This protocol is significant as it shows a step-by-step methodology for isolating highly pure uterine epithelium from the mouse uterus for the culture of endometrial organoids. This method efficiently utilizes enzymatic and mechanical dissociation to isolate uterine epithelium. The techniques were streamlined to establish, treat, and analyze endometrial epithelium organoids derived from the isolated epithelium.
Begin by thawing the gel matrix on ice for approximately one to two hours before use. Then dissect the euthanized mouse by making a midline incision on the abdomen using scissors, and gently peeling back the skin to expose the underlying peritoneal layer. Locate the uterine horns by gently moving the abdominal fat pads aside and hold the uterine horns at the cervical junction before cutting them along the mesenteric fat.
Following the dissection of the mouse uterus, thoroughly remove fat tissue from the uterine horn with small scissors. Once done, cut each uterine horn into small fragments, each measuring approximately four to five millimeters. Place all the uterine fragments from one uterus into one well of a 24 well plate containing 0.5 milliliters of 1%trypsin.
Then incubate the 24 well plate in a 37 degree Celsius humidified tissue culture incubator for approximately one hour for enzymatic separation of the endometrial epithelium from the underlying stroma. After the incubation, transfer the uterine fragments to a 35 millimeter tissue culture plate containing one milliliter of Dulbecco's phosphate buffered solution or DPBS. Next, separate the uterine epithelium from the uterine tube under a dissection microscope by holding one end of a uterine fragment with the forceps and gently running the pipette tip longitudinally across the fragment, squeezing the epithelium out of the other end of the uterine tube.
Then observe the separated epithelial sheets from the uterine fragment under the dissection microscope. Next, gently transfer all the epithelial sheets into the same 1.5 milliliter tube using the one milliliter pipette. And pellet the dissociated epithelial sheets by centrifugation at 375 x g for five minutes.
Carefully remove the supernatant without disturbing the cell pellet and resuspend the cell pellet in 0.5 milliliters of 2.5 milligrams per milliliter collagenase plus two milligrams per milliliter of DNA solution. Pipette up and down approximately 10 times or until a single cell suspension is achieved. Next, add 0.5 milliliters of DMEM F/12 plus 10%fetal bovine serum or FBS plus antibiotics and centrifuge the cells for five minutes at 375 x g.
Remove the supernatant, resuspend the cells in one milliliter DMEM/F12 plus 10%FBS plus antibiotics and centrifuge the cells. Place the gel matrix on ice until it is ready. Next, remove the supernatant from the centrifuge cells and resuspend the cell pellet using 20 times the volume of the gel matrix without introducing the bubbles.
Allow the gel matrix cell suspension to settle at room temperature for approximately 10 minutes. Once the gel matrix cell suspension becomes a semi-solid gel, use a P200 micropipette with a wide bore 200 microliter tip and gently aspirate 25 microliters of the gel matrix cell suspension. Dispense three separate 25 microliter domes per well of a 12 well plate and allow the gel matrix to cure for 15 minutes and a 37 degree Celsius humidified tissue culture incubator.
After the gel matrix is cured, add 750 microliters of organoid medium to each well containing gel matrix dome, and incubator at 37 degrees Celsius in a humidified tissue culture incubator. Phase contrast images of mouse endometrial organoids showed that the epithelial and stromal cell separation yielded samples with no more than 10%to 15%contamination of the opposite cell type. The endometrial epithelial cells were assembled into organoids within three to four days.
The sectioned formalin fixed paraffin embedded endometrial organoids were visualized by hematoxylin and eosin stains, which displayed the single layer of epithelium and hollow centers, the lumens of the organoids. The fluorescence microscope imaging showed that all the cells in the organoids were Cytokeratin 8-positive and contained DAPI, indicating that fixation, embedding, and processing of the endometrial organoids is compatible with antibody-based immunostaining. The realtime quantitative PCR showed that the stimulation with 10 nanomolar estradiol increased the expression of lipocalin 2, lactoferrin, and progesterone receptor in the epithelial organoids, indicating that endometrial epithelial organoids can be successfully used to measure the gene expression changes in response to estradiol.
It's important to ensure that clear separation of the endometrial epithelium is obtained during the mechanical dissociation step. One should be able to observe epithelium emerging from the uterine tube. Other methods include establishing the epithelial organoids by encapsulation into a gel matrix.
If desired, the stromal compartment can be further digested to create 3D epithelial stromal co-cultures. The method can help answer questions about the signals controlling the renewal of the endometrium. It could help treat disease in women, such as endometriosis, pregnancy loss, and cancer.
