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><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&amp;amp;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&amp;prime;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 &amp;amp; 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&amp;amp;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&amp;amp;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.

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>

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

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.

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 ...

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

Research

JoVE Journal

Biology

1.2K Views.  University College Cork. 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.

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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cancer Research

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Bioengineering

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

Medicine

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Quantifying Cell Death Initiation Through Continuous Imaging

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells

Source: Stedman, A. et al., <a href="https://app.jove.com/v/62880">Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.</a> J. Vis. Exp. (2021).
This video describes the technique of visualizing oxidative stress in cultured organoids. This work helps in understanding the level of oxidative stress due to ROS accumulation in the various cell types present in an organoid after treatment with different ROS modulators.

Source: Stedman, A. et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. ...

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

Summary

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Chapters in this video

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8⁺ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Quantification of Proliferative and Dead Cells in Enteroids

Studying Cell Death Initiation Using a Digital Microscope

Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells