The authors would like to thank the patients for their informed consent and participation in the research study, and the clinical personnel for their excellent assistance. Figure 1A was created with BioRender.com.&#160;This work was supported by grants from Science Foundation Ireland-namely a career development award (CDA) to K.N. (SFI-13/CDA/2171), a research centre grant (SFI-12/RC/2273), and a research centre spoke award (SFI-14/SP/2710) to APC Microbiome Ireland. P.F. also received funding from SFI/20/RP/9007.

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

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K.N. was in receipt of research funding from AbbVie Inc. during the time the work was completed. This funding was in the context of a research centre spoke award (SFI-14/SP/2710) to APC Microbiome Ireland.

Several methods have been developed for quantitative analyses of cell death in intestinal organoids. Examining the disruption of intestinal organoid morphology by light microscopy is a straightforward approach to quantifying the effects of cytotoxic substances11. However, morphological changes are not a direct measurement of cell death, and the method is only semiquantitative. Another method is to evaluate organoid metabolic activity using an MTT or ATP assay10,11. It is important to note that these assays can only determine changes in cell viability and must be validated with a cell death assay. Other fluorometric cell death assays using DNA binding dyes have been reported12,13. A non-imaging approach using a fluorescent microplate reader is possible and allows for high throughput12. However, this method measures the average signal of an entire well, making it unsuitable for heterogeneous populations. It also requires the use of a microplate reader with Z-height adjustment. Fluorescent imaging-based techniques can be used for single-organoid analysis and capture cellular/subcellular and morphological data. Automated confocal high-content imaging (HCI) systems can generate large amounts of data at a high throughput13. Unfortunately, confocal HCI needs specialized equipment, uses complex protocols, typically requires commercial image analysis software, and is expensive.
Our protocol for quantitative analysis of colonoid cell death at multiple time points is straightforward, simple, and inexpensive. However, compared to automated HCI and plate reader systems, it is time-consuming and has reduced throughput. Another limitation of our method is the use of widefield as opposed to confocal microscopy. Confocal microscopy is more suitable for imaging thick 3D samples such as organoids as it reduces out-of-focus signal and can acquire serial optical sections (Z-stacks). However, confocal imaging typically requires longer acquisition times and high-intensity lasers that increase phototoxicity/photobleaching. It is critical to note that fluorescent cell death dyes like SYTOX Green are only suitable for measuring forms of cell death where there is loss of cell membrane integrity such as necrosis, late apoptosis-associated secondary necrosis, necroptosis, and pyroptosis21. There are some forms of regulated cell death where the cell membrane remains impermeable at least during the early phases of cell death, such as caspase-dependent apoptosis. However, this protocol could be easily modified to also incorporate imaging of a caspase 3/7 activity fluorescent reporter22. This would provide additional data to help characterize the specific cell death modality.
