This protocol has been used for over 40 years and remains the only reliable method for mammary cell transplantation assays in the rat. This method is widely used to interrogate mammary cell autonomous versus host effects in breast cancer susceptibility research. By grafting in the interscapular fat pad, this method circumvents having to clear the endogenous membrane epithelium which is not effective in rats.
The method is therefore less invasive and the endogenous mammary glands can be used as an internal control. To begin this procedure, place a euthanized female rat on its back and secure it with pins. Spray the entire ventral surface with 70%ethanol.
Make a sagittal Y-shaped incision allowing access to thoracic, abdominal, and inguinal mammary glands. Use scissors to dissect all the mammary gland tissue from the donor rat and remove all visible lymph nodes. Next, use forceps to extract the mammary tissue.
Place it in a labeled 60 millimeter dish containing 500 microliters of serum-free DMEM F12 media and keep it on ice. Then use scissors to finely mince the mammary tissue. First, carefully turn the body of the donor animal over to place it in a prone position and secure it with pins.
Spray the head and upper back with 70%ethanol. Locate the base of the skull and make an incision beneath the occipital condyles, insert sharp scissors beneath the skin and cut the skin away from the skull including the sides of the head. Next, use bone cutters or strong scissors to cut the skull along the midline from the occipital to frontal bones while making sure to keep the blade as superficial as possible and angled upward to prevent the destruction of the underlying brain tissue.
Use Rongeurs or strong forceps to peel away the bone. Insert the tool lateral to the cerebellum to break the bone on either side. Sever the connections to the meninges by fully exposing the auditory canal.
Use curved fine-tipped forceps to lift the brain out of the skull and record its weight. Use a fresh start of supplies for additional donor brains. Immediately transfer it to a 15 milliliter tube containing an equal amount of media that is stored on ice.
Homogenize the brain for 10 to 15 seconds on a low speed. Then filter the homogenate to remove large pieces. Store the liquid on ice until needed.
First, cut one centimeter off the end of a 1, 000 microliter pipette tip and manually place it on the pipettor. Use a cut pipette tip to transfer the minced donor tissue from each 60 millimeter dish to the collagenase digestion tube. Gently mix the minced tissue by pipetting it up and down one to two times.
Then secure the samples in a horizontal position in the shaking incubator. Allow the samples to digest for 1.5 to two hours at 37 degrees Celsius while shaking at a speed between 200 and 220 RPM. When the donor mammary gland tissue is fully digested, centrifuge the suspension at 1, 200 times g and at four degrees Celsius for 10 minutes.
After ensuring a pellet has formed, use a pipette to remove the fat layer or carefully pour off the supernatant. Gently resuspend the pellet in 10 milliliters of fresh DMEM F12 media. Repeat this process to thoroughly wash the cells.
Prepare one 50 milliliter tube with a 40 micrometer strainer for every donor animal. Pre-wet the strainer with at least one milliliter of DMEM F12 media. Then use a pipette to pass the cell suspension through the filter to collect ductal fragments or organoids.
For organoids, keep only the cells that remain in the filter. Invert the cell strainer over a new 50 milliliter tube and rinse with any volume of sterile media required to collect all the cells. After this, pellet the cells once more and resuspend in a small volume of media for counting.
Prepare single batches of donor material for all the transplant recipients by combining equal volumes of the cell suspension with 50%brain homogenate for every site of transplantation. First, identify the base of the skull and start of the vertebral column on a recipient animal. Approximately one-third of the way down the spine, shave a three centimeter by two centimeter area on the upper thoracic portion of the back.
Next, prepare the sterile field that will be used for surgery and arrange all needed supplies. Flush the Hamilton syringes with sterile DMEM F12 media to prevent the loss of cells. Load the entire volume of donor material into a separate syringe for each condition.
After the syringe is fully loaded, invert it and press the plunger slightly to remove air bubbles at the tip of the needle. Keep the syringe on ice throughout the procedure. Ensure the animal remains unresponsive to deep stimuli with a firm toe pinch.
Then use a sharp surgical blade to make a small interscapular incision. Locate the medial blood vessel for orientation and use forceps to lift the skin on one side of the incision. Hold the skin away from the fat pad and avoid the underlying brown fat while the transplant is performed.
Next, insert the needle into the graft site. Carefully inject 40 microliters of the cell mixture into the interscapular white fat pad tissue. Remove the needle slowly.
Hold the tissue in place and allow the transplanted cells to settle for three to five seconds. Repeat the entire injection procedure as previously described at the second site of transplantation. After this, close the surgical wound using wound clips or sutures and then discontinue the anesthesia.
After sacrificing the animal, prepare it for dissection. Identify the medial blood vessel that separates the graft sites in the interscapular fat pad. Excise the entire pad as a single piece of tissue.
Place the tissue on a positively charged microscope slide for whole mount. Place the slides in 70%ethanol for seven to 10 days to de-fat the tissue. Then prepare the alum carmine stain and process the slides when the tissue is sufficiently opaque.
When processing is complete and the tissue on the slide has cleared, use low powered light microscopy or high resolution digital photography to acquire images of the slides for analysis. In this study, mammary epithelial cells are transplanted using a technique optimized for working with rats where the isolated mammary epithelial organoids from donor rats are grafted into the interscapular white fat pad of recipient rats. The presence, absence, or abundance of epithelium can be evaluated to determine success of the experiment as well as the autonomy of effects related to experimental variables.
A reciprocal transplantation experiment where each factor has two levels such as wild type versus knockout will create four transplant groups for hypothesis testing. The representative whole mounted interscapular fat pad shown here contains positive epithelial outgrowth at both sites of transplantation. When analyzing the slides, lack of epithelium in the interscapular fat pad across many samples may indicate a technical problem with the procedure and should be treated as a negative outcome.
Donor epithelial outgrowth can also be compared to the endogenous mammary gland and may facilitate additional conclusions. In the example shown here, the results of reciprocal transplantation using wild type and CDKN1B knockout rats suggested non-mammary cell autonomous effects on mammary gland development. Remember donor epithelial cells and organoids must be prepared carefully and kept alive until injected into the recipient animal.
It is imperative to plan ahead and to perform the procedure carefully and without any interruptions. Primary carcinogenesis assays can be performed following transplantation to interrogate mammary cell autonomy in the etiology or treatment of breast cancer. Gene editing can also be incorporated to genetically engineer the donor cells or the recipient rat.
As the sole method of performing mammary epithelial cell transplantation in rats, this procedure facilitates mammary gland development as well as carcinogenesis studies. This technique is particularly important for researching interactions between breast cancer cells and/or mammary epithelial cells with the host microenvironment. It enables studies in mammary gland development as well as breast cancer research.
Those attempting this technique for the first time should optimize donor cell preparations to ensure maximal cell yield and viability, then perform small pilot experiments to practice surgical procedures and determine cell numbers to inject and also determine post-surgical incubation times required for experiments.
