An Ex vivo Model to Study Hormone Action in the Human Breast

The overall goal of this procedure is to provide an ex vivo model of the human breast that maintains tissue architecture, intercellular interactions, and hormone responsiveness to study hormone action. The model is based on human tissue microstructures that are prepared by first dissecting breast parenchyma, which consists of ducts and lobules from the fatty mammary stroma. In addition to adipocytes, the stroma contains fibroblasts immune cells and endothelial cells.

Once the parenchymal tissue has been freed of the fat, it is mechanically dissociated into pinhead sized pieces using opposing scalpels. Subsequently, the tissue mass is subjected to enzymatic digestion with collagenase hormone stimulation can be associated with this step if desired. The tissue microstructures are then recovered by centrifugation, followed by removal of the remaining fat tissue fibroblast enriched fraction and red blood cells.

After a recovery procedure, the suspension of remaining digested breast tissue is enriched for tissue microstructures containing luminal and myoepithelial cells with the surrounding basal lamina, fibroblast endothelial cells, and immune cells. Ultimately, ex vivo culturing is used to evaluate microstructure morphology. Fluorescence activated cell sorting is used to separate the breast cell subpopulations and protein and transcriptomic analysis is used for hormone responsiveness studies.

Hormones control breast development and are important in breast tumorgenesis. Hormone signaling has mostly been studied in breast cancer, cell lines in tissue culture, plastic disease. Yet when primary mammary epithelial cells are culture in vitro, they lose hormone receptor expression.

Now, the tissue microstructures we obtain from surgical discard material remain hormone responsive, and patient samples reflect in the individual diversity. Visual demonstration of this method is important as tissue digestion steps and obtaining microstructures from heterogeneous. Human mammary gland are complex.

Collagenous concentration and digestion time are critical to avoid partial digestion or over digestion. This ex vivo model can help us to understand how the reproductive hormones act on the human breast and by what mechanisms they impedance on cancer development and progression. To begin transfer the bottles containing the tissue samples to a laminar flow cabinet in a P two cell called room, working on a sterile chopping board.

Hold the material with forceps and scrape the remaining fat and vessels away from the breast parenchyma with a scalpel. Place the white material in phosphate buffered saline or PBS in a 10 centimeter cell culture dish. After removing the fatty tissue from all pieces and disposing of them according to the safety rules of the laboratory, place the white parts containing the epithelial tissues back on the chopping board using opposing scalpels, cut the tissue and chop it into pieces of less than two millimeter diameter.

Place the minced material into tubes of adequate size for paraffin, embedment and hormone stimulations. Use one milliliter of material in a five milliliter tube. When performing fluorescence activated cell sorting or facts, use 2.5 milliliters of material in a 15 milliliter tube.

If freezing aliquots, use 10 milliliters of minced material in a 50 milliliter tube. Then add digestion. Master mix containing collagenase A at a volume, up to four times the amount of the digested material to ensure adequate agitation and aeration.

Next, add hormones at the desired concentration for stimulation with 17 beta estradiol and progest stone. Use a final concentration of 20 nanomolar. Add the vehicle only to the control, agitate or mix the samples very well to ensure that all material distributes along the centrifuge tubes.

Then place the tubes on the roller mixer inside an incubator at 37 degrees Celsius and 40 RPM overnight. Allow the digestion to take place for at least 12 hours to recover the tissue. Microstructures first centrifuge the tubes at 400 Gs for five minutes.

After aspirating the fat from the top of the centrifuge tube, transfer the remaining aqueous supernatant, which is enriched in fibroblasts to a new centrifuge tube. Then centrifuge the aqueous supernatant at 1, 250 Gs for five minutes. As the breast tissue microstructures are very sticky for all subsequent except reus, the pellet by gentle agitation pipetting is not recommended due to the risk of losing material.