We used our protocol to demonstrate the cytotoxic synergistic interaction between the cytokines IFN-&#947; and TNF-&#945; (Figure 2C), which we have previously reported in CD patient-derived organoids9,10. The physiological relevance of this form of synergism has also been demonstrated in murine models of hemophagocytic lymphohistiocytosis and sepsis23. Several mathematical reference models and approaches have been implemented for quantifying synergy between combinations of biological agents24,25. They differ in terms of their complexity, the number of factors they consider, and the threshold for considering an interaction to be synergistic24,25. Some models need prior knowledge of the biological agents tested, make certain assumptions about the activity of agents, and can require comprehensive dose-response curves for each single and combination treatment25. The method we selected to measure synergy is a modification of the coefficient of drug interaction (CDI) model, which has previously been used to measure the inhibitory effects of chemotherapy drug combinations on cancer cell line proliferation26. The CDI is a Bliss independence model; when calculating the predicted combined effect of two perturbagens Bliss independence assumes that they target separate pathways and have independent mechanisms of action27. For an interaction between perturbagens to be synergistic the actual combined effect must be greater than the predicted effect. This model is appropriate for our experimental setup as IFN-&#947; and TNF-&#945; are known to have different receptors and downstream signaling components. Further, Bliss independence allows for the calculation of a coefficient of interaction to quantify synergism and does not require dose-response datasets.
There are a few key factors that must be considered to ensure optimal results for this protocol. It is important that colonoids are propagated to a high density (Figure 1Bi), that they are approximately 25-50 &#181;m in diameter, and are actively proliferating before attempting to seed cells. The use of suboptimal cultures of colonoids for assays may result in insufficient cell numbers, low colonoid recovery, and inconsistent experiments. For reproducible results, it is also important to seed the density of colonoids consistently between experiments. It has previously been demonstrated that the in vitro response to inflammatory cytokines can be influenced by cell seeding density28,29. Another common issue is the formation of air bubbles in the BME dome, which can affect imaging. This can be prevented by using the reverse pipetting technique. This technique also results in more consistent seeding.
Further, if imaging multiple time points, prepare a Max Toxicity condition for each time point. Triton-X 100, a nonionic surfactant, is commonly used as a positive control (Max Toxicity condition) for cytotoxicity assays. The addition of Triton-X 100 lyses and kills the colonoids, allowing the fluorescent cell death dye to enter the cells. Using a Max Toxicity condition from an earlier time point will result in inaccurate and inconsistent normalization of data due to the fluorescent signal decaying over time.
A final point to consider is the choice of BME used for colonoid culture. There are several commercial producers of BME; however, for our protocol, we have only tested the brand included in the Table of Materials. A recent study using patient-derived pancreatic cancer organoids found that the commercial source of BME altered cell proliferation rates but had no significant effect on response to chemotherapy drugs or gene expression30. With this in mind, we expect the trend of results to be similar between BME brands for our protocol, but we recommend using the same brand consistently.
We demonstrated how this protocol can be used for the analysis of IFN-&#947; and TNF-&#945; induced cell death using CD patient-derived colonoids. Patient-derived intestinal organoids are a powerful tool to study CD as they retain many characteristics of the disease, including increased sensitivity to the cytotoxic effects of TNF-&#945;31. However, the protocol could be easily modified to investigate cytotoxic effects of perturbagens other than cytokines or disease states other than IBD such as colorectal cancer (we have successfully tested the protocol using non-IBD colonoids). We believe this method is useful for any research area concerned with cell death mechanisms, epithelial barrier function, or intestinal mucosal immunology.

