This is the first protocol that allows human breast tissue to be maintained alive ex vivo for prolonged periods of time directly allowing the study of interactions between human breast tissue and human breast cancer. The main advantage of this technique is that it allows macroscopic pieces of human breast tissue including breast adipocytes, immune cells, vascular structures, ductal structures and extracellular matrix to be kept alive ex vivo. This technique has major implications for several areas of breast cancer research including personalized medicine, pharmaceutical development and the pathophysiology of tumor initiation and slower breast cancer processes such as fibrosis and extracellular matrix remodeling.
Demonstrating this procedure are Katherine Hebert, a Tulane graduate student and Rakesh Gurrala a Tulane medical student from my lab. To begin, melt the prepared gelatin solution in a 37 degrees Celsius water bath. Use a five or 10-milliliter serological pipette to dispense 2.5 milliliters of this solution onto each plunger for wells of a six well plate.
Once the gelatin has solidified, move the pNIPAAm coded ASC plates to the bio-safety cabinet. Gently place the plungers into the wells of these plates so that the gelatin contacts the ASCs. Place a metal washer on the plunger to weigh it down so that the gelatin is in direct contact with the ASC sheet for 30 minutes at room temperature.
Gently move the plate with the plungers into the sterile box in the bio-safety cabinet and place the lid on it. Move the box to a four degree fridge or place the plate on ice and an ice bucket in the bio-safety cabinet for 30 minutes. In a bio-safety cabinet, wash the human breast tissue three times with 10 milliliters of sterile PBS.
Use sterile forceps and a razor blade to coarsely mince the BC-MPS and try to remove as much fascia and connective tissue as possible. Once the connective tissue has been removed use a sterile razor blade to finally mince the tissue until it has a homogeneous liquid consistency. Cut the tip of a P1000 pipette tip to assist in pipetting the minced tissue.
In a 1.5 milliliter tube, combine the mince tissue, cancer cell lines and BC-MPS media as described in the text manuscript. Move the ACS plate that will be used for the bottom cell sheet from the incubator to the bio-safety cabinet and aspirate the media from the plate. Pipette the prepared breast tissue mixture onto the center of the well of the bottom ACS plate using the cut pipette tip, move the box containing the upper ACS plate with plungers to the bio-safety cabinet.
Gently remove the gelatin plungers from the pNIPAAm coated plate and place them on top of the tissue mixture. Add BC-MPS media to the well and carefully move the bottom ACS plates with the BC-MPS mixture and plungers to the sterile plastic box. Place the lid of the box on for transport, taking care to avoid contamination of the culture.
Incubate the bottom plate in the box and the 37 degrees Celsius incubator until the gelatin is melted and the top ACS layer has begun to adhere to the bottom layer. Then move the box with the plates to the bio-safety cabinet, gently remove the plungers from the bottom plates to observe the tissue with the cancer cells anchored to the bottom of the well. Place the lid of the six-well plate back onto the bottom plate and incubate at 37 degrees to completely melt the gelatin and allow the top layer to anchor to the bottom layer.
Gently move the plates to a bio-safety cabinet and aspirate the media from the edge of the well with a 10 milliliter serological pipette. Add two milliliters of fresh media onto the edge of each well to avoid dislodging the tissue. Maintain the BC-MPS at 37 degrees Celsius and 5%carbon dioxide for the desired length of time.
Changing the media every two to three days. Move the plate to the bio-safety cabinet when the BC-MPS is ready to be analyzed. Remove media with a serological pipette to avoid accidental dislodging of any tissue.
Add one volume of PBS to each well. Then remove the PBS with a serological pipette. Add one milliliter of cell dissociation solution to each well and move the plate back to the incubator for five minutes to allow this cells to detach.
After incubation, use a cell scraper to completely detach the cells and the tissue from the culture plate in a bio-safety cabinet. Transfer the solution with the tissue to a 15 milliliter conical tube and collect any remaining cells by adding two milliliters of PBS. Wrap the tube with aluminum foil if the cells are fluorescent.
Incubate the tube at 37 degrees under constant agitation in an orbital shaker at one times G for 10 to 20 minutes to completely dissociate the cells from the tissue. In a bio-safety cabinet, use a serological pipette to disrupt any remaining clumps of cells in the tube. And filter the sample through a 250 micro meter tissue strainer into a new 15 milliliter tube.
Rinse the strainer with one milliliter of PBS to collect any remaining cells. Centrifuge the samples at 500 times g at room temperature for five minutes to separate the adipocytes which will be floating in the top layer from the cancer cells and ASCs mixed together in a pellet. Transfer the adipocyte layer to a new tube using a blunt cut pipette tip.
Centrifuge the sample again and use a syringe and a needle to remove the remaining solution from below the adipocytes. Aspirate the remaining solution from the tube containing the ASCs and cancer cells without disrupting the cell pellet. Re-suspend the pellet in PBS or BC-MPS media and use flow cytometry to sort the cells based on fluorescence.
The stri-aided pattern of the confluent ASC sheet is shown here. The buoyant human breast tissue was still stable anchored by the ASC cell sheets to the bottom of the well after 14 days of culture. Fluorescent microscopy images demonstrate the stability of the BC-MPS with MDA-MB-231 expressing RFP cell lines after 14 days and with triple negative tumor explant PDX for at least six days.
BC-MPS containing breast tissue was cultured in vitro for 14 days, stained and image to demonstrate the native elements of the tissue at 100X and 20X magnification. Staining of the macrophage markers was used to show the preservation of primary macrophages after three and seven days in culture. Flow cytometry analysis of Bodipy stained MDA-MB-231 cells after 14 days indicate minimal lipid accumulation in 2D culture and extensive lipid accumulation and BC-MPS.
The proportion of lipid positive MDA-MB-231 cells was 26.2 fold greater in BC-MPS than 2D culture. Cells cultured in BC-MPS displayed increase lipid droplets compared to cells cultured in standard 2D culture. Time-lapse images of RFP labeled BC-MPS with MDA-MB-231 cells showed amoeboid movement of 231 cells with pseudo pods and high motility.
The breast tissue must be minced small enough and separated enough to be distributed across the bottom of the well. Breast tissue is very buoyant and if the pieces are too large or clustered too closely together, the ASC cell sheets won't be able to anchor the breast tissue to the bottom of the well. The resulting breast tissue cancer constructs can undergo enzymatic digestion to isolate particular cell types of interest.
This will allow key questions to be answered such as how cancer cells in breast tissue respond differently to chemotherapy compared to traditional 2D culture or organoids.