Next, resus suspend the pellets enriched in microstructures in PBS 2%FCS also resuspend the pellets enriched in fibroblasts derived from the aqueous supernatant in PBS 2%FCS following centrifugation of both tubes. As before, discard the supernatant and resus, suspend the pellets in three to five milliliters of red blood cell lysis buffer, and incubate them for five minutes at room temperature. Then add PBS 2%FCS two times the volume of red blood cell lysis, buffer, and centrifuge after centrifugation.

Resus, suspend the pellet in P-B-S-F-C-S 2%To prepare samples for flow cytometry, add one to two milliliters of 0.25%trypsin to the pellet and mix it by pipetting up and down very gently with a 1000 microliter pipette man for two minutes to dissociate the cells after inhibiting trypsin activity by addition of nine milliliters of PBS 2%FCS centrifuge at 450 gs for five minutes. Resus suspend the pellet in 10 milliliters of PBS 2%FCS and transfer the cell suspension onto a 40 micron cell strainer placed on top of a 50 milliliter centrifuge tube to prepare single cells After centrifugation, separate the immune cells fibroblasts and endothelial cells from microstructures with a cocktail of antibodies before sorting them with facts to prepare cells for histology. Wash and digest the samples as before, and then fix the pellet in one milliliter of 4%Paraform aldehyde in PBS at room temperature for 30 minutes.

Centrifuge at 1, 250 GS for five minutes and wash with one to two milliliters of PBS two times after washing centrifuge at 16, 000 GS for 10 minutes. During this time, prepare 1%multipurpose aros in tris acetate EDTA buffer and heat the solution in the microwave oven until the solution comes to a boil. Remove the solution from the microwave oven and swirl gently to mix the solution and resus suspend any remaining aros particles.

Cool the aro solution to 50 degrees Celsius before pouring one milliliter in a paraffin base mold. Next, discard the supernatant and place the pellets on the top of the solidified layer of aros with a spatulas flat spoon end. Cover them all with an additional layer of agros along with the aro samples.

Place a piece of white office paper in the cassette with information about the reduction number experimental condition and date written in pencil to withstand all subsequent treatment steps. After the agarro solidifies, put the aros blocks into the histology cassettes for paraffin embedment. Put the cassettes in PBS overnight or in 70%ethanol for storage over the weekend at four degrees Celsius before transferring the histology cassettes to histology facilities for paraffin embedding to prepare samples for freezing resus suspend microstructures in freezing medium consisting of FCS with 10%dimethyl sulf oxide and freeze one milliliter aliquots.

In cryo vials, the number of cryo vials is dependent on the amount of primary material obtained from reduction. Mammoplasty tissue from fresh reduction. Mammoplasty is mechanically and enzymatically dissociated.

The resulting tissue microstructures have ducts and lobules characteristic of the breast tissue of origin. Immunohistochemical analysis of aros and paraffin embedded tissue microstructures stained for the myoepithelial marker delta NP 63 reveal that microstructures retain not only the morphological characteristics of the normal breast tissue, but also the molecular profile of the cell populations to assess the hormone response to tissue. Microstructures were treated either with a synthetic progesterone receptor agonist, promega stone, or ethanol control to enrich for the hormone receptor positive luminal cells.

Flow cytometry was performed based on epithelial and myo epithelial markers after depletion for endothelial fibroblasts and immune cells with a cocktail of anti CD 31 anti FAP and anti CD 45 antibodies. Q-R-T-P-C-R analysis of EPAM positive CD 10 negative cells showed upregulation of the progesterone target gene wind four in the luminal cell enriched population from the Promega stone exposed tissue microstructures Once master this technique can be done in two days if it is performed properly. Importantly, collagenous concentration, digestion, temperature, duration, and rotation speed during tissue digestion are all significant details that are specified in the protocol.

This method can be extended to pharmacological studies, in particular, a better characterization of selective estrogen receptor modulators or progestins. In fact, progestins are widely used in the context of hormonal contraception. However, little is known about its action on the human breast After its development.

This x vivo tissue microstructure model paved the way for the researcher in the field of breast development and carcinogenesis to explore hormonal contribution and action in the human breast.