Exploring cell death mechanisms in spheroid cultures: A novel application of the RIP3-Caspase3-Assay

Exploring cell death mechanisms in spheroid cultures: A novel application of the RIP3-Caspase3-Assay | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring cell death mechanisms in spheroid cultures: A novel application of the RIP3-Caspase3-Assay Clara Isabell Philippi, Johanna Hagens, Kim Marili Heuer, Hans Christian Schmidt, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3866340/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract This study explores the application of the RIP3-Caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-Caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research. Health sciences/Medical research/Paediatric research Health sciences/Medical research/Stem cell research Biological sciences/Immunology/Cell death and immune response Biological sciences/Stem cells/Intestinal stem cells Organoids Cell death mechanisms RIP3-Caspase3 Assay TNFα -induced stress Apoptosis and necroptosis Figures Figure 1 Figure 2 1. Introduction The intricate processes of cell death, including apoptosis and necroptosis, are fundamental to our understanding of various pathological conditions. In particular, the balance between these processes is crucial in conditions of inflammation, neurodegenerative diseases, or cancer [ 1 – 3 ]. Despite advancements in cellular biology, the accurate delineation of these processes remains a significant challenge, especially in physiologically relevant models like organoids. Traditional methods often fall short in providing a detailed analysis of these complex pathways, especially in heterogeneous cell populations like those found in organoid cultures. This limitation hinders our ability to fully understand and effectively target dysregulated cell death mechanisms in disease settings [ 4 , 5 ]. Recent advances in organoid technology offer promising avenues for more accurate disease modeling, providing three-dimensional structures that closely mimic in vivo conditions [ 6 ]. However, applying existing cell death assays to these models is challenged by their cellular and structural complexity. There is a critical need for assays that can differentiate between various cell death pathways in a single, cohesive analysis. Addressing these challenges is crucial for the development of targeted therapies and enhancing our understanding of disease mechanisms at a cellular level [ 7 , 8 ]. In this context, our study introduces the RIP3-Caspase3-assay, a novel approach employing directly conjugated monoclonal antibodies to analyze regulated cell death mechanisms in spheroid cultures. This assay is designed to overcome the limitations of existing methods, offering a detailed and nuanced analysis of cell death pathways in heterogeneous organoid samples. By providing insights into RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, the RIP3-Caspase3-assay aims to advance our understanding of cell death in disease processes and aid in the development of more effective therapeutic strategies [ 9 ]. 2. Material and Methods 2.1 Cultivation of Organoids The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board Hamburg ethics committee. Parents or guardians provided informed consent for collection and analyses of the tissues. Tissue samples were harvested from patients who underwent surgery for Morbus Hirschsprung pull-through procedure (n = 3), intestinal atresia repair (n = 1) or colostomy closure (n = 1) (patient characteristics see table 2s, supplementary material). Samples were transported in Iscove’s Modified Dulbecco’s medium (IMDM, #12440053, Gibco ThermoFisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum (FBS, #0500 − 064, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (P/S, #PS/B, Capricorn Scientific, Ebsdorfergrund, Germany) for viability preservation and processed within 24 h. Samples were washed in sterile Dulbecco’s phosphate-buffered saline (DPBS; #37350, Gibco Thermo Fisher Scientific, USA). The colonic mucosa was mechanically separated from the rest of the tissue and sliced into pieces measuring 1–2 mm. The pieces were incubated in IMDM containing 5 mM ethylenediaminetetraacetic acid (#15575-038, Invitrogen, Waltham, MA, USA) and 2 mM DL-Dithiothreitol (DTT, #D9779-5G, Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 4°C. Crypt isolation was verified using an inverse microscope (Olympus IX50-S8F; Olympus, Tokyo, Japan). The medium containing the mucosa was pipetted up and down several times with a 25 ml serologic pipette, followed by another incubation at 4°C for 20 min. Adult stem cells (AdSCs) were isolated using a 70 µm cell strainer (#352350, Corning, Corning, NY, USA) and rinsed with a washing buffer containing IMDM with 2% FBS and 1% P/S to collect all cells. The cells were centrifuged at 500 × g at 4°C for ten minutes and washed twice with Advanced Dulbecco’s Modified Eagle Medium (Advanced DMEM, #12491-015, Gibco ThermoFisher Scientific, USA) containing 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, #H3537-100ML, Sigma-Aldrich, St. Louis, MO, USA), 1% GlutaMAX (#35050-061, Gibco ThermoFisher Scientific, Waltham, MA, USA), and 1% P/S (further referred to as Adv. +++). 20 µl cell suspension was pipetted into 40 µl of growth factor-reduced, phenol red-free Matrigel Matrix (#356231, Corning, Corning, NY, USA) and mixed carefully. 30 µl of the mixture was pipetted into a prewarmed flat bottom 24-well-plate (#3526, Costar, Corning, NY, USA). Domes solidified upon incubation at 37°C and 5.0% CO 2 for 30 min. 500 µL of Seeding Medium consisting of IntestiCult Organoid Growth Medium Human (OGM-h, #060610, Stemcell Technologies, Canada) supplemented with 5 mM of ROCK-Pathway Inhibitor (ROC, #72302, Stemcell Technologies, Vancouver, Canada), 1% P/S, and 0.02% Primocin (#ant-pm-05, InvivoGen, San Diego, CA, USA) was added to the wells. Successful seeding was confirmed by light microscopy. Organoids were cultivated at 37°C and 5.0% CO 2 . The seeding medium was changed every 2–3 days. After 7–10 days, organoids were ready for the first passage. 2.2 Maintenance of the Organoids Passaging of organoids was performed as follows. The medium was removed and the Matrigel domes were broken up by pipetting with 1 ml Adv.+++. The wells were washed with Adv.+++ and cell suspension was centrifuged at 300 × g and 4°C for 5 min. Organoid cells were separated into smaller cell clusters by pipetting up and down several times, and again centrifuged at 400 × g . After removing the supernatant, disrupted organoids were resuspended with Adv.+++ and reseeded in Matrigel, as described in section 1.2.. Medium was changed every two to three days. After the fifth passage, organoids were considered mature and used for the experiments. After 4–6 days, OGM-h was replaced with IntestiCult Organoid Differentiation Medium Human (ODM-h, #100–02114, StemCell Technologies, Vancouver, Canada) containing 5 mM Daptomycin (DAPT), a notch pathway inhibitor (# 72080, StemCell Technologies, Vancouver, Canada). Organoids were cultured for 3–5 days in ODM-h, which was changed every 2 days as well. Once the epithelia of the organoids started to thicken and form bud-like structures, organoids were prepared for the experiment. Organoid growth and differentiation were monitored using light microscopy (Leica DM IL LED, Leica Microsystems, Wetzlar, Germany). 2.3 Determination of TNFα concentration using flow cytometry The purpose of this experiment was to assess the effect of Tumor Necrosis Factor-α (TNFα) on differentiated organoids. Ten wells containing 30 µl Matrigel dome were needed from each culture of organoids to perform the experiment. Organoids were maintained for five days in OGM-h. On day 5, the medium was replaced with supplemented ODM-h to initiate differentiation. After 24 hours, TNFα (TNFα, #300-01A, PeproTech London, UK) was added to the ODM-h in the concentrations 0,1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, and an unstimulated control, always treating two wells the same. Organoids were maintained in ODM-h with TNFα for a total of 72 h, with one medium change in between (Fig. 3s, supplementary material). Organoid growth was monitored using light microscopy (Fig. 1 ). For flow cytometry analysis of the cells, the medium was removed and Matrigel containing the organoids was pipetted up and down with Adv.+++, transferred into a 15 ml tube (#188271, Greiner Bio-One, Kremsmünster, Austria), and incubated with TrypLE (#12605028, Gibco ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 4 min to separate the organoids into smaller cell clusters. The organoid fragments were further separated into single cells by pipetting. Cell isolation was verified using a light microscope. The cells were centrifuged in Adv.