to promote differentiation. Miniaturized 3D cultures were prepared as described previously [6] (link), [7] (link). Bottom wells of ibidi Angiogenesis μ-slides were filled with 10 µl of 50% Matrigel-medium (typically 3–5 mg/ml protein, depending on the batch), and incubated at +37°C for 30–60 min. Cells were placed on top of the polymerised bottom gel at a density of 700–1500 cells/well (depending on the cell line), and incubated at +37°C for 1–2 h. Medium was discarded, and cell layers covered with 20 µl of 25% Matrigel (1.5–2.5 mg/ml depending on the batch). The μ-slides were humidified by adding 15 µl drops of sterile water between the wells. The upper gel was allowed to polymerize at +37°C for 3–4 h or overnight. Wells were then filled with medium, and changed every second day.
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