This murine extrahepatic bile duct organoid system can address the limited access to preclinical cholangiopathies models and the limitations of pluripotent stem cell and liver-derived cholangiocyte organoid models. Our model is adult-tissue specific, reductionist, reproducible, and time and cost efficient. It will be of special benefit to laboratories that do not have access to human tissues.
Our technique allows the culturing of an almost unlimited number of extrahepatic bile duct cholangiocytes to study tissue regeneration and cell-to-cell interactions. Demonstrating the procedure will be Junya Shiota, a post-doc from my laboratory. For intrahepatic bile duct isolation, place the adult mouse in a supine position and open the abdominal cavity along the midline.
Retract the liver to rest on the diaphragm and use a hemostat to gently pull the proximal duodenum to reveal the common bile duct immediately below the liver hilum. Use a scalpel to separate the extrahepatic bile duct from the surrounding tissues. Holding the proximal end of the common bile duct with forceps, dissect the duct distally just above its juncture with the duodenum before dissecting the proximal end of the duct from the liver.
Immediately place the isolated extrahepatic bile duct into a glass plate containing cold washing buffer on ice, then clean the bile duct from the surrounding tissue and mince into 0.5-millimeter sections. When all of the tissue has been minced, transfer the sections into a tube containing 500 microliters of dissociation buffer for a 20-minute incubation at 37 degrees Celsius. At the end of the dissociation, neutralize the buffer with 500 microliters of ice-cold cell culture medium and triturate the tissue suspension 20 times through an 18-gauge needle and then 20 times through a 20-gauge needle.
Then filter the resulting cell suspension through a 70-micrometer cell strainer into a 50-milliliter tube. To establish a biliary organoid culture, collect the cells by centrifugation and carefully remove the supernatant. Resuspend the pellet in one milliliter of ice-cold, sterile PBS and transfer the cells to a 1.5-milliliter tube for a second centrifugation.
Resuspend the washed pellet in 120 microliters of liquified, ice-cold basement matrix and plate 40 microliters of cells into the center of each of three wells of a 37 degrees Celsius-warmed, 24-well plate, then place the plate in a 37 degrees Celsius tissue culture incubator for about 15 minutes. When the basement matrix has solidified, add 600 microliters of 37 degrees Celsius seeding medium to each well before returning the plate to the incubator. After three days and every three days thereafter, replace the seeding medium with 600 microliters of fresh organoid culture medium, monitoring the organoid growth regularly with a inverted microscope.
To passage the extrahepatic bile duct cultures, pipette the organoids in each well with 400 microliters of ice-cold PBS 10 times per well before transferring the well contents to individual 1.5-milliliter tubes. Passage each mixture through a 25-gauge needle four times to dissociate the organoids and collect the cells by centrifugation. Then resuspend the cells in basement matrix at the appropriate ratio for re-plating.
For long-term extrahepatic bile duct organoid storage, wash each well of organoids with room-temperature PBS without disturbing the basement matrix drops and add 500 microliters of ice-cold freezing medium to each well. Gently resuspend the organoids and transfer the mixture into individual cryogenic vials, then place the vials at minus 80 degrees Celsius for 48 hours before transferring the organoids to a nitrogen tank for long-term storage in a vapor phase. To prepare the organoids for paraffin embedding, replace the medium with 500 microliters of four degrees Celsius PBS and transfer the suspension from each well into individual 1.5-milliliter tubes containing liquified basement matrix.
Collect the organoids by centrifugation and use a modified P1000 pipette tip to carefully remove the supernatants without disturbing the pellets. Then add one milliliter of ice-cold, 4%paraformaldehyde to the organoids for an overnight incubation at four degrees Celsius. The next morning, replace the fixative with one milliliter of room temperature PBS for a five-minute incubation at room temperature and wash the organoids by centrifugation three times.
After the last wash, resuspend the organoids in one milliliter of 30%ethanol for a five-minute incubation at room temperature, followed by a five-minute incubation in one milliliter of 70%ethanol at room temperature. At the end of the 70%ethanol incubation, collect the organoids by centrifugation and resuspend the organoids in one milliliter of 100%ethanol for five minutes at room temperature. At the end of the 100%ethanol incubation, heat specimen processing gel in a microwave for 20 seconds or until liquified, and add 50 microliters of the liquified gel to each tube of organoids.
Place the tubes on ice until the specimen processing gel is solidified and transfer the drop of organoids from each tube to between the blue sponge pads in a cassette for further processing in the paraffin embedder. Place the cassette into a tissue processor programmed for 15 minutes per step, then section the paraffin-embedded organoids in specimen processing gel at four micrometers per section for immunohistochemical staining and analysis according to standard protocols. Extrahepatic bile duct organoid plating efficiency is approximately 2%when isolated from either neonatal or adult mice.
After passage two, the plating efficiency of extrahepatic bile duct organoids derived from adult mice increases to 11%and remains stable. The majority of organoids demonstrate a cystic morphology throughout all of the passages with rare irregular organoids. The organoids reach a growth peak at five to seven days, after which they begin accumulating intraluminal debris and deteriorate.
Therefore, for maintenance of the organoid culture, the organoids should be split every seven to 10 days. When analyzed with immunofluorescence, extrahepatic bile duct organoids consist of a pure population of epithelial cells marked by E-cadherin. Organoid cells also demonstrate markers of biliary progenitor cells as well as markers of biliary differentiation.
Importantly, a high percentage of organoid cells possess a primary cilium marked by acetylated alpha Tubulin, which is a feature of normal cholangiocytes and suggests an appropriate organoid cell polarization. Close adherence to the described temperature condition is required. Meticulous bile duct dissection prevents pancreas cell contamination.
The loss of cellular material can be avoided by careful manipulation after centrifugation. These organoids can be used as preclinical models, genetically and pharmacologically manipulated, or used to test drugs and the effects of infectious agents. This method can be used by labs that want to take advantage of genetically modified mouse models to further study the mechanisms of cholangiocyte biology.