+++ at 300 × g and 4°C for five minutes and the supernatant was discarded. The cells were incubated with Zombie NIR fluorescent dye (#423105, dilution 1:2000, BioLegend, San Diego, CA, USA) for 30 min at 4°C in the dark. Next, the cells were washed and fixed with an intracellular fixation buffer (eBioFix, #00-8222-49, BD Biosciences, San Jose, CA, USA) for 20 min at 4°C. Cells were resuspended in 200 µl DPBS and ready for analysis with LSR Fortessa and BD FACS Diva Software (BD Biosciences, San Jose, CA, USA). For each tube, 10,000 single cells were analyzed. Small debris at the origin was removed using a gate in the FSC-A vs. SSC-A dot plot. Single cells were gated on SSC-W versus SSC-H dot plots. These were gated on a dot plot of Zombie NIR vs. SSC-A showing all zombie-positive dyed cells. Zombie staining indicated a compromised cell membrane, these cells were defined as dead. 2.4 RIP3-Caspase3-Analysis of apoptosis, necroptosis and RIP1-dependent apoptosis To quantify RIP1-independent apoptosis, RIP1-dependent apoptosis, necroptosis, and other forms of cell death in the epithelial cells, the intracellular cell-death proteins RIP3 and Caspase3 were stained in addition to Zombie NIR and measured by flow cytometry, as described by Lee et al. (2018). Sensitivity to the stressor was measured by the extent of cell death for each sample and condition. Untreated samples served as negative controls. For this experiment, four wells containing a 30µl Matrigel dome were used from each culture. The organoids were maintained in OGM-h for five days. On day 5, the medium was replaced with ODM-h. 48h before flow cytometry, two of the four wells were treated with TNFα at a concentration of 100 ng/ml. The other two wells were maintained in ODM-h without TNFα (Fig. 4s, supplementary material). Organoids were prepared for flow cytometry as described in Section 2.3 .. After the fixation with eBioFix, cells were permeabilized with PBS containing 0.25% Triton X-100 (#T8787-50ML, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4°C under movement to expose intracellular targets. The cells were incubated with fluorescent-conjugated antibodies anti-active Caspase-3-BV650 (#564096, dilution 1:50; BD Biosciences, San Jose, CA, USA) and anti-RIP3-Alexa Fluor 488 (clone B-2, #sc-374639, dilution 1:125, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at 4°C in the dark. Cells were washed with 500 µl PBS containing 0.25% Triton X-100 and then resuspended in 200 µl PBS. In each tube, 5.000 single cells were analyzed using LSR Fortessa and BD FACS Diva Software. Small debris was removed using a gate in the FSC-A vs. SSC-A dot plot. Single cells were gated on SSC-W versus SSC-H dot plots. These were gated on a dot plot of Zombie NIR vs. SSC-A showing all zombie-positive dyed cells that were defined as dead. Cells unstained by Zombie NIR were gated in the FSC-A vs. SSC-A dot plot and were defined to be living cells. Using Caspase-3-BV650 vs. RIP3-Alexa Fluor 488 dot plots, live and dead cells were gated separately with the same gate configuration for each plot. Caspase3-positive cells indicate a RIP1-independent apoptotic process, and RIP3-positive cells indicated a necroptotic process. Caspase3- and RIP3-positive cells were determined to undergo RIP1-dependent apoptosis. Cells with neither Caspase3 nor RIP3 signals were assumed to have undergone other forms of cell death (Table 1 ). Table 1 Cell death status definitions. Caspase3-positive signal indicating an apoptotic cell, RIP3-positive signal indicating a necroptotic cell, Caspase3- and RIP3-positive signal indicating a RIP1-dependent Apoptosis and no Caspase3- or RIP3-signal indicating other forms of cell death. RIP3-Status Caspase3-Status RIP1-dependent Apoptosis positive positive Apoptosis negative positive Necroptosis positive negative Other cell death negative negative 2.5 Statistical analysis Statistical analyses were generated using GraphPad Prism 9 (San Diego, CA, USA). Simple comparisons were performed by descriptive statistics and student’s t-test. Mean values of multiple variables were compared using two-way analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant. 3. Results 3.1 Determination of TNFα concentration This experiment was performed to determine the effect of increasing TNFα concentrations on overall cell survival, using Zombie NIR staining only. Stimulation with TNFα showed stable percentages of Zombie + cells between 0 ng/ml and 10 ng/ml, ranging from 39.06% (± 8.51%) to 42.68% (± 7.35%). Cell death rate increased to 45.30% (± 7.05%) after treatment with 100 ng/ml. The FSC-A vs. SSC-A dot plot detected an increase in cell debris after 72 h treatment with TNFα as a display of organoid damage. Consequently, only 5,000 single cells were analyzed in each tube, ensuring comparability between the different treatments and samples. The destruction was already visible at 48 h of treatment during daily monitoring (Fig. 1 ). Therefore, for the final next experimental analysis setup, 100 ng/ml TNFα for 48 h was considered the optimal condition to induce stress in the cells. 3.2 Cell death markers RIP3, Caspase3 and Zombie can be detected in organoids using the RIP3-Caspase3-Assay As exemplary seen in Fig. 2 B, stimulation with TNFα caused morphological changes indicating destruction within the cultures. As shown in Fig. 2 A, the assay was able to detect Zombie staining as well as RIP3- and Caspase3-status. Treatment with TNFα caused a slight increase in Zombie-cells (29.71% vs. 32.89%, p = 0.3451) and Caspase3 expression (63.00% vs. 69.91%, p = 0.2038) while RIP3 was detected less after stimulation (16.61% vs. 12.13%, p = 0.3482). 3.3 RIP3-Caspase3-assay reveals apoptosis as the leading cell death mechanism in living cells For further analysis, cell death measurements were separated into living (Zombie − ) and dead cells (Zombie + ). Living cells represent cells with intact cell membranes not stained by Zombie NIR and were gated as described in section 2.4 .. As seen in Figs. 2 C-D, apoptosis was the leading cause of cell death. Comparing cell death mechanisms, predominance of RIP1-dependent apoptosis reached significance against all other groups pre and post treatment ( p < 0.0001). Also, RIP1-independent apoptosis was significantly higher compared to necroptosis after stimulation ( p = 0.0002), but not before ( p = 0.0644). Comparison between RIP1-independent apoptosis and other forms of cell death also did not reach significance neither pre ( p = 0.8027) nor post treatment ( p = 0.0559) with TNFα. Stimulation caused an increase in RIP3 − /Caspase3 + cells from 16.06–28.91% ( p = 0.1572), while RIP3 + /Caspase3 + cells showed less change (64.08% vs. 59.47%, p = 0.9101). There were near to no detectable RIP3 + /Caspase3 − cells (0.33% vs. 0.13%, p > 0.9999). RIP3 − /Caspase3 − cells decreased from 21.54–12.79% ( p = 0.4987) after the treatment. 3.4 Dead cells show a significant rate of RIP1-independent apoptosis after TNFα stimulation Zombie-positive cells, representing cells with compromised membranes, were also gated as described in section 2.4 and are displayed in Figs. 2 C and 2 E. Comparison between the different cell death mechanisms groups before and after treatment revealed significant differences ( p < 0.0001) between all except three combinations: Before stimulation, RIP1-independent and RIP1-dependent apoptosis did not differ ( p = 0.1307), but difference reached significance after TNFα treatment ( p = 0.0003), showing a growth of the first and decrease of the latter one. Moreover, RIP1-independent apoptosis nearly reached the level of other cell deaths after stimulation ( p = 0.8368). While apoptosis overall remained a prominent cause of cell death, RIP3 − /Caspase3 + cells increased significantly from 19.72–38.05% ( p 0.9999) after treatment. RIP3 − /Caspase3 − cells presented the leading cause of cell degradation in this cohort, but also significantly decreased after treatment with TNFα (52.66% vs. 42.17%, p = 0.0255). 