Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

The inflammatory cytokines interferon gamma and TNF alpha have existed for hundreds of millions of years and are critical for effective host defense against intercellular pathogens. Our research group is trying to understand how these cytokines work to kill cells such as epithelial cells in the gut and cancer cells. Commonly used systems to study intestinal epithelial cell death mechanisms include mouse models and immortalized cancer cell lines.

Human test organoids are advantageous as they're generated directly from patient biopsies and retain many physiological and morphological characteristics of the pair in tissue. This means they have increased translational value. Organ research has issues with experiments reproducibility and has several technical challenges compared to traditional cell culture.

The current techniques for measuring organoid cell death also have limitations. Some are only semi-quantitative, do not measure cell death directly, cannot measure single organoid responses, or require expensive equipment and complex protocols. Our protocol for quantitative analysis of organoid cell death is straightforward, robust, and inexpensive.

We used our protocol to measure single organoid responses to cytotoxic cytokine combinations, but it can be easily adapted to study any type of perturbagen. This method is useful for research on cell death, epithelial barrier function, or mucosal immunology. Recently we have reported that interferon gamma and TNF alpha synergize to induce inflammatory cell death in gut epithelial cells and colon cancer cells.

They do this via the JAK one two stat one signaling pathway. In future work, we wish to understand what type of cell death this is and how JAK one and JAK two kill the cells. After expanding Crohn's disease patient-derived colonoids in a 48 well plate, gently remove the medium from the edge of the well without damaging the colonoid dome.

Add 300 microliters of enzymatic dissociation reagent supplemented with 10 micromolar ROCK1 and two inhibitor Y-27632 to each well. Using a P1000 pipette tip, scrape the surface of the well to break the colonoid dome and pipette the cell suspension up and down. Transfer the cell suspension into a 15 milliliter tube.

Incubate the collected colonoids in a 37 degrees Celsius water bath for five minutes. Centrifuge the colonoids at 400 G for three minutes. And remove the supernatant, leaving approximately 1.2 milliliters in the tube.

Next, place the tip of the P1000 pipette into the suspension, holding it just above the bottom of the tube and rapidly pipette the suspension in and out of the tip to dissociate the colonoids. Observe the tube under the microscope to ensure no whole colonoids remain, and colonoid fragments are approximately 30 to 40 micrometers in size. Next, add 10 milliliters of ice cold organoid wash media to the 15 milliliter tube.

Centrifuge the tube at 400 G for three minutes. Remove the supernatant and resuspend pellet in one milliliter of ice cold organoid wash media. Transfer the suspension to a 1.5 milliliter micro centrifuge tube and label it as a colonoid fragment tube.

After mixing, transfer 50 microliters of the sample to a new tube and label it as a cell count tube. Centrifuge the tube at 400 G for three minutes. Remove the supernatant and resuspend the pellet in 500 microliters of enzymatic dissociation reagent supplemented with 10 micromolar Y-27632.

Incubate the single cell count tube in a 37 degrees Celsius water bath for five minutes. Using a P1000 pipette set to 400 microliters, rapidly pipette the sample. Then observe the sample under a microscope to ensure a single cell suspension.

Add one milliliter of organoid wash media to the single cell count tube. Then centrifuge the suspension and remove the supernatant before resuspending the pellet in 50 microliters of organoid wash media. Add 50 microliters of Trypan blue and count the cells using a hemocytometer.

Based on this, calculate the concentration of cells in the colonoid fragment tube. Centrifuge the required volume in a new 1.5 milliliter microcentrifuge tube. Then remove the supernatant.

Resuspend the pellet in basement membrane extract, or BME. Now, in a pre incubated 96 well microtiter plate, reverse pipette 10 microliters of the colonoid BME solution per well and carefully mix the solution regularly to prevent uneven seeding. Invert the plate and incubate at 37 degrees Celsius and 5%carbon dioxide.

After 20 minutes, overlay the domes with 200 microliters of prewarmed organoid proliferation media, and continue incubation for three days. After incubating Crohn's disease patient-derived colonoids for three days, tilt the plate and remove the medium from the edge of the wells. Add 200 microliters per well of cytokine treatment media containing 2.5 micromolar fluorescent cell death dye.

Incubate the colonoids at 37 degrees Celsius and 5%carbon dioxide for the required treatment time. After incubation, transfer the plate to the stage of a digital inverted epifluorescence microscope and confirm that the max toxicity condition colonoids are fully liced. Using the transmission channel, focus on a colonoid with SYTOX positive cells.

Then switch to the GFP channel. Adjust the light intensity and exposure time to maximize the fluorescent signal while minimizing the background. Using optimized image settings, observe the no dye and max toxicity conditions to ensure the samples are not over or underexposed.

Acquire transmission and GFP images of colonoids using a random sampling approach by selecting fields of view that follow a fixed grid pattern covering the colonoid dome. Save images in portable network graphic format and export them. Drag and drop the image dataset files onto the ImageJ toolbar and sequentially click Image, Stacks, and Images to Stack to combine the files.

Then click Image followed by Type and 8-bit to convert the image stack to an 8-bit file. Click the freehand selections tool and manually select the region of interest on the transmission image. Then toggle through the image stack to the corresponding GFP channel image.

From the toolbar, click Analyze and Set Measurements. In the Set Measurements dialogue window, tick the mean gray value and leave other boxes unticked. With the GFP image selected, click Analyze and Measure.

Once the dataset is analyzed, copy all the data in the results window and paste them into a spreadsheet. Calculate the mean of the technical replicate mean gray values for each condition. Subtract the mean of the untreated condition from each treatment condition.

Then divide each treatment condition by the background subtracted mean of the max toxicity condition. To calculate cell viability after treatment, subtract the normalized values from one. Using the given equation, calculate the coefficient of perturbant interaction.

Transmission fluorescent overlay images of colonoids at eight hours indicated that only the interferon gamma plus TNF alpha-treated colonoids were positive for fluorescent signal. At 24 hours, colonoids treated with interferon gamma plus TNF alpha displayed large regions positive for fluorescent signal with a clear breakdown in colonoid morphology. Cell death levels at eight hours were low for BSA control colonoids with a slight increase in TNF alpha-treated conditions.

At 24 hours, interferon gamma plus TNF alpha-treated colonoids showed the highest cell death levels. CPI values indicated slight synergism at eight hours and substantial synergism at 24 hours. This confirmed a time-dependent synergistic interaction between interferon gamma and TNF alpha.