Experimental strategies involving the three-dimensional (3D) culture of epithelial cells recapitulate numerous features of epithelium in vivo, overcoming monolayer cell growth limitations. Indeed, monolayer cells do not provide the optimal system to understand, for instance, how proliferation, cell death and differentiation influence epithelium formation and functions, both in physiological state and during tumor formation.
Here we show mammary epithelial cells grown in 3D. In particular, MCF-10A cells (an immortalized non-transformed human epithelial cell line derived from the breast tissue of a 36-year-old patient with fibrocystic changes) has been grown for 7 days in culture medium HUMEC with 2% Matrigel (for further method details see Debnath et al.,Methods, 2003) and subsequently stained by Hoechst 33342 to visualize nuclei. Under these growth conditions, 3D organized and polarized acinar units have been formed starting from single cells seeded on a basement membrane gel. Noteworthy, in literature it has been reported that this 3D epithelial culture method helps to recapitulate numerous features of breast epithelium in vivo, such as the formation of acini-like spheroids, cell polarization, and, in some cases, the production of milk proteins. Moreover, it provides an important tool to characterize how cancer genes disrupt glandular architecture during carcinoma progression.
Acini images have been acquired with a Nikon Ti2 inverted microscope equipped with CrestOptics confocal spinning disk X-Light V3, camera Photometrics BSI (6.5 um pixel size) and Celesta laser source (Lumencor). In Figure A, we shown a maximum intensity projection of a Z stack acquired with a 20x objective (Nikon, Plan Apo Lambda NA 0.75) displaying different size and shapes of acini in culture and a movie with 3D acini reconstruction. Focusing on the 3D structures of interest, we used a 60x objective (Nikon,Plan Apo Lambda 60X, Oil, NA 1.4) to better appreciate cellular organization along the Z axis (Figure B_1) and in 3D volume and orthogonal views (Figure B_2). Finally, we were interested in morphological nuclei analysis and by thresholding based on nuclei fluorescence intensity it was possible to identify each single object (nucleus; Figure C) and consequently perform measurements such as nucleus volume, orientation, sphericity, elongation (data not shown).
To conclude, 3D culture system provides the appropriate context for examining the biological activities not only during physiological epithelial growth but also in cancer onset and development, even representing a tool in the search for new diagnostic markers and therapeutic drug tests in cancer treatment.
maximum intensity projection and movie with 3D reconstruction
sequence of single Z plans (from top to bottom of the acini)
Figure B_2: 3D volume and orthogonal views
Figure C: 3D volume view and corresponding objects (nuclei) identification