Protocol for biomimetic tumoroid models by plastic compression using centrifugation

Abstract

Here, we present a protocol for engineering biomimetic tumoroid models by plastic compression using centrifugation. We describe steps for generating multi-compartment tumor-stroma models by mixing cells into a collagen hydrogel crosslinked at 37°C and centrifuging the hydrogel. We then detail procedures for generating compartmentalized models and encapsulating the final layered hydrogel containing a 96-well tumor mass in a 24-well stroma. This protocol increases collagen density and improves mechanical properties of collagen hydrogels.

Keywords: Cancer; Cell-based Assays; Tissue Engineering.

Graphical abstract

Figure 1

Expected color change during neutralization of Collagen mixture The approximate color change expected following the dropwise addition of the neutralizing solution. The color should change from orange (A) to salmon pink (B).

Figure 2

A compressed tumoroid with TM and surrounding stromal compartment, following the centrifugation process

Figure 3

Biomechanical profiles of collagen hydrogel, absorbed gels and centrifuged gels (A) Quantification of collagen density in absorbed (n = 5, sd = 0.9485) and centrifuged (n = 8, sd = 0.2966) gels as percentages (w/v), p = 0.0095. (B) Average shear elastic modulus of different gels: Hydrogel, n = 3. Centrifuged, n = 3. Absorbed, n = 3. For ∗∗p = 0.0068. For ∗∗∗∗p < 0.0001. Error bars represent SEM. (C) Phase angle graph of gels, for significant data, for ∗∗∗∗p < 0.0001. (D) Amplitude sweep graphs of different gels with end of LVE region annotated with a red dotted line. All tests were conducted using a roughened immersion well geometry and a 20 mm roughened steel plate top geometry.

Figure 4

Cancer cell growth within tumoroids (A–C) Prestoblue fluorescence of centrifuged and absorbed tumoroids measured every 7 days for 21 days of MCF7 (A), n = 4, and MDA-MB-231 cells (B), n = 4. Spheroid size of MCF-7 cells (C) centrifuged and absorbed tumoroids at day 1 and day 7 (n = 4) quantified in Image J from phase contrast images. (D) Representative phase contrast images of morphology of MDA-MB-231, and MCF-7 cells in absorbed and centrifuged tumoroids respectively (Scale bar represents 1,000 μm). ∗p < 0.05, ∗∗p <0.01, ∗∗∗p < 0.001.

Figure 5

Cancer invasion in tumoroid models (A) Representative images (n = 3) at days 7, 14 and 21 of centrifuged and absorbed tumoroids with 50K MDA-MB-231 cells seeded in the TM embedded in an acellular stroma. All images in this figure were fixed, blocked, stained with Phalloidin (red) and DAPI (blue), imaged on an Axio Observer Zeiss microscope, and quantified using ImageJ. Scale bar represents 500 μm. (B) Quantifications of MDA-MB-231 invasion distance and surface area into the stromal compartment. Invasion markers were confirmed by qPCR (data not shown). (C) Representative images (n = 3) at days 7, 14 and 21 of centrifuged and absorbed tumoroids with 50K MCF-7 cells seeded in the TM embedded in an acellular stroma. (D) Quantifications of MCF-7 invasion distance and surface area into the stromal compartment. ∗∗p < 0.01.