The overall goal of this physiologically-relevant 3D culture method, is to test the action of the main mammogenic hormones on mammary epithelial morphogenesis. This method can help answer key questions in the endocrinology and mammary gland biology fields, such as how do estrogen, progesterone, and prolactin influence mammary epithelial morphogenesis. The main advantage of this standardized method is that hormone-sensitive mammary epithethial cells form 3D structures in response to hormones, in a manner similar to what is observed in vivo.

The implications of this technique extend toward breast cancer therapies, as it is a physiologically-relevant culture system of the complex human breast epithelium. We first had the idea for this method because we notice how lack of standardized in vitro models for studying how human breast morphogenesis is influenced by hormones. Begin by counting the number of cells in a single cell suspension of T47D cells.

The way the cells are maintained is critical for retention of hormone-responsive phenotypes. We recommend frequent checks for a trial responsiveness in the T47D cells, on the fourth passage after thawing a new vial, and every ten passages thereafter. Then spin down 75, 000 cells per gel.

And use a cold pipette to gently resuspend the pellet in 1.5 milliliters of collagen per gel without bubbles. Next, use a static-reducing brush to wipe the borders top and bottom of a 12-well plate. And place the plate on top of another empty static-reduced 12-well plate.

The presence of static electricity during the preparation of the gel may affect the distribution of the cells, causing cells to sink to the bottom during gel laying, and grow as a 2D model layer. When the plate is ready, gently more 1.5 milliliters of the cell collagen mixture into each well. And place the plate in a 37 degree Celsius incubator.

After 30 minutes, add 1.5 milliliters of freshly-prepared cdFBS medium supplemented with hormones to each well. And use a sterile P200 pipette tip to carefully detach each gel from the border of the well with a gentle circular motion. Then, place the 3D cultures in a 37 degree Celsius, 5%CO2, 100%humidity tissue culture incubator for one to two weeks, changing the media every two days.

To process the gels for whole mounting, replace the medium in each well with 1.5 milliliters of PBS. Then, transfer one gel at a time to a slide, and use a curved surgical blade to cut each gel in half. Transfer one half of the gels to individual slides to try.

After five minutes, fix the gels in 10%formalin on a shaker overnight. The next morning, wash the gels two times with PBS for ten minutes each on the shaker. After the second wash, incubate the gels in 70%ethanol for one hour on the shaker, followed by an overnight incubation in filtered carmine alum overnight.

The next day, dehydrate the gels in progressive ethanol solutions while shaking for one hour each, followed by two one-hour shaking incubations in xylene. After the final xylene incubation, mount the gels with toluene-based mounting medium, and cover them with 1.5 millimeter thick cover slips. To process the gels for histological analysis, place the saved gel halves in individual 20 milliliters glass vials of 10%formalin for overnight fixation on the shaker.

The next morning, wash the gels two times in PBS for ten minutes while shaking, as just demonstrated. Then, place the gels in processing cassettes for paraffin embedding. Next, dehydrate the gels in progressive ethanol incubations for one hour each as indicated, using fresh ethanol for each incubation.

Then, incubate the gels in xylene for two one-hour incubations, and transfer the cassettes into paraffin for three one-hour incubations at 60 degrees Celsius in a vacuum incubator, using fresh paraffin each time. After the last vacuum incubation, remove the gel from the cassette and place in a plastic mold. Fill the mold with fresh paraffin, and cover the mold with the cassette top.

After cooling, remove the plastic molds and use a microtome to obtain 5-micrometer thick sections of the gels. To extract the cells, carefully transfer whole gels from the 12-well plate into a Petri dish of PBS, and briefly rinse the gels. Then, place the gels in individual 15 milliliters tubes containing 2 milliliters of 0.125%collagenase, and pipette up and down four to five times to break the gels into small pieces.

Incubate the gel fragments at 37 degrees Celsius for 25 to 30 minutes. When the gels have fully dissolved, add two milliliters of serum-supplemented culture medium to each cell suspension, and centrifuge the samples. Then, resuspend the pellets and count the number of viable cells.

Epithelial structures are observed in whole mounts of gels cultured for two weeks in the presence of E2 alone, or in combination with other hormones. When no hormones are added, only single cells or groups of two to three cells are observed. Rat tail collagen type one is most frequently used for the gels, as seeding in bovine collagen type one may result in the formation of disorganized cell groups.

E2 treatment results in thick, rounded, or elongated structures that exhibit more budding structures with the addition of prolactin. E2 and promegestone treatment, however, induces irregular structures, with cellular projections reminiscent of side branching. Here, the effect of an anti-estrogen on E2-dependent cell proliferation in the 3D cultures was quantified after collagenase cell extraction as just demonstrated.

While attempting this procedure, it's important to remember to keep the neutralized collagen on ice, otherwise it will rapidly solidify at room temperature. After watching this video, you should have a good understanding of how to prepare hormone-sensitive 3D cultures of the breast epithelium, and how to extract data on the action of hormones on epithelial morphogenesis and cell proliferation.