Culture medium
For culture and passaging medium, Dulbecco’s Modified Eagle Medium F-12 Nutrient Mixture (DMEM/F12) + GlutaMAX (Gibco) with 10% heat inactivated Fetal Bovine Serum (Gibco), 1% Penicillin Streptomycin (Gibco), 1% HEPES 1M (Invitrogen), 5% Rspondin1 and 5% WNT was used. Complete medium was kindly provided by the Hogeschool Utrecht, Utrecht, The Netherlands.
Crypt Isolation
A female mouse C57/BL6, 6 months of age was sacrificed using carbon dioxide. The intestine was removed and the ileum isolated. Villi were removed from the ileum by scraping using a scalpel and the tissue was cut into small pieces, transferred to a tube with ice-cold PBS and washed three times with PBS. A Polter-Elvehjem tube was then used to fragment the tissue and the cell suspension was filtered through a 100 µm filter. The now isolated crypts were spun down at 335x g for 5 minutes at room temperature and supernatant was removed. The crypts were resuspended in Matrigel (Basement membrane Matrix Growth Factor Reduced Low concentration, Phenol-red-free #356231, Corning), and drops of 20 µl suspension were added to each well (Corning Costar). One ml of culture medium with Y27632 (Selleckchem), CHIR (Sigma) and 2-Propylpentanoic acid (Sigma) was added to the wells. After 3 days of incubation at 37˚C and 5% CO2, medium was replaced by normal culture medium and cultures were incubated for 4 days in the same conditions.
Intestinal Organoid Culture
Matrigel droplets containing isolated crypts were cultured in 24 wells culture plate (Corning Costar) and organoid medium was added to the culture. Organoids were grown for 7 days at 37˚C and 5% CO2 and their morphology and size were checked every other day. For passaging, organoids were first counted and then split using a ratio between 1:3 wells or 1:5 wells, depending on the density of the culture. Organoids were split by placing the tip of the pipette on the bottom of the well and resuspended in the organoid-containing medium. The cells suspension was centrifuged at 335xg for 5 minutes at room temperature and supernatant removed. New fresh Matrigel was added to the cell pellet and 20 µL drops of the resultant suspension were added each well.
Development Of The Mucositis Model
MTX dose finding
To select an optimal MTX dose and incubation time, 7 days post-seeding organoids were used. Prior to MTX insult, organoids were counted and only droplets containing a similar number of organoids per well were included in the experiments. Medium was then removed and replaced by fresh medium (900 µL). MTX was diluted in new medium until the appropriate concentration. 100 µL of the resultant dilutions were added to the organoids with an end concentration of 0, 10, 100 and 1000 ng/ml. Organoids were incubated for different time periods (24, 48, 72 and 96 hours) at 37˚C and 5% CO2. After incubation, 200 µL of medium were removed and stored at -20˚C for citrulline and cytokine/chemokine measurements and a metabolic activity assay was assessed with staining WST-1.
Recovery assay
To assess the ability of MTX-treated organoids to recover from MTX insult, 7-days seeded organoids were first treated with 0, 100 and 1000 ng/ml MTX for 96 hours. MTX-containing medium was then removed and the same amount of fresh MTX-free medium was added. Organoid cultures were incubated for 24, 48, 72 and 96 hours at 37˚C and 5% CO2. At the correspondent time points, 200 µL of medium was collected and stored for cytokine/chemokine analysis and metabolic activity was assessed with WST-1.
Folinic acid challenge
To optimize the dose of Folinic Acid (FA) to be used in the experiment, different concentrations ranging from 0.005 and 50 µg/ml were added in combination with MTX (100 ng/ml). Following optimization, organoids were treated with MTX (100 ng/ml) for 96 hours and FA (5 µg/ml) was added simultaneously (0 hours) or at different time points (24, 48, 72 and 96 hours). As previously described, 200 µL were removed from the cultures and stored and WST-1 was performed in order to assess metabolic activity.