4. Discussion Our study's successful application of the RIP3-Caspase3-assay in heterogeneous organoids highlights its methodological strengths. By employing directly conjugated monoclonal antibodies, our assay provides a nuanced analysis of cell death pathways, revealing the complexity of these processes in greater detail. This approach aligns with Lee et al.’s findings, which distinguished between apoptosis, necroptosis, and RIP1-dependent apoptosis in both viable and non-viable cell populations [ 9 ]. Our extension of these advancements to spheroid cultures marks a first in the field, showcasing the assay's reliability and its capability to discern between cell death mechanisms. In our study, the treatment of isolated intestinal epithelial cells from spheroids with TNFα accelerated the cell death rate, as indicated by Zombie-stained cells. The study observed an initial lower response to TNFα that increased with higher concentrations. In this context, this pattern suggests a possible adaptive or threshold response of the cells to TNFα-induced stress. TNFα is known to activate multiple signaling pathways, including NF-κB, MAPKs, and the apoptotic pathway [ 10 ]. The activation of NF-κB, for instance, can lead to the expression of survival genes, which might help cells adapt and survive under increasing TNFα concentrations [ 11 ]. However, in this study we had a linear response to the TNFα concentration with relevant damage of the organoids at 100ng/ml after 48h. This finding was in line with observed morphological changes of treated organoids (Fig. 1 ). Upon TNFα stimulation, we observed an increase in Caspase3 expression and a decrease in RIP3 expression across all samples. FACS analysis further revealed that TNFα-treated cells showed a significant increase in apoptosis, while simultaneously shifting away from RIP1-dependent apoptotic mechanisms, without involving necroptosis (Fig. 2 C-E). This finding suggests a preference for RIP1-independent cell death in both living cells undergoing cell death and those that had already died. The shift towards apoptosis in the presence of TNFα is a key finding. Apoptosis is a programmed and orderly cell death process, often considered immunologically silent, while necroptosis is a form of programmed necrosis that is inflammatory in nature. The decision between apoptosis and necroptosis is often determined by the availability and activity of key regulatory proteins like RIP1, RIP3, and Caspase8 [ 11 ]. This shift in the type of cell death can also be attributed to the inflammatory context, where the co-sensing of multiple cytokines and the activation of various pathways, such as the non-canonical NF-κB pathway, lead to different forms of cell death. For instance, the combination of TNFα with other cytokines can result in TNFR1-induced RIP1 kinase activity-dependent apoptosis [ 11 ]. Also, the balance between apoptosis and necroptosis is depended on the concentration of TNFα [ 12 ]. In contrast, TNFα treatment did not impact the proportion of "other" cell death types substantially. However, cells sorted into this category showed a higher proportion in the dead cell section than in the living cells, which may be attributable to the disruption of cell membrane integrity and subsequent cell death during assay preparation. Another important finding is the differential response in living versus dead cells (Fig. 2 D-E). The significant changes in the dead cell population, as opposed to the living cells, suggest that TNFα effects are more pronounced in cells already compromised or stressed. This could imply that TNFα is a more potent inducer of cell death pathways in cells that are already damaged. This is because stressed or damaged cells might have compromised defense mechanisms (for example reduced expression of anti-apoptotic proteins), making them more prone to TNFα cytotoxic effects [ 11 ]. Also, the absence of necroptotic cells in both living and dead cells suggests that, under these conditions, TNFα does not induce necroptosis and underscores TNFα role in promoting apoptosis under these experimental conditions in colonic epithelial organoids. The assay's ability to detect both major and minor cell populations undergoing various forms of cell death is particularly valuable in the context of spheroid cultures, where cell heterogeneity and complexity are inherent [ 7 ]. Notably, unlike the original technique described by Lee et al., our approach did not use CalTag fixative, known to upregulate RIP3. This modification is noteworthy because the detection of RIP3 in our results is not influenced by the fixative. Since we were still able to detect RIP3 as an individual cell death marker in all organoids, suggests that RIP3 is inherently present in the organoids and is not an artifact of the fixation process. Interestingly, necroptotic RIP3 + /Caspase3 − cells were not identified in downstream analysis, regardless of stimulation or Zombie status. Since RIP3 was present in RIP1-dependent apoptosis, as indicated by RIP3 + /Caspase3 + status, the reasons why the assay did not determine necroptosis remain unclear. This could be due to several factors, such as the specific cellular context, the sensitivity of the assay, or other regulatory mechanisms in the cells that may inhibit necroptosis. To further explore this, our ongoing investigations are utilizing a cytokine cocktail containing IL-6 and IL-1ꞵ to simulate intestinal inflammation in a more holistic manner. This study, while showcasing several strengths, also acknowledges certain limitations. Colonic organoid cultures, composed of different cell types like the colonic epithelium, provide insights into the composition and interaction of diverse cell populations in organoids. However, our analysis did not include cell type determination, which may mask specific dysregulations in signaling pathways. Additionally, for FACS analysis, organoids must be dissociated into single cells, a process that can inevitably cause cell damage and produce debris. The amount of “other” cell death observed may partly result from mechanical damage during this process. To mitigate these challenges, we employed lower centrifugal accelerations and carefully separated cell clusters using a dissociation reagent, which positively influenced cell death rates. Moreover, cell damage and death, accompanied by an increase in debris, were noted after differentiation periods exceeding 96 hours. To ensure sufficient differentiation of the organoids while minimizing these effects, we precisely matched the supplement addition and time points of differentiation. Considering the varied growth rates of organoid cultures derived from primary patient material, ensuring comparability of our results required measuring the same number of single cells in each tube, leading to a smaller number of analyzed cells. The challenges in reaching the necessary cell count during assay preparation led to the exclusion of samples with lower cell numbers, with minimal cell loss achieved by transferring organoids twice during preparation. Despite these challenges, the robustness of our results across a highly heterogeneous set of organoids highlights the substantial broader implications of this methodological innovation. The assay’s adaptability to spheroid cultures significantly enhances our understanding of diseases marked by cell death dysregulation. By providing detailed insights into the mechanisms of cell death in these physiologically relevant 3D models, our approach paves the way for more accurate disease modeling and the potential development of targeted therapies [ 7 ]. In conclusion, the RIP3-Caspase3-assay represents a significant advancement in organoid research. The application of this assay to spheroid cultures, a first in this field, has demonstrated its robustness and reliability in discerning between various cell death mechanisms, such as apoptosis and necroptosis. Abbreviations AdSC Adult stem cells FACS Fluorescence activated cell sorting FSC-A Forward scatter area IL-1ꞵ Interleukin 1 beta IL-6 Interleukin 6 NF-κB nuclear factor kappa-light-chain-enhancer of activated B-cells TNFα Tumor necrosis factor-alpha RIP1 Receptor-interacting serine-threonine protein kinase 1 RIP3 Receptor-interacting serine-threonine protein kinase 3 ROCK Rho-kinase SSC-A Side scatter area Declarations 6.1 Competing interests The authors declare no competing interests. Supplementary information Supplementary material provides one additional table and two additional figures. 6.2 Funding The authors would like to acknowledge the Department of Pediatric Surgery of the University Medical Center Hamburg-Eppendorf and especially Prof. Konrad Reinshagen for the financial support. Pauline Schuppert and Hans Christian Schmidt were financially supported by the Else Kröner-Fresenius-Stiftung iPRIME Scholarship (2021_EKPK.10), UKE, Hamburg. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Author Contribution Conceptualization C.I.P., J.H. and C.T.; methodology C.I.P., J.H., K.M.H., L.P.-R. and M.T.; formal Analysis C.I.P. and J.H.; writing – original draft preparation C.I.P., J.H. and C.T.; writing – review and editing K.R., H.