This protocol is significant for the production of memory epithelial organoids that are generated without passaging. The main advantage of this technique is that organoids can be isolated from human and mouse breast and memory tissue after enzymatic digestion. We perform a series of differential centrifugation steps that isolate epithelial organoids without having to passage cells.
An individual performing this technique may face challenges when embedding the tissues in collagen or matrix and plating them within a 96-well plate. Furthermore, using properly polymerized collagen and plating stable domes is the key to seeing invasion. To begin, collect the mammary tissue of a euthanized mouse by splaying mouse limbs and using four 19 gauge needles, pin the mouse by the paws to a board covered with an absorbent pad with the ventral side facing upward.
Spray with 70%ethanol to smooth the fur down and sanitize the skin and wipe away feces with a gauze pad or tissue. Starting just above the anogenital region, cut upward from the midline using surgical scissors taking care not to pierce through the peritoneum. Upon reaching the chin, make lateral cuts down both clavicles and down the hind legs and pin the skin of the mouse taut to the board to expose mammary fat pads.
After locating the inguinal and thoracic mammary fat pads on wild type mice, use forceps to elevate the mammary fat pad. Using the blunt end of sharp blunt scissors, create a pocket underneath the mammary fat pad away from the skin and cut away the mammary fat pad in one complete piece. Once mammary fat pads have been removed, rinse in PBS before placing them in a sterile tissue culture dish and then quickly transfer to a tissue culture hood.
Mince the mammary tumors with a number 10 or number 11 scalpel to loosen tissue until it reaches a paste-like consistency. Transfer the minced tissue into a conical tube containing 10 to 30 milliliters of collagenase solution using a scalpel. To ensure all tissue is collected, pipette one milliliter of collagenase solution onto the tissue culture plate and back into the conical tube.
Place the conical tube into a benchtop shaking incubator until the tissue becomes stringy and the collagenase solution becomes cloudy. Spin down 50 milliliters of solution for five to 10 minutes at 1, 500 RPM. Aspirate supernatant, and add 12 milliliters of the basal medium and mix by pipetting up and down three to four times or gently rotate the wrist while holding the tube 15 times.
Again, spin down aspirate supernatant, add basal medium, and mix by pipetting or tube rotation as demonstrated earlier. Once the heaviest tissue fragments settle to the bottom collect the supernatant with a serological pipette and transfer it to a 15 milliliter conical tube. After each centrifugation, ensure that the pellet becomes increasingly opaque.
Aliquot appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of BECM per well. Centrifuge the microcentrifuge tubes for 10 minutes at 300 G at room temperature and discard the supernatant from the tubes. Move the tubes with organoid pellets to ice and add the appropriate volume of BECM to each microcentrifuge tube.
Gently pipette up and down to resuspend the organoids in BECM, taking care not to produce bubbles. Slowly and carefully pipette the BECM suspended organoids onto the plating surface and fill all the empty wells with PBS to maintain humidity. Place the plate into a 37 degrees Celsius incubator for one hour to allow BECM to solidify and then cover with the appropriate volume of media.
To prepare the collagen solution, combine 375 microliters of 10 times the concentration of DMEM, 100 microliters of one normal sodium hydroxide, and three milliliters of rat tail collagen I solution in a 15 milliliter conical tube. Pipette mix, taking care not to make bubbles, and titrate the solution to a pH of 7.2 to 7.4 with a small amount of sodium hydroxide or 10 times the concentration of DMEM as needed. Coat the bottom of the wells with the minimum amount of collagen required to fully cover the bottom of the well by placing a small amount of collagen and rock the plate from side to side to coat.
Allow the collagen underlays to set at 37 degrees Celsius for 30 minutes to two hours. Aliquot the appropriate suspension of organoids into microcentrifuge tubes according to the organoid density relative to the amount of collagen per well. Store the collagen solution at four degrees Celsius and monitor polymerization by checking for fiber formation under a microscope every 10 minutes.
Centrifuge the microcentrifuge tubes at 300 G for 10 minutes at room temperature and discard the supernatant from the tubes. Move the organoid pellets to ice and add the appropriate volume of collagen to each microcentrifuge tube. Gently pipette up and down to resuspend organoids in collagen, taking care not to produce bubbles.
Slowly and carefully pipette the collagen suspended organoids onto the plating surface. The immunofluorescent images of embedded organoids in either basement extracellular matrix or collagen were obtained to study the inter-species tissue composition similarities. Organoids embedded in collagen matrix can be used for an invasion assay and analyzed by tracking the expansion of tendrils branching out from the organoid itself either through membrane tomato labeling or phalloidin staining of actin.
Lastly, hematoxylin and eosin staining of paraffin-embedded organoids show that organoids maintain the same histology of breast cancer. It is crucial to keep BECM and collagen cold while pipetting gelled domes to avoid premature polymerization. Additionally, prolonged fixing of domes in PFA will lead to gelled solution if performing immunofluorescent staining.
Organoids can also be used in in vitro and in vivo procedures such as drug screens and mouse models of metastasis. These methods can provide insights into therapeutic sensitivity of tumor tissue, metastatic properties, and immune interactions.