Citrulline Levels
For citrulline measurements, stored culture supernatants were used in this assay. Proteins and polypeptides were precipitated with perchloric acid and the culture medium was centrifuged. Amino acids, including citrulline, were determined in the supernatant by fully automated High-Performance Liquid Chromatography (HPLC) [10].
Metabolic Activity
WST reagent (Cell Proliferation Reagent, Roche Life Science) was added to the medium (1:10) and incubated at 37 °C. At time points 0, 3 and 5 hours 100 µl were collected and absorbance was measured at 450 nm and background at 650 nm using Flexstation3 Multi-mode Microplate Reader (Molecular Devices). Absorbance of the samples was corrected for the background.
Cytokine/chemokine Release
Stored culture supernatants were used in this assay. A wide range of cytokines/chemokines were tested in the medium of the organoids including Eotaxin, GM-CSF, Gro-olpha/KC/CINC1, TNF-α, IL-10, IL12p70, IL-13, IL-17A, IL-18, IL-1beta, IL-2, IL-22, IL-23, IL-27, IL-4, IL-5, IL-6, IL-9, IP-10, MCP-1, MCP-3, MIP1-α, MIP1-β, MIP-2, RANTES, CXCL10 (IP-10) and CCL5. The Multiplex Immunoassay was performed according to the instructions of the manufacturer (Cytokine/Chemokine Panel 1 (26plex), #EPX260-26088-901, eBioscience). Analysis of the samples was performed using Flexmap3D (Luminex). Recovery of the standards between 70–130% was accepted, values beyond these percentages were considered as invalid.
Short-chain Fatty Acids Challenging
Butyrate, propionate and acetate were used in this study were obtained commercially (Sigma). To assess the ability of different SCFAs to improve relative metabolic activity of MTX-treated organoids, 7-days seeded organoids were treated simultaneously with MTX (100 ng/ml) and butyrate (0.25-2 mM), acetate (0.25-8 mM) or propionate (0.25-4 mM) for 96 hours. Medium (200 µL) was removed from the wells and WST-1 assay was performed on the remaining media in order to assess metabolic activity.
Rna Extraction And Quantitative Real-time Pcr (qpcr)
Total RNA was isolated from cells using a combination of TRI-Reagent (Sigma) and RNeasy Mini Kit columns (Qiagen). In short, organoids were solubilized in TRI-Reagent mixed with chloroform and centrifuged to allow for phase separation. The aqueous phase was collected and mixed with one volume of 70% ethanol after which the mixture was transferred to a RNeasy column. RNA was isolated according to the manufacturer’s instructions. Contaminating DNA was removed using the RNase-free DNase Set (Qiagen) according to manufacturer’s protocol. To synthesize first-strand cDNA, 1000 pg of total RNA was reverse transcribed at 25 °C for 5 min 46 °C for 30 min and 95 °C for 1 min using iScript cDNA Synthesis Kit (Bio-Rad). The PCR reaction contained cDNA equivalent to 30 pg of RNA input, 200 nM primer sets, and SYBR Select Master Mix (Applied Biosystems), and was run in a QuantStudio 6 Flex real-time PCR system (Life Technologies). PCR products were quantified and normalized relative to the amount of GAPDH cDNA products as described by Schmittgen et al. [11]. Relative quantification of gene expression was calculated using the ΔΔCt (Ct, threshold cycle of real-time PCR) method according to the formula: ΔCT = CtHPRT − Cttarget, ΔΔCt = ΔCtcontrol – ΔCtTreatment, Ratio = 2−ΔΔCt. Primers used were RT² qPCR Primer Assay for Mouse HPRT, Slc19a1, Abcb10, Abcc1, Abcc2, Abcc3, Abcg5, Abcg8 and Alc46a1, all from Qiagen.
Statistical analysis
Data were compared using Prism version 8.0.1 (GraphPad® Software, San Diego, USA). Significant differences between different groups was determined by repeated measured Two-Way ANOVA with Turkey’s multiple comparison test or one-way ANOVA with Dunnett’s multiple comparisons test. A p-value < 0.05 was considered statistically significant.