-C.S., P.S., M.J.B. and Z.L.; visualization C.I.P. and J.H.. All authors have read and agreed to the published version of the manuscript. Acknowledgements The authors highly appreciate the technical assistance provided by the FACS Sorting Core Facility of the University Medical Center Hamburg-Eppendorf. Furthermore, the authors thank the laboratory of Dr. Markus Geißen for providing the microscopic equipment for organoid imaging. The responsibility for the content and any remaining errors, omissions, and inaccuracies is our own. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Apr, 2024 Reviews received at journal 27 Apr, 2024 Reviewers agreed at journal 28 Mar, 2024 Reviews received at journal 05 Mar, 2024 Reviewers agreed at journal 14 Feb, 2024 Reviews received at journal 07 Feb, 2024 Reviewers agreed at journal 25 Jan, 2024 Reviewers invited by journal 20 Jan, 2024 Editor assigned by journal 20 Jan, 2024 Editor invited by journal 20 Jan, 2024 Submission checks completed at journal 20 Jan, 2024 First submitted to journal 15 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3866340","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268557339,"identity":"678884e0-f4ef-44a3-b095-cee05c8bb2cd","order_by":0,"name":"Clara Isabell Philippi","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"Isabell","lastName":"Philippi","suffix":""},{"id":268557340,"identity":"765d970c-ef06-422f-9f80-ad86fea4d6c4","order_by":1,"name":"Johanna Hagens","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"","lastName":"Hagens","suffix":""},{"id":268557341,"identity":"30da6190-4d0c-4a83-92cf-a7bf400efc69","order_by":2,"name":"Kim Marili Heuer","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Kim","middleName":"Marili","lastName":"Heuer","suffix":""},{"id":268557342,"identity":"9cc0cecb-3822-4a12-bfe9-25576afc7ea1","order_by":3,"name":"Hans Christian Schmidt","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"Christian","lastName":"Schmidt","suffix":""},{"id":268557343,"identity":"97104ae0-6f95-4193-8ce5-38ddb6c97815","order_by":4,"name":"Pauline Schuppert","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Schuppert","suffix":""},{"id":268557344,"identity":"64dde1e0-1191-4311-85cd-23bdbf5391db","order_by":5,"name":"Laia Pagerols Raluy","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Laia","middleName":"Pagerols","lastName":"Raluy","suffix":""},{"id":268557345,"identity":"80456291-b755-4561-967f-de1422c56dee","order_by":6,"name":"Magdalena Trochimiuk","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Trochimiuk","suffix":""},{"id":268557346,"identity":"92652809-0471-4885-ba44-543b318ebc25","order_by":7,"name":"Zhongwen Li","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Zhongwen","middleName":"","lastName":"Li","suffix":""},{"id":268557347,"identity":"c3bff411-30df-4c9a-b23e-c6de416de0c0","order_by":8,"name":"Madeleine J. Bunders","email":"","orcid":"","institution":"Research Department of Virus Immunology, Leibniz Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Madeleine","middleName":"J.","lastName":"Bunders","suffix":""},{"id":268557348,"identity":"02476a86-8acd-453c-ac6f-ee5ac9ba1a38","order_by":9,"name":"Konrad Reinshagen","email":"","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Konrad","middleName":"","lastName":"Reinshagen","suffix":""},{"id":268557349,"identity":"65c3268f-55b4-4ded-ad60-add97ce946ec","order_by":10,"name":"Christian Tomuschat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDADAwYGxgcMB4CsAwTVMsO0MDMbkKyFTYIoLfwN/Acf3WyrkzdnP3+smueMDQPf8Qb8WiQOMDMb57YdNtzZk8x2m+dGGoPkGQLWgNwjndt2IMHgAEjLh8MMBjcSiNJSl2Bw/jFbMc+H/wwG9x8QpYU5weBGMhszz40DQFvw62CQOMxsbJxz7rDhhhuPjSXnnEnmkTxDwGH87Y0PH+eU1ckbnE98+OHNMTs5vuMHCFjDjMbnIaB+FIyCUTAKRgExAABjfEIrKPzIxQAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf","correspondingAuthor":true,"prefix":"","firstName":"Christian","middleName":"","lastName":"Tomuschat","suffix":""}],"badges":[],"createdAt":"2024-01-15 11:44:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3866340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3866340/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50022072,"identity":"1876e7b8-18ad-4d25-861a-279a207ecda6","added_by":"auto","created_at":"2024-01-23 08:44:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":559701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLight microscopy imaging of two exemplary organoid cultures (a, b) after stimulation with different TNFα concentrations. The enteroids present visible morphological changes and cell death with increasing amounts of the cytokine.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.1OrganoidswithdifferentTNFconcentrations.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3866340/v1/1f7004761462dbb718ec0d75.jpg"},{"id":50022073,"identity":"7cba7eb2-19c6-4d20-b231-bbbcf1e676de","added_by":"auto","created_at":"2024-01-23 08:44:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":698049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSummary of experimental findings using the RIP3-Caspase3-assay. \u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cem\u003e Measurement of assay markers Zombie NIR, Caspase3 and RIP3. \u003c/em\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cem\u003e Visible organoid impairment after treatment with TNFα for 48 hours. \u003c/em\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cem\u003e FACS dot plots for two exemplary samples (a, b) for living (upper row, blue) and dead (lower row, red) cells with and without TNFα treatment. The dot plots visualize the overall shrinkage of cell amount after the treatment with TNFα as well as a shift from the RIP1-dependent apoptotic cells (RIP3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Caspase3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) to the non-dependent apoptotic cells (RIP3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Caspase3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e). \u003c/em\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cem\u003e Living cells undergoing cell death at measurement timepoint were defined by a Zombie-negative status. RIP1-dependent apoptosis represents the predominant cell death type. \u003c/em\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003cem\u003e Dead cells were defined by a Zombie-positive status. Other cell death types represent the largest group of dead cells. However, analysis reveals a significant shift to RIP1-independent apoptosis after treatment (p\u0026lt;0.0001) with a loss in other forms of cell death (p=0.0255).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2SummaryofResults.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3866340/v1/8905173bd602155d2ea97897.jpg"},{"id":50022417,"identity":"a33ec1b4-74f1-449f-b6ff-58ab3c742e8c","added_by":"auto","created_at":"2024-01-23 08:52:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":706952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866340/v1/1f6674c4-e542-4061-bcb0-8410fb5d5ac2.pdf"},{"id":50022074,"identity":"81c1c97b-2ce9-48de-9207-3955c802a027","added_by":"auto","created_at":"2024-01-23 08:44:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":132374,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3866340/v1/a295d5ac518ef7a79a4625fa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring cell death mechanisms in spheroid cultures: A novel application of the RIP3-Caspase3-Assay","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe intricate processes of cell death, including apoptosis and necroptosis, are fundamental to our understanding of various pathological conditions. In particular, the balance between these processes is crucial in conditions of inflammation, neurodegenerative diseases, or cancer [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite advancements in cellular biology, the accurate delineation of these processes remains a significant challenge, especially in physiologically relevant models like organoids. Traditional methods often fall short in providing a detailed analysis of these complex pathways, especially in heterogeneous cell populations like those found in organoid cultures. This limitation hinders our ability to fully understand and effectively target dysregulated cell death mechanisms in disease settings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advances in organoid technology offer promising avenues for more accurate disease modeling, providing three-dimensional structures that closely mimic in vivo conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, applying existing cell death assays to these models is challenged