The intestinal epithelium creates a physical semipermeable barrier between the contents of the gut lumen and the underlying tissues. To maintain this barrier effectively, intestinal epithelial cells (IECs) undergo an extremely high cellular turnover, with a continuous cycle of cell death and regeneration. However, during inflammatory disorders, such as inflammatory bowel disease (IBD), higher levels of aberrant cell death occur1. This may promote a breakdown in barrier function and activation of the immune system, triggering further inflammation. In Crohn&#39;s disease (CD), a form of IBD, it has been shown that cytokine signaling contributes to the increased levels of IEC death2. By studying how cytokine signaling induces cell death of IECs, it is hoped that improved treatments can be developed for patients with IBD and other intestinal inflammatory disorders1.
In biology and drug target discovery research, synergy is generally understood to occur when a biological system treated with combinations of individual stimuli shows a response to the combination that is greater than the combined additive effects of the single stimuli alone. Synergistic interactions between cytokines have been well documented in driving innate antiviral responses3. Cytokines have also been known to induce cell death synergistically, including in IECs4. However, the role synergistic cytotoxic cytokine signaling plays in intestinal inflammatory disorders such as IBD is understudied.
Human intestinal organoids are 3D microtissues produced in vitro that are generated from intestinal epithelial stem cells. Intestinal organoids can be grown from gut mucosal biopsies obtained from patients with IBD and retain many characteristics of the disease5,6. Organoids have proven to be an ideal model system for studying cytokine cytotoxicity in the context of intestinal inflammation7,8. Previously, our group has characterized the synergistic killing effects of the IBD-relevant cytokines IFN-&#947; and TNF-&#945; in CD patient-derived colonic organoids (colonoids)9,10. However, the exact mechanisms involved in mediating this form of synergistic cell death remain elusive. There are also potentially many more uncharacterized cytotoxic cytokine interactions that are relevant to intestinal inflammatory disorders.
Several protocols are available to study cell death in intestinal organoids10,11,12,13; however, they each have drawbacks. Some of these techniques only measure cell viability and do not measure cell death directly, are incapable of evaluating single-organoid responses, or require expensive equipment and complex protocols. Robust and straightforward methodologies are needed to quantify organoid cell death and perturbagen interactions in intestinal organoids. The protocol we present is a simple and inexpensive approach to measure single-organoid responses to cytotoxic cytokines but can be used for any type of stimulus or perturbagen. We also demonstrate how to use the Bliss independence synergy model to calculate a coefficient of perturbagen interaction (CPI) that describes cytotoxic cytokine interactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B Solution</td>
			<td>Sigma-Merck</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Sigma-Merck</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender</td>
			<td>Science Suite Inc.</td>
			<td>N/A</td>
			<td>Scientific illustration software</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Merck</td>
			<td>A2058</td>
			<td>Essentially IgG-free, low endotoxin</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Sigma-Merck</td>
			<td>SML1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 48-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Basement Membrane Extract, Type 2, Pathclear</td>
			<td>R&#38;D Systems</td>
			<td>3532-010-02</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Merck</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline</td>
			<td>Sigma-Merck</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Digital Inverted Fluorescence Microscope</td>
			<td>Invitrogen</td>
			<td>AMF4300</td>
			<td>Digital inverted epifluorescence microscope</td>
		</tr>
		<tr>
			<td>EVOS 40x Objective, fluorite, LWD, phase-contrast</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4683</td>
			<td>Long working distance 40x fluorescence objective</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ (Windows version)</td>
			<td>Open-source software</td>
			<td>N/A</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Sigma-Merck</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Solution</td>
			<td>Sigma-Merck</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>100-0485</td>
			<td>Enzyme-free cell dissociation reagent</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanyl-L-glutamine dipeptide supplement</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5 (Windows version)</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Data graphics and statistics software</td>
		</tr>
		<tr>
			<td>Greiner 15 mL Polypropylene Centrifuge Tube, Sterile with conical bottom &#38; Screw Cap</td>
			<td>Cruinn</td>
			<td>188261CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant EGF (animal free)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Merck</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Merck</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-05</td>
			<td>Broad range antimicrobial reagent</td>
		</tr>
		<tr>
			<td>Nunc Edge 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>ThermoFisher Scientific</td>
			<td>15543115</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-gamma Protein</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Resuspend in sterile filtered 0.1% PBS/BSA</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha Protein</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Resuspend in sterile filtered 0.1% PBS/BSA</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Merck</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap Low Retention Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>3451</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Green Nucleic Acid Stain - 5 mM Solution in DMSO</td>
			<td>Invitrogen</td>
			<td>S7020</td>
			<td>Fluorescent cell death dye, protect from light</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Merck</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Merck</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple Express</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>Enzymatic dissociation reagent</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>MedChemExpress</td>
			<td>HY-10071</td>
			<td>Inhibitor of ROCK-I and ROCK-II</td>
		</tr>
	</tbody>
</table>

