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 10 min 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% CO2 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% CO2. The seeding medium was changed every 2–3 days. After 7–10 days, organoids were ready for the first passage.
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. 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).
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 5 days in OGM-h. On day 5, the medium was replaced with supplemented ODM-h to initiate differentiation. After 24 h, 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).
Light microscopy imaging (magnification ×4) of two exemplary organoid cultures (a,b) after stimulation with increasing TNFα concentrations (0, 0.1, 1, 10 and 100 ng/ml). Images were taken immediately before processing for flow cytometry after 72 h of proinflammatory stimulation. Without cytokine influence, enteroids present complex budding structures without accelerative debris. With increasing concentrations of TNFα, the enteroids present visible morphological changes with rounded buds and cell detritus inside the lumen as a sign for cell destruction, especially following concentrations 10 ng/ml and 100 ng/ml.
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 5 min 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 (version 8.0.2, firmware version 1.13, BD Biosciences, San Jose, CA, USA). Zombie NIR was excited by a 633 nm laser and collected in a 780/60 nm detector. Compensation was not necessary using only one laser for this analysis. Gating was set using unstained samples. 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.
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)16. 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 30 μl Matrigel dome were used from each culture. The organoids were maintained in OGM-h for 5 days. On day 5, the medium was replaced with ODM-h. 48 h 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 Sect “Determination of TNFα concentration using flow cytometry”. 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, 5000 single cells were analyzed using LSR Fortessa and BD FACS Diva Software. Zombie NIR was excited by a 633 nm laser and collected in a 780/60 nm detector. Caspase-3-BV650 was excited by a 405 nm laser and collected at 675/30 nm. RIP3-Alexa Flour 488 was excited by a 488 nm laser and collected at 585/40 nm. The lasers were compensated with unstained samples using the BD FACS Diva Software. Gates were set by using fluorescence minus one controls as a standard procedure. 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).
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.
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- Source: https://www.nature.com/articles/s41598-024-66805-4