by their cellular and structural complexity. There is a critical need for assays that can differentiate between various cell death pathways in a single, cohesive analysis. Addressing these challenges is crucial for the development of targeted therapies and enhancing our understanding of disease mechanisms at a cellular level [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, our study introduces the RIP3-Caspase3-assay, a novel approach employing directly conjugated monoclonal antibodies to analyze regulated cell death mechanisms in spheroid cultures. This assay is designed to overcome the limitations of existing methods, offering a detailed and nuanced analysis of cell death pathways in heterogeneous organoid samples. By providing insights into RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, the RIP3-Caspase3-assay aims to advance our understanding of cell death in disease processes and aid in the development of more effective therapeutic strategies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cultivation of Organoids\u003c/h2\u003e \u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board Hamburg ethics committee. Parents or guardians provided informed consent for collection and analyses of the tissues. Tissue samples were harvested from patients who underwent surgery for Morbus Hirschsprung pull-through procedure (n\u0026thinsp;=\u0026thinsp;3), intestinal atresia repair (n\u0026thinsp;=\u0026thinsp;1) or colostomy closure (n\u0026thinsp;=\u0026thinsp;1) (patient characteristics see table 2s, supplementary material). Samples were transported in Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s medium (IMDM, #12440053, Gibco ThermoFisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum (FBS, #0500\u0026thinsp;\u0026minus;\u0026thinsp;064, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (P/S, #PS/B, Capricorn Scientific, Ebsdorfergrund, Germany) for viability preservation and processed within 24 h. Samples were washed in sterile Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS; #37350, Gibco Thermo Fisher Scientific, USA). The colonic mucosa was mechanically separated from the rest of the tissue and sliced into pieces measuring 1\u0026ndash;2 mm. The pieces were incubated in IMDM containing 5 mM ethylenediaminetetraacetic acid (#15575-038, Invitrogen, Waltham, MA, USA) and 2 mM DL-Dithiothreitol (DTT, #D9779-5G, Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 4\u0026deg;C. Crypt isolation was verified using an inverse microscope (Olympus IX50-S8F; Olympus, Tokyo, Japan). The medium containing the mucosa was pipetted up and down several times with a 25 ml serologic pipette, followed by another incubation at 4\u0026deg;C for 20 min. Adult stem cells (AdSCs) were isolated using a 70 \u0026micro;m cell strainer (#352350, Corning, Corning, NY, USA) and rinsed with a washing buffer containing IMDM with 2% FBS and 1% P/S to collect all cells. The cells were centrifuged at 500 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for ten minutes and washed twice with Advanced Dulbecco\u0026rsquo;s Modified Eagle Medium (Advanced DMEM, #12491-015, Gibco ThermoFisher Scientific, USA) containing 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, #H3537-100ML, Sigma-Aldrich, St. Louis, MO, USA), 1% GlutaMAX (#35050-061, Gibco ThermoFisher Scientific, Waltham, MA, USA), and 1% P/S (further referred to as Adv. +++). 20 \u0026micro;l cell suspension was pipetted into 40 \u0026micro;l of growth factor-reduced, phenol red-free Matrigel Matrix (#356231, Corning, Corning, NY, USA) and mixed carefully. 30 \u0026micro;l of the mixture was pipetted into a prewarmed flat bottom 24-well-plate (#3526, Costar, Corning, NY, USA). Domes solidified upon incubation at 37\u0026deg;C and 5.0% CO\u003csub\u003e2\u003c/sub\u003e for 30 min. 500 \u0026micro;L of Seeding Medium consisting of IntestiCult Organoid Growth Medium Human (OGM-h, #060610, Stemcell Technologies, Canada) supplemented with 5 mM of ROCK-Pathway Inhibitor (ROC, #72302, Stemcell Technologies, Vancouver, Canada), 1% P/S, and 0.02% Primocin (#ant-pm-05, InvivoGen, San Diego, CA, USA) was added to the wells. Successful seeding was confirmed by light microscopy. Organoids were cultivated at 37\u0026deg;C and 5.0% CO\u003csub\u003e2\u003c/sub\u003e. The seeding medium was changed every 2\u0026ndash;3 days. After 7\u0026ndash;10 days, organoids were ready for the first passage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Maintenance of the Organoids\u003c/h2\u003e \u003cp\u003ePassaging of organoids was performed as follows. The medium was removed and the Matrigel domes were broken up by pipetting with 1 ml Adv.+++. The wells were washed with Adv.+++ and cell suspension was centrifuged at 300 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 5 min. Organoid cells were separated into smaller cell clusters by pipetting up and down several times, and again centrifuged at 400 \u0026times; \u003cem\u003eg\u003c/em\u003e. After removing the supernatant, disrupted organoids were resuspended with Adv.+++ and reseeded in Matrigel, as described in section 1.2.. Medium was changed every two to three days.\u003c/p\u003e \u003cp\u003eAfter the fifth passage, organoids were considered mature and used for the experiments. After 4\u0026ndash;6 days, OGM-h was replaced with IntestiCult Organoid Differentiation Medium Human (ODM-h, #100\u0026ndash;02114, StemCell Technologies, Vancouver, Canada) containing 5 mM Daptomycin (DAPT), a notch pathway inhibitor (# 72080, StemCell Technologies, Vancouver, Canada). Organoids were cultured for 3\u0026ndash;5 days in ODM-h, which was changed every 2 days as well. Once the epithelia of the organoids started to thicken and form bud-like structures, organoids were prepared for the experiment. Organoid growth and differentiation were monitored using light microscopy (Leica DM IL LED, Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of TNFα concentration using flow cytometry\u003c/h2\u003e \u003cp\u003eThe purpose of this experiment was to assess the effect of Tumor Necrosis Factor-α (TNFα) on differentiated organoids. Ten wells containing 30 \u0026micro;l Matrigel dome were needed from each culture of organoids to perform the experiment. Organoids were maintained for five days in OGM-h. On day 5, the medium was replaced with supplemented ODM-h to initiate differentiation. After 24 hours, TNFα (TNFα, #300-01A, PeproTech London, UK) was added to the ODM-h in the concentrations 0,1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, and an unstimulated control, always treating two wells the same. Organoids were maintained in ODM-h with TNFα for a total of 72 h, with one medium change in between (Fig.\u0026nbsp;3s, supplementary material). Organoid growth was monitored using light microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor flow cytometry analysis of the cells, the medium was removed and Matrigel containing the organoids was pipetted up and down with Adv.+++, transferred into a 15 ml tube (#188271, Greiner Bio-One, Kremsm\u0026uuml;nster, Austria), and incubated with TrypLE (#12605028, Gibco ThermoFisher Scientific, Waltham, MA, USA) at 37\u0026deg;C for 4 min to separate the organoids into smaller cell clusters. The organoid fragments were further separated into single cells by pipetting. Cell isolation was verified using a light microscope. The cells were centrifuged in Adv.+++ at 300 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for five minutes and the supernatant was discarded. The cells were incubated with Zombie NIR fluorescent dye (#423105, dilution 1:2000, BioLegend, San Diego, CA, USA) for 30 min at 4\u0026deg;C in the dark. Next, the cells were washed and fixed with an intracellular fixation buffer (eBioFix, #00-8222-49, BD Biosciences, San Jose, CA, USA) for 20 min at 4\u0026deg;C. Cells were resuspended in 200 \u0026micro;l DPBS and ready for analysis with LSR Fortessa and BD FACS Diva Software (BD Biosciences, San Jose, CA, USA). For each tube, 10,000 single cells were analyzed. Small debris at the origin was removed using a gate in the FSC-A vs. SSC-A dot plot. Single cells were gated on SSC-W versus SSC-H dot plots. These were gated on a dot plot of Zombie NIR vs. SSC-A showing all zombie-positive dyed cells. Zombie staining indicated a compromised cell membrane, these cells were defined as dead.