quantification of cytokine-induced cell death in human colonic organoids using live fluorescence microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

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

The inflammatory cytokines interferon-gamma and TNF-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 parent 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-synergize to induce inflammatory cell death in gut epithelial cells and colon cancer cells.They do this via the JAK1/2-STAT1 signaling pathway. In future work, we wish to understand what type of cell death this is and how JAK1 and JAK2 kill the cells.

Using this protocol, we demonstrated how CD patient colonoids can be used to study the cytotoxic effects of the IBD-relevant cytokines IFN-&#947; and TNF-&#945; on primary epithelium. We used a commercially available fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), which can only enter cells that have a compromised cell membrane where it is then activated by binding to nucleic acids. We co-treated colonoids with cytokines and the fluorescent cell death dye and performed live cell imaging at 8 h and 24 h with an inverted epifluorescence&#160;microscope. Representative transmission/fluorescent overlay images at 8 h indicate that only the IFN-&#947; + TNF-&#945;-treated colonoids are positive for fluorescent signal; however, there are only a small number of fluorescent cells (Figure 2A). Cell blebbing, a morphological indicator of cell death20, can also be observed in the IFN-&#947; + TNF-&#945; condition. At 24 h, colonoids treated with IFN-&#947; + TNF-&#945; display large regions positive for fluorescent signal (Figure 2A). There is also a clear breakdown in the morphology of the colonoid-the central lumen is no longer visible, and the epithelial barrier has been completely disrupted.
To quantify the cell death dye signal, we used open-source image analysis software to calculate the fluorescent intensity of each colonoid. We then normalized the data by expressing the mean of each condition as a percentage of the Max Toxicity treatment. At 8 h, homeostatic or background cell death in the BSA control colonoids was relatively low (7.7% of Max Toxicity) (Figure 2B). There were no statistically significant changes in cell death levels at this time point; however, conditions treated with TNF-&#945; displayed a small increase in cytotoxicity (Figure 2B). After 24 h, cell death levels had increased for all cytokine-treated conditions. However, there was minimal change in cell death for the BSA Control condition between time points (7.5% of Max Toxicity at 24 h). The colonoids treated with IFN-&#947; + TNF-&#945; had the largest increase in cell death levels compared to BSA control (29.4% of Max Toxicity). The difference in cell death levels between the combined treatment and the single cytokine treatments (IFN-&#947;, TNF-&#945;) was highly significant. These results suggest the possibility of a cytotoxic synergistic interaction between IFN-&#947; and TNF-&#945; at 24 h.
We used the CPI to quantify the cytotoxic interactions between cytokine treatments and to determine if they were synergistic. Interactions between cytokines are considered synergistic when the CPI value is &#60;1, additive when =1, or antagonistic when &#62;1. We calculated CPI values per time point (Figure 2C). At 8 h, the CPI value indicated slight synergism (0.99), with the CPI value decreasing substantially at 24 h (0.83). This analysis confirmed that the interaction between IFN-&#947; and TNF-&#945; at 24 h was synergistic. Further, it illustrates how in this context synergism between IFN-&#947; and TNF-&#945; is time-dependent.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66759/66759fig01v2.jpg" /> 
Figure 1: Schematic of experimental workflow and troubleshooting. (A) Schematic overview of protocol. (B) Representative images. (Bi) Light microscopy image illustrating the optimal density of the culture and optimal size of colonoids prior to passaging for an assay. Scale bar = 500 &#181;m. (Bii) Light microscopy image illustrating optimal size of colonoid fragments after dissociation; fragments highlighted in red. Scale bar = 100 &#181;m. (Biii) Light microscopy image of necrotic colonoid morphology after MT treatment (with Triton X-100). Scale bar = 25 &#181;m. (Biv) Light microscopy image of two colonoids overlapping in the same focal plane. Scale bar = 25 &#181;m. (Bv) Light microscopy image of colonoid cell debris present after passaging; debris highlighted in red. Scale bar = 25 &#181;m. Abbreviations: ROI = region of interest; MFI = mean fluorescence intensity; MT = Max Toxicity. <a href="https://www.jove.com/files/ftp_upload/66759/66759fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66759/66759fig02v2.jpg" /> 
Figure 2: Quantitative analysis of cytokine-induced cell death in human CD colonoids. (A) Representative live microscopy images of CD colonoids treated with SYTOX Green Nucleic Acid Stain (fluorescent cell death dye) and cytokines at 8 h and 24 h; transmission and GFP (green color) channels overlayed. Colonoids were treated as follows: 1) PBS/BSA, 2) 10 ng/mL IFN-&#947;, 3) 10 ng/mL TNF-&#945;, 4) 10 ng/mL IFN-&#947; + 10 ng/mL TNF-&#945;. Scale bars = 25 &#181;m. (B) Quantitative analysis of CD colonoids treated with the fluorescent cell death dye and cytokines at 8 and 24 h; data are expressed as a % of the MT condition. N = 2 CD colonoid lines, 11-16 colonoids imaged per condition. (C) CPI calculated per time point using the dataset from B, N = 2 CD colonoid lines. Data are expressed as means &#177; SE. In B, two-way ANOVA analysis was performed followed by Bonferroni post-tests, *P &#60; 0.05, ***P &#60; 0.001 as indicated. Abbreviations: CD = Crohn&#39;s disease; GFP = green fluorescent protein; CPI = coefficient of perturbagen interaction; PBS = phosphate-buffered saline; BSA = bovine serum albumin; TNF-&#945; = tumor necrosis factor-alfa; IFN-&#947; = interferon-gamma; MT = Max Toxicity. <a href="https://www.jove.com/files/ftp_upload/66759/66759fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of culture media for protocol. To prepare Organoid Proliferation Media, combine L-WRN conditioned media and Serum-free Media 1:1, then add supplements. Organoid Proliferation Media should be used within 2 weeks of preparation. Please note all complete media should be stored at 4 &#176;C. <a href="https://www.jove.com/files/ftp_upload/66759/66759_Table 1 (1).xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Experimental 96-well plate layout and cytokine treatments. <a href="https://www.jove.com/files/ftp_upload/66759/66759_Table 2 (1).xlsx" target="_blank">Please click here to download this Table.</a>

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

quantification-cytokine-induced-cell-death-human-colonic-organoids