Fibrillar collagen I, the most abundant structural protein in mammary glands, plays an important role in normal development as well as in breast cancer. Our previous studies have demonstrated that collagen I induces a conserved response of protrusive invasion in both normal and tumor organoids [23 (link)]. This section describes how to properly prepare preassembled collagen I and embed mammary organoids in a 3D gel.

Use a plate with a glass bottom for time-lapse imaging.

Use 20-30 μL of neutralized collagen to make a thin underlay on the cover glass of the well at room temperature (Fig. 5e). The underlay helps the top collagen/organoid suspension attach better to the cover glass.

Incubate the plate with the underlays at 37 °C until ready for plating.

Preincubate the neutralized collagen I solution (used for the top gel) at 4 °C for 60–120 min for preassembly [24 (link)] (seeNote 14). The collagen I solution will turn cloudy and fibrous (Fig. 5d1–6) (seeNote 15), a state we term preassembled collagen I.

Set up the tissue culture hood in preparation for plating (Fig. 4a).

Set the heating block to 37 °C, and place the plate on top, in direct contact with the block (Fig. 4b–c′) (seeNote 10).

Always keep the collagen I solution on ice. Add the desired amount of preassembled collagen I to the organoid pellet in a microcentrifuge tube. Since collagen I is quite viscous, first pipette up and down slowly a few times to coat the tip and ensure an accurate volume.

Keep the tube on ice or in a cold block. Resuspend the organoid pellet gently to avoid introducing air bubbles. Do not try to take up the entire volume into the pipette tip while mixing.

Plate the appropriate volume of collagen/organoid suspension (see table in Subheading 3.4) on top of the underlay (Fig. 5e′). Pipette up and down to resuspend the organoids before plating each sample, and pipette out only until the first stop.

Keep the plate on the heating block for several minutes to allow further gelation before returning it to the incubator (seeNote 16).

Incubate the plate at 37 °C, 5 % CO2, for 45–60 min. After gelation, collagen I fibrils are visible under the microscope at 10× and 40× (Fig. 5f-f′).

Gently add pre-warmed organoid medium supplemented with growth factor to the wells. A variety of growth factors may be used, including EGF ligands (EGF, TGF-α, amphiregulin, heregulin, neuregulin), FGF ligands (FGF2, FGF7), and HGF. We most commonly use 2.5 nM FGF2.

Add sterile water or PBS to the empty wells to prevent desiccation.

Label the wells. Return the plate to the incubator.

If the plate will be used for DIC imaging, use a glass plate cover for better image quality.