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RIP3-Caspase3-Analysis of apoptosis, necroptosis and RIP1-dependent apoptosis\u003c/h2\u003e \u003cp\u003eTo quantify RIP1-independent apoptosis, RIP1-dependent apoptosis, necroptosis, and other forms of cell death in the epithelial cells, the intracellular cell-death proteins RIP3 and Caspase3 were stained in addition to Zombie NIR and measured by flow cytometry, as described by Lee et al. (2018). Sensitivity to the stressor was measured by the extent of cell death for each sample and condition. Untreated samples served as negative controls.\u003c/p\u003e \u003cp\u003eFor this experiment, four wells containing a 30\u0026micro;l Matrigel dome were used from each culture. The organoids were maintained in OGM-h for five days. On day 5, the medium was replaced with ODM-h. 48h before flow cytometry, two of the four wells were treated with TNFα at a concentration of 100 ng/ml. The other two wells were maintained in ODM-h without TNFα (Fig.\u0026nbsp;4s, supplementary material).\u003c/p\u003e \u003cp\u003eOrganoids were prepared for flow cytometry as described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e.. After the fixation with eBioFix, cells were permeabilized with PBS containing 0.25% Triton X-100 (#T8787-50ML, Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4\u0026deg;C under movement to expose intracellular targets. The cells were incubated with fluorescent-conjugated antibodies anti-active Caspase-3-BV650 (#564096, dilution 1:50; BD Biosciences, San Jose, CA, USA) and anti-RIP3-Alexa Fluor 488 (clone B-2, #sc-374639, dilution 1:125, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at 4\u0026deg;C in the dark. Cells were washed with 500 \u0026micro;l PBS containing 0.25% Triton X-100 and then resuspended in 200 \u0026micro;l PBS. In each tube, 5.000 single cells were analyzed using LSR Fortessa and BD FACS Diva Software. Small debris was removed using a gate in the FSC-A vs. SSC-A dot plot. Single cells were gated on SSC-W versus SSC-H dot plots. These were gated on a dot plot of Zombie NIR vs. SSC-A showing all zombie-positive dyed cells that were defined as dead. Cells unstained by Zombie NIR were gated in the FSC-A vs. SSC-A dot plot and were defined to be living cells. Using Caspase-3-BV650 vs. RIP3-Alexa Fluor 488 dot plots, live and dead cells were gated separately with the same gate configuration for each plot. Caspase3-positive cells indicate a RIP1-independent apoptotic process, and RIP3-positive cells indicated a necroptotic process. Caspase3- and RIP3-positive cells were determined to undergo RIP1-dependent apoptosis. Cells with neither Caspase3 nor RIP3 signals were assumed to have undergone other forms of cell death (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell death status definitions. Caspase3-positive signal indicating an apoptotic cell, RIP3-positive signal indicating a necroptotic cell, Caspase3- and RIP3-positive signal indicating a RIP1-dependent Apoptosis and no Caspase3- or RIP3-signal indicating other forms of cell death.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRIP3-Status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase3-Status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRIP1-dependent Apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecroptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther cell death\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were generated using GraphPad Prism 9 (San Diego, CA, USA). Simple comparisons were performed by descriptive statistics and student\u0026rsquo;s t-test. Mean values of multiple variables were compared using two-way analysis of variance (ANOVA). P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Determination of TNFα concentration\u003c/h2\u003e \u003cp\u003eThis experiment was performed to determine the effect of increasing TNFα concentrations on overall cell survival, using Zombie NIR staining only. Stimulation with TNFα showed stable percentages of Zombie\u003csup\u003e+\u003c/sup\u003e cells between 0 ng/ml and 10 ng/ml, ranging from 39.06% (\u0026plusmn;\u0026thinsp;8.51%) to 42.68% (\u0026plusmn;\u0026thinsp;7.35%). Cell death rate increased to 45.30% (\u0026plusmn;\u0026thinsp;7.05%) after treatment with 100 ng/ml.\u003c/p\u003e \u003cp\u003eThe FSC-A vs. SSC-A dot plot detected an increase in cell debris after 72 h treatment with TNFα as a display of organoid damage. Consequently, only 5,000 single cells were analyzed in each tube, ensuring comparability between the different treatments and samples. The destruction was already visible at 48 h of treatment during daily monitoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, for the final next experimental analysis setup, 100 ng/ml TNFα for 48 h was considered the optimal condition to induce stress in the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cell death markers RIP3, Caspase3 and Zombie can be detected in organoids using the RIP3-Caspase3-Assay\u003c/h2\u003e \u003cp\u003eAs exemplary seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, stimulation with TNFα caused morphological changes indicating destruction within the cultures. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the assay was able to detect Zombie staining as well as RIP3- and Caspase3-status. Treatment with TNFα caused a slight increase in Zombie-cells (29.71% vs. 32.89%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3451) and Caspase3 expression (63.00% vs. 69.91%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2038) while RIP3 was detected less after stimulation (16.61% vs. 12.13%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3482).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RIP3-Caspase3-assay reveals apoptosis as the leading cell death mechanism in living cells\u003c/h2\u003e \u003cp\u003eFor further analysis, cell death measurements were separated into living (Zombie\u003csup\u003e\u0026minus;\u003c/sup\u003e) and dead cells (Zombie\u003csup\u003e+\u003c/sup\u003e). Living cells represent cells with intact cell membranes not stained by Zombie NIR and were gated as described in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e.. As seen in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D, apoptosis was the leading cause of cell death. Comparing cell death mechanisms, predominance of RIP1-dependent apoptosis reached significance against all other groups pre and post treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Also, RIP1-independent apoptosis was significantly higher compared to necroptosis after stimulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002), but not before (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0644). Comparison between RIP1-independent apoptosis and other forms of cell death also did not reach significance neither pre (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8027) nor post treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0559) with TNFα.\u003c/p\u003e \u003cp\u003eStimulation caused an increase in RIP3\u003csup\u003e\u0026minus;\u003c/sup\u003e/Caspase3\u003csup\u003e+\u003c/sup\u003e cells from 16.06\u0026ndash;28.91% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1572), while RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e+\u003c/sup\u003e cells showed less change (64.08% vs. 59.47%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.9101). There were near to no detectable RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells (0.33% vs. 0.13%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.9999). RIP3\u003csup\u003e\u0026minus;\u003c/sup\u003e/Caspase3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells decreased from 21.54\u0026ndash;12.79% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4987) after the treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Dead cells show a significant rate of RIP1-independent apoptosis after TNFα stimulation\u003c/h2\u003e \u003cp\u003eZombie-positive cells, representing cells with compromised membranes, were also gated as described in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e and are displayed in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. Comparison between the different cell death mechanisms groups before and after treatment revealed significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) between all except three combinations: Before stimulation, RIP1-independent and RIP1-dependent apoptosis did not differ (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1307), but difference reached significance after TNFα treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003), showing a growth of the first and decrease of the latter one. Moreover, RIP1-independent apoptosis nearly reached the level of other cell deaths after stimulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8368).