The endometrium is a tissue that changes every month in response to hormones. Hormone imbalance can prevent embryo attachment and also cause diseases like cancer. Our protocol rebuilds the endometrium so that it can be studied outside of the woman's body in a physiological way.
The main advantage of this technique is that it provides a 3D structure of cells comprised of two hormonally responsive cell types, the epithelial and stroma cells that specifically organize and behave like they do in the body. Since the organoids can better mimic tissue behavior, they can eventually be used to test different drugs. The endometrial organoids can be used to study direct effects of currently known risk factors for cancer, including obesity and polycystic ovarian syndrome.
Our organoids emphasize the importance of paracrine actions between two cell types. It's important to start with an adequate amount of tissue. Getting the right density of epithelial and stromal cells can be tricky.
Also, the organoids are quite small and can be difficult to visualize with IHC staining. Visualization of this procedure is important because there are some steps that will be much easier to understand after seeing them. These include getting the right tissue layer of the uterus, getting the right density of cells to combine, and seeding the agarose molds.
Before beginning cell isolation, cast and equilibrate 1.5%agarose micro-molds according to the manufacturer's instructions to house the organoids. In a biosafety cabinet, use aseptic techniques to prepare an enzyme solution at 37 degrees Celsius and sterile filter the solution using a 0.2 micrometer syringe filter into a 15-milliliter conical tube. Using a scalpel, scrape off endometrium from the uterine biopsies and mince the tissue into very small pieces.
Place the freshly minced tissue into the 15-milliliter conical tube containing the enzyme solution. Close the cap and wrap the top of the tube with a wax film to prevent contamination. Place the tube into a water bath or incubator at 37 degrees Celsius for 30 minutes with gently shaking.
After this, stack a 100-micron cell strainer on top of a 20-micron cell strainer on a 50-milliliter conical tube. Filter the solution through the two strainers. Rinse the 15-milliliter conical tube with HBSS and put this wash through the strainer to ensure that all cells are collected.
Then, invert the 20-micron cell strainer onto a new 50-milliliter conical tube and wash the epithelial cells off the strainer with 20 milliliters of organoid media supplemented with 1%penicillin and streptomycin. Centrifuge the conical tubes at 500 G for five minutes. For the collected stromal cells, remove the supernatant and resuspend the pellet in 10 milliliters of red blood cell lysis buffer.
Incubate at 37 degrees Celsius for 10 to 15 minutes. Centrifuge these cells at 500 G for five minutes. Remove the supernatant and resuspend the pellet with 200 to 300 microliters of organoid media.
For collected epithelial cells, remove the supernatant and resuspend the pellet in 100 microliters of organoid media. After the 1.5%agarose molds are equilibrated, remove the organoid media on the outside of the agarose molds and tilt the tissue culture plate so that the medium from cell seeding chamber of the agarose dishes can also be carefully removed. View the epithelial and stromal cell suspensions under a microscope and add more organoid media to the suspensions, making sure to add only 100 microliters at a time so that the suspension are roughly equal in density.
Then combine one part of the stromal cells with three parts of the epithelial cells by volume. Pipette 50 microliters of the combined cell suspension into the cell seeding chamber of the agarose mold. Once the agarose molds are filled with cells, carefully add 400 microliters of fresh organoid media into the wells of the 24-well plate.
Incubate at 37 degrees Celsius with 5%carbon dioxide, making sure to change the medium every second day with 500 microliters of organoid media. After seven days, change the medium to one that is supplemented with 0.1 nanomolar estradiol and 0.8 nanomolar testosterone to promote organization of the epithelial and stromal cells. First, dissolve 1.5%agarose in PBS by boiling.
Place the liquid agarose into a beaker of hot water and allow it to cool to approximately 50 degrees Celsius. Under a dissecting microscope, tilt the 24-well plate and carefully pipette out the medium from the outside of the agarose mold. Then carefully pipette out the medium from the interior of the agarose mold to avoid disrupting the organoids.
Carefully add between 70 and 75 microliters of warm, 1.5%agarose into the chamber of the agarose dish, being careful not to disturb the organoids. Let the plate cool at four degrees Celsius for five minutes. After this, add 4%paraformaldehyde into each well of the 24-well plate and fix the entire sealed agarose mold overnight at four degrees Celsius.
The next day, store the plate in 70%ethanol at four degrees Celsius until ready to process for paraffin embedding. To begin RNA isolation, view the plate under a dissecting microscope. Tilt the plate and carefully pipette out the medium from the chamber of the agarose mold.
Forcefully pipette one milliliter of fresh organoid medium directly into the agarose mold so that the organoids are flushed out of the microwells, being careful not to create too many bubbles. Repeat this forceful pipetting process once using the same medium. After this, collect all of the medium containing the organoids.
Centrifuge at 500 G to collect the organoids, and proceed to RNA extraction. In this study, human endometrial organoids comprised of epithelial and stromal cells of the endometrium are generated without the use of exogenous scaffold materials. For histological processing, the agarose molds containing the endometrial organoids are sealed with agarose, followed by fixation with 4%paraformaldehyde overnight.
These molds are processed for standard paraffin embedding sectioned and stained for histology, immunofluorescence, or immunohistochemistry. The hematoxylin in eosin staining reveals a spheroid-like structure with a single layer of cells lining the outside and cells in the center. Cell-specific markers for endometrial, epithelial, and stromal cells reveal epithelial cells surrounding the organoid with stromal cells in the center.
Markers of endometrial physiology confirm that the endometrial organoids retained certain characteristics of the native tissue. Tri-chrome staining, which stains collagen blue and cells red, shows the presence of collagen within the center where stromal cells resided, demonstrating active production and secretion of collagen by stromal cells similar to the native tissue. In addition, immunohistochemical staining reveals the presence of estrogen receptors, androgen receptors, and progesterone receptors in both the epithelial and stromal cells.
It's important to plate the cells densely so that we have enough organoids for downstream experiments. This will take some practice. Endometrial organoids can be cultured under different conditions and then harvested for experiments like immunohistochemistry or immunofluorescence staining to study protein expression or harvested for RNA to study gene expression.
Our lab investigates how the endometrium responds to altered hormone levels, which can lead to endometrial hyperplasia and cancer. We can use these organoids for long-term treatments of hormones and add pathological conditions such as excess testosterone found in polycystic ovarian syndrome, or signals from adipocytes to study obesity-induced endometrial changes. Human tissues are considered to be biohazardous, and it will be important to take appropriate precautions.
