This protocol provides a significant tool to study the interactions between intestinal immune cells and the epithelium and to dissect how these interactions may regulate in intestinal homeostasis and inflammation. The main advantage of this technique is the exquisite amount of experimental control facilitated by the reduction in vitro model, which can be used to address questions in epithelial and immune biology. This system has already been used to determine the role of ILC1 in the expansion of CD44-positive epithelial crypts and has the potential to further advance our understanding of inflammation-mediated gastrointestinal diseases.
To begin, place thermal cross-linking basal extracellular matrix on ice to thaw and pre-warm a standard tissue culture-treated 24-well plate by placing it in a 37-degree-Celsius incubator. Afterward, remove the plate containing organoids from the incubator and discard the media from the well to be passaged. Then, add 500 microliters of ice-cold advanced DMEM F12 to the wells, and using a P-1000 tip, harvest the organoids from all the wells into a 15-milliliter tube.
Rinse the bottom of the wells with 250 to 300 microliters of ice-cold advanced DMEM F12, ensuring no organoids remain in the well and pool into the 15-milliliter tube containing harvested organoids. Resuspend the one-to two-day-old harvested organoid pellet in one milliliter of ice cold advanced DMEM F12. Pre-coat one 1.5-milliliter tube with PBS2 per innate lymphoid cells replicate sample, and then using a pre-coated PBS2 tip, distribute approximately 100 to 200 organoids into the tube.
Using a PBS2-coated P-1000 tip, add a volume of no less than 500 ILC1s as calculated from the sorting step to the PBS2-coated 1.5-milliliter tube containing the organoids. Spin down ILC1 and organoids at 300 G for five minutes at four degrees Celsius and ensure to avoid small-diameter tabletop centrifuges, as they will pull the cells along the edge of the tube interior instead of creating a pellet at the tip of the tube. Then, remove the supernatant slowly and gently without disturbing the pellet and place the sample on ice.
While holding the tube on a cold surface, resuspend the organoids and ILC in 30 microliters of thermal-crosslinking basal extracellular matrix at least 10 to 15 times to ensure even distribution. Apply 30 microliters per well of ILC organoids in the thermal-crosslinking basal extracellular matrix to a pre-warmed 24-or 48-well plate to form a single dome. Afterward, place the plate directly in the incubator for 10 to 20 minutes at 37 degrees Celsius and 5%carbon dioxide.
Then, add 550 microliters of complete ILC1 medium per well with any desired experimental cytokines or blocking antibodies and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. After 24 hours, gently remove the plate from the incubator and allow the plate to sit in a tissue culture hood for one minute to ensure that the lymphocytes are settled. Remove 200 to 250 microliters of media and place it into an empty well of a 24-well plate.
Using an inverted microscope, check the supernatant to ensure that no lymphocytes were removed. If the supernatant is clear, add 300 microliters of fresh ILC1 medium to the co-culture in the original well. If lymphocytes are present in the supernatant, centrifuge at 300 to 400 G for three to five minutes at four degrees Celsius and resuspend the pellet in 300 microliters of fresh ILC1 medium.
Then, add the cell suspension to the remaining 200 to 250 microliters of media in the original well. Perform downstream analysis using immunofluorescence, flow cytometry, or FACS purification of target populations into lysis buffer for gene expression analysis by a single-cell or bulk RNA seek or RT-qPCR as described in the text. After successful completion of passage, healthy and robust organoid cultures revealed that the freshly isolated crypts should form budding crypt structures within two to four days.
The flow cytometric analysis was performed to isolate small intestinal ILC1 from the ROR gamma-T murine transgenic reporter line, and the expected ILC count range was found to be 350 to 3, 500 isolated cells. After being seeded with organoids, co-cultures can be visualized by immunochemistry cytochemistry. Confocal microscopy revealed the images of small intestinal organoids that are cultured alone and in the presence of ILC1s.
Immuno-cytochemical staining reveals presence of CD45-positive ILC1 in close proximity to Zonula occludens protein-1-positive epithelial cells in organoid ILC1 co-cultures. Flow cytometry demonstrated that epithelial cellular adhesion molecule marks intestinal epithelial cells in organoids, while CD45 marks ILC1s. The flow cytometric plot and immunochemistry revealed increased expression of CD44 in intestinal epithelial cells when co-cultured with ILC1.
The RT-qPCR studies show that ILC1 co-culture specifically upregulated CD44 variant 6, which was inhibited by TGF-beta 1, 2, 3 neutralizing antibody, and upregulated by recombinant TGF-beta 1 in small intestinal-organoids-only cultures. It is important to be gentle during the manipulation of the organoids to ensure the whole structures are maintained and to check that the structures have pelleted sufficiently after centrifugation. By increasing the system complexity by adding other immune and non-immune components, this in vitro system can be used to address further questions in intestinal biology.