\u003c/p\u003e \u003cp\u003eWhile apoptosis overall remained a prominent cause of cell death, RIP3\u003csup\u003e\u0026minus;\u003c/sup\u003e/Caspase3\u003csup\u003e+\u003c/sup\u003e cells increased significantly from 19.72\u0026ndash;38.05% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) while RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e+\u003c/sup\u003e cells decreased slightly from 28.31\u0026ndash;21.31% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2212). RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells represented the smallest group with 0.47% before versus 0.13% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.9999) after treatment. RIP3\u003csup\u003e\u0026minus;\u003c/sup\u003e/Caspase3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells presented the leading cause of cell degradation in this cohort, but also significantly decreased after treatment with TNFα (52.66% vs. 42.17%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0255).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study's successful application of the RIP3-Caspase3-assay in heterogeneous organoids highlights its methodological strengths. By employing directly conjugated monoclonal antibodies, our assay provides a nuanced analysis of cell death pathways, revealing the complexity of these processes in greater detail. This approach aligns with Lee et al.\u0026rsquo;s findings, which distinguished between apoptosis, necroptosis, and RIP1-dependent apoptosis in both viable and non-viable cell populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our extension of these advancements to spheroid cultures marks a first in the field, showcasing the assay's reliability and its capability to discern between cell death mechanisms. In our study, the treatment of isolated intestinal epithelial cells from spheroids with TNFα accelerated the cell death rate, as indicated by Zombie-stained cells. The study observed an initial lower response to TNFα that increased with higher concentrations. In this context, this pattern suggests a possible adaptive or threshold response of the cells to TNFα-induced stress. TNFα is known to activate multiple signaling pathways, including NF-κB, MAPKs, and the apoptotic pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The activation of NF-κB, for instance, can lead to the expression of survival genes, which might help cells adapt and survive under increasing TNFα concentrations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, in this study we had a linear response to the TNFα concentration with relevant damage of the organoids at 100ng/ml after 48h. This finding was in line with observed morphological changes of treated organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUpon TNFα stimulation, we observed an increase in Caspase3 expression and a decrease in RIP3 expression across all samples. FACS analysis further revealed that TNFα-treated cells showed a significant increase in apoptosis, while simultaneously shifting away from RIP1-dependent apoptotic mechanisms, without involving necroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). This finding suggests a preference for RIP1-independent cell death in both living cells undergoing cell death and those that had already died. The shift towards apoptosis in the presence of TNFα is a key finding. Apoptosis is a programmed and orderly cell death process, often considered immunologically silent, while necroptosis is a form of programmed necrosis that is inflammatory in nature. The decision between apoptosis and necroptosis is often determined by the availability and activity of key regulatory proteins like RIP1, RIP3, and Caspase8 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This shift in the type of cell death can also be attributed to the inflammatory context, where the co-sensing of multiple cytokines and the activation of various pathways, such as the non-canonical NF-κB pathway, lead to different forms of cell death. For instance, the combination of TNFα with other cytokines can result in TNFR1-induced RIP1 kinase activity-dependent apoptosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Also, the balance between apoptosis and necroptosis is depended on the concentration of TNFα [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, TNFα treatment did not impact the proportion of \"other\" cell death types substantially. However, cells sorted into this category showed a higher proportion in the dead cell section than in the living cells, which may be attributable to the disruption of cell membrane integrity and subsequent cell death during assay preparation.\u003c/p\u003e \u003cp\u003eAnother important finding is the differential response in living versus dead cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). The significant changes in the dead cell population, as opposed to the living cells, suggest that TNFα effects are more pronounced in cells already compromised or stressed. This could imply that TNFα is a more potent inducer of cell death pathways in cells that are already damaged. This is because stressed or damaged cells might have compromised defense mechanisms (for example reduced expression of anti-apoptotic proteins), making them more prone to TNFα cytotoxic effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Also, the absence of necroptotic cells in both living and dead cells suggests that, under these conditions, TNFα does not induce necroptosis and underscores TNFα role in promoting apoptosis under these experimental conditions in colonic epithelial organoids.\u003c/p\u003e \u003cp\u003eThe assay's ability to detect both major and minor cell populations undergoing various forms of cell death is particularly valuable in the context of spheroid cultures, where cell heterogeneity and complexity are inherent [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Notably, unlike the original technique described by Lee et al., our approach did not use CalTag fixative, known to upregulate RIP3. This modification is noteworthy because the detection of RIP3 in our results is not influenced by the fixative. Since we were still able to detect RIP3 as an individual cell death marker in all organoids, suggests that RIP3 is inherently present in the organoids and is not an artifact of the fixation process. Interestingly, necroptotic RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e\u0026minus;\u003c/sup\u003e cells were not identified in downstream analysis, regardless of stimulation or Zombie status. Since RIP3 was present in RIP1-dependent apoptosis, as indicated by RIP3\u003csup\u003e+\u003c/sup\u003e/Caspase3\u003csup\u003e+\u003c/sup\u003e status, the reasons why the assay did not determine necroptosis remain unclear. This could be due to several factors, such as the specific cellular context, the sensitivity of the assay, or other regulatory mechanisms in the cells that may inhibit necroptosis. To further explore this, our ongoing investigations are utilizing a cytokine cocktail containing IL-6 and IL-1ꞵ to simulate intestinal inflammation in a more holistic manner.\u003c/p\u003e \u003cp\u003eThis study, while showcasing several strengths, also acknowledges certain limitations. Colonic organoid cultures, composed of different cell types like the colonic epithelium, provide insights into the composition and interaction of diverse cell populations in organoids. However, our analysis did not include cell type determination, which may mask specific dysregulations in signaling pathways. Additionally, for FACS analysis, organoids must be dissociated into single cells, a process that can inevitably cause cell damage and produce debris. The amount of \u0026ldquo;other\u0026rdquo; cell death observed may partly result from mechanical damage during this process. To mitigate these challenges, we employed lower centrifugal accelerations and carefully separated cell clusters using a dissociation reagent, which positively influenced cell death rates. Moreover, cell damage and death, accompanied by an increase in debris, were noted after differentiation periods exceeding 96 hours. To ensure sufficient differentiation of the organoids while minimizing these effects, we precisely matched the supplement addition and time points of differentiation. Considering the varied growth rates of organoid cultures derived from primary patient material, ensuring comparability of our results required measuring the same number of single cells in each tube, leading to a smaller number of analyzed cells. The challenges in reaching the necessary cell count during assay preparation led to the exclusion of samples with lower cell numbers, with minimal cell loss achieved by