Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

So progenitor cells interact with the environment to maintain tissue homeostasis. And using this protocol we can begin to understand how fibroblasts affect esophagal progenitor cell behavior. The co-culture system uses firstly isolated cells that mimics the normal esophageal progenitor niche.

Clearing of the organoids before imaging enables the analysis of both cell interactions and organoid morphology. This method could be applied to human esophageal organoids. For example using materials from healthy or cancer patients.

In doing so, we can obtain insights into the specific function of cancer associated fibroblasts. Dissociation of epithelial and stromal cells is critical to obtain viable organoids. Under digestion will result in a low cell yield, where over digestion will reduce cell viability and organoid forming capacity.

To begin, mechanically remove the muscularis externa of the esophagus using a dissection microscope and forceps. Hold the distal end of the dissected esophagus using one pair of forceps and use the other forceps to grab and pull the muscle from the distal to the proximal end of the esophagus. Remove and discard the muscle layer.

To open the esophagus longitudinally, hold one end of the esophagus and insert the ball of the micro dissection spring scissors into the lumen to prevent tissue damage. Cut open the esophagus while holding onto the end and place it in a 1.5 milliliter microcentrifuge tube, or a 24 well plate. Submerge the opened esophagus in 0.5 milligrams per milliliter thermolysin in HBSS and incubate at 37 degrees Celsius on a rocker shaker for 15 minutes.

After removing the esophagus from the thermolysin solution carefully separate the esophageal epithelium from the stroma. Transfer the epithelial and stromal layer to two separate 1.5 milliliter microcentrifuge tubes with 200 microliters of dissociation solution in HBSS, and place them on ice. Mince the esophageal epithelium using a sharp scalpel and collect the minced tissue from the Petri dish with 200 microliters of dissociation solution.

Transfer it to a 1.5 milliliter microcentrifuge tube. Add 800 microliters of fresh dissociation solution and place the tube with the minced epithelial layer on a rocker shaker at 37 degrees Celsius for 60 minutes. Pipette the solution up and down approximately 20 times using a 200 microliter pipette tip every 15 minutes.

Cut the stromal layer into fine pieces in a 1.5 milliliter tube containing 200 microliters of dissociation solution using dissection scissors and add 800 microliters of fresh dissociation solution. Place the tube on a rocker shaker at 37 degrees Celsius for 30 minutes. After 30 minutes of incubation for stromal solution and 60 minutes of incubation for epithelial solution, pipette the solution up and down for another 20 times.

Pass the stromal solution through a 70 micrometer cell strainer, and the epithelial solution through a 40 micrometer cell strainer into new 1.5 millimeter micro centrifuge tubes. Centrifuge at 300G for 10 minutes at four degrees Celsius and discard the supernatant by removing the excess liquid with a one milliliter pipette. Resuspend the pellet in 200 microliters of antibody mix.

After transferring the mixture to a flow cytometry tube, incubate the cells for 30 minutes at four degrees Celsius. Add three milliliters of 1%FBS and centrifuge again for five minutes. Then resuspend the cells in a minimum of 200 microliters of 1%FBS.

Add one to 10, 000 diluted dead cell stain marker, five minutes prior to fax sorting to isolate the live cells. Mix the sorted epithelial cells and fibroblasts at a ratio of one to two in a tube. After centrifuge at 300 G for five minutes, discard the supernatant by carefully removing it with a 200 microliter pipette.

Wash the cells once by resuspending the pellet in cold, basic organoid medium and centrifuge again for five minutes. Place the cells on ice and discard the supernatant by carefully removing it with a 200 microliter pipette. Resuspend the cells in 10 microliters of matrix mix per dome and put the tube back on ice.

Take the pre-warmed 48 well plate from the 37 degrees Celsius incubator and make one matrix dome per well. Using a 20 microliter pipette add 200 microliters of pre-warmed, ER low medium to the matrix domes containing organoid co-cultures and ENR medium to the respective matrix domes containing only epithelial organoids. Place the plate in an incubator at 37 degrees Celsius and 5%carbon dioxide.

And for the first two days, supplement the medium with 10 micromolar rock inhibitor. Remove the organoid medium and add 200 microliters of ice cold PBS to the matrix domes. After placing the plate on ice for five to 10 minutes, pipette up and down and transfer the solution to a 0.6 milliliter non-adherent tube.

Centrifuge shortly for 30 to 60 seconds at 100G to let the organoids settle in the bottom. After removing the excess liquid, add ice cold PBS to the tube and centrifuge again for 30 to 60 seconds. Remove the excess liquid as shown earlier and fix the organoid with 200 microliters of cold, 4%formaldehyde in PBS solution for 30 minutes on ice.

Place the tube upright to let the organoid sink and remove the formaldehyde. Add 500 microliters of cold PBS to wash away the remaining formaldehyde. After removing the excess PBS, add 500 microliters of blocking buffer.

Place the tube on a rocker shaker for 60 minutes at room temperature. Remove the blocking buffer and resuspend the organoids in 200 microliters of blocking buffer with primary antibodies. Place the organoids again on a rocker shaker overnight at four degrees Celsius.

Remove the primary antibody mix and wash the organoids using 500 microliters of 0.02%Triton X 100 in PBS for 60 minutes at room temperature. Repeat this three times. Similarly, remove the washing buffer and add 200 microliters of fluorescence conjugated secondary antibody in the blocking buffer overnight at four degrees Celsius.

Rewash the organoids using 0.02%Triton X 100 in PBS followed by 500 microliters of PBS. After removing all the excess liquid add 10 microliters of clearing solution to the organoids and incubate for 15 minutes at room temperature. Place a 0.05 millimeter double-sided sticky four well spacer on a microscope slide.

Add the 10 microliters of clearing solution with the organoids in one well and place a cover slip on top of the spacer. Acquire images using a confocal microscopy system. Esophageal progenitor cells are sorted based on their high integrated beta four and e-cadherin expression.

The representative flow cytometry plot of epithelial cell isolation shows the percentage of live cells from all single cells. The percentage of isolated integrin beta four and e-cadherin progenitors cells from all live cells is shown here. The flow cytometry plots shown here represent the percentage of isolated DPP4+and Pdgf receptor alpha positive fibroblasts.

The bright field images with the organoids co-cultured with Pdgf receptor alpha H2 BEGFP positive fibroblasts show the nuclear EGFP signal. The bright field images of the whole matrix dome on day six are shown here. The organoid forming efficiency is presented by this graphical image.

Each dot represents a matrix dome, and the bar represents the mean of all dots per condition. The bright field and fluorescent images of day six organoids co-culture with Pdgf receptor alpha positive fibroblasts are shown here. The whole mount images of the co-cultured organoids show 3D surfaces of the organoids with vimentin positive fibroblasts wrapped around and in close contact with the organoid.

The whole mount staining of mono and co-cultured organoids with Pdgf receptor alpha positive fibroblasts reveal distinct basal and suprabasal cell populations. The organoid forming efficiency as well as the organoid size are influenced by the fibroblast included into the co-culture. Variations and results are to be expected when using different fibroblast subpopulations.

The same strategy can be employed to characterize tumor associated fibroblasts in both mice and humans.