transferring organoids twice during preparation. Despite these challenges, the robustness of our results across a highly heterogeneous set of organoids highlights the substantial broader implications of this methodological innovation. The assay\u0026rsquo;s adaptability to spheroid cultures significantly enhances our understanding of diseases marked by cell death dysregulation. By providing detailed insights into the mechanisms of cell death in these physiologically relevant 3D models, our approach paves the way for more accurate disease modeling and the potential development of targeted therapies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, the RIP3-Caspase3-assay represents a significant advancement in organoid research. The application of this assay to spheroid cultures, a first in this field, has demonstrated its robustness and reliability in discerning between various cell death mechanisms, such as apoptosis and necroptosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAdSC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Adult stem cells\u003c/p\u003e\n\u003cp\u003eFACS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fluorescence activated cell sorting\u003c/p\u003e\n\u003cp\u003eFSC-A\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Forward scatter area\u003c/p\u003e\n\u003cp\u003eIL-1ꞵ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Interleukin 1 beta\u003c/p\u003e\n\u003cp\u003eIL-6\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Interleukin 6\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;nuclear factor kappa-light-chain-enhancer of activated B-cells\u003c/p\u003e\n\u003cp\u003eTNF\u0026alpha;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tumor necrosis factor-alpha\u003c/p\u003e\n\u003cp\u003eRIP1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Receptor-interacting serine-threonine protein kinase 1\u003c/p\u003e\n\u003cp\u003eRIP3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Receptor-interacting serine-threonine protein kinase 3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROCK\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Rho-kinase\u003c/p\u003e\n\u003cp\u003eSSC-A\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Side scatter area\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003e6.1 Competing interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary information\u003c/h2\u003e \u003cp\u003eSupplementary material provides one additional table and two additional figures.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003e6.2 Funding\u003c/h2\u003e \u003cp\u003eThe authors would like to acknowledge the Department of Pediatric Surgery of the University Medical Center Hamburg-Eppendorf and especially Prof. Konrad Reinshagen for the financial support. Pauline Schuppert and Hans Christian Schmidt were financially supported by the Else Kr\u0026ouml;ner-Fresenius-Stiftung iPRIME Scholarship (2021_EKPK.10), UKE, Hamburg. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization C.I.P., J.H. and C.T.; methodology C.I.P., J.H., K.M.H., L.P.-R. and M.T.; formal Analysis C.I.P. and J.H.; writing \u0026ndash; original draft preparation C.I.P., J.H. and C.T.; writing \u0026ndash; review and editing K.R., H.-C.S., P.S., M.J.B. and Z.L.; visualization C.I.P. and J.H.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors highly appreciate the technical assistance provided by the FACS Sorting Core Facility of the University Medical Center Hamburg-Eppendorf. Furthermore, the authors thank the laboratory of Dr. Markus Gei\u0026szlig;en for providing the microscopic equipment for organoid imaging. The responsibility for the content and any remaining errors, omissions, and inaccuracies is our own.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBock, F. J. \u0026amp; Riley, J. S. When cell death goes wrong: inflammatory outcomes of failed apoptosis and mitotic cell death. Cell Death Differ 30, 293\u0026ndash;303 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErekat, N. S. Apoptosis and its therapeutic implications in neurodegenerative diseases. Clin Anat 35, 65\u0026ndash;78 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParseh, B. \u003cem\u003eet al.\u003c/em\u003e 3-Dimensional Model to Study Apoptosis Induction of Activated Natural Killer Cells Conditioned Medium Using Patient-Derived Colorectal Cancer Organoids. Front Cell Dev Biol 10, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLui, K. N.-C. \u0026amp; Ngan, E. S.-W. Human Pluripotent Stem Cell-Based Models for Hirschsprung Disease: From 2-D Cell to 3-D Organoid Model. \u003cem\u003eCells\u003c/em\u003e 11, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, C., Kang, R. \u0026amp; Tang, D. Organoids Models of Pancreatic Duct Adenocarcinoma. Methods Mol Biol 2712, 45\u0026ndash;60 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMead, B. E. \u003cem\u003eet al.\u003c/em\u003e Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation. Nat Biomed Eng 6, 476\u0026ndash;494 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRauth, S., Karmakar, S., Batra, S. K. \u0026amp; Ponnusamy, M. P. Recent advances in organoid development and applications in disease modeling. Biochim Biophys Acta Rev Cancer 1875, 188527 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwarzer, R., Laurien, L. \u0026amp; Pasparakis, M. New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8. Curr Opin Cell Biol 63, 186\u0026ndash;193 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, H. L., Pike, R., Chong, M. H. A., Vossenkamper, A. \u0026amp; Warnes, G. Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis. Methods 134\u0026ndash;135, 56\u0026ndash;66 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLork, M., Verhelst, K. \u0026amp; Beyaert, R. CYLD, A20 and OTULIN deubiquitinases in NF-κB signaling and cell death: so similar, yet so different. Cell Death Differ 24, 1172\u0026ndash;1183 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Loo, G. \u0026amp; Bertrand, M. J. M. Death by TNF: a road to inflammation. Nat Rev Immunol 23, 289\u0026ndash;303 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou, K., Gu, H., Yuan, Z. \u0026amp; Xu, X. Tumor Necrosis Factor Alpha Signaling and Organogenesis. Front Cell Dev Biol 9, 727075 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Organoids, Cell death mechanisms, RIP3-Caspase3 Assay, TNFα -induced stress, Apoptosis and necroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3866340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3866340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the application of the RIP3-Caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-Caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.\u003c/p\u003e","manuscriptTitle":"Exploring cell death mechanisms in spheroid cultures: A novel application of the RIP3-Caspase3-Assay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-23 08:44:36","doi":"10.21203/rs.3.rs-3866340/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-29T05:10:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-27T11:28:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64c102b8-0679-4df1-98c2-8ac075bf564a","date":"2024-03-28T04:01:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-05T21:12:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d6c19ea8-fa64-4509-a7a2-f16d42bca3a9","date":"2024-02-14T20:18:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-07T08:02:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d5173d43-c374-4bfc-ad9a-db1d8d7814fd","date":"2024-01-26T02:46:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-20T13:30:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-20T13:10:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-20T09:42:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-20T09:40:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-15T11:32:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c8b9c35-d053-44d1-bdc8-a72951d93a88","owner":[],"postedDate":"January 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28287606,"name":"Health sciences/Medical research/Paediatric research"},{"id":28287607,"name":"Health sciences/Medical research/Stem cell research"},{"id":28287608,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":28287609,"name":"Biological sciences/Stem cells/Intestinal stem cells"}],"tags":[],"updatedAt":"2024-07-04T04:40:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-23 08:44:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3866340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3866340","identity":"rs-3866340","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}