2.5.2. LC-MS Analysis

Data was generated using an Alliance e2695 HPLC system coupled to a 2998 Photodiode array detector and an Acquity QDa detector mass spectrometer equipped with an electrospray interphase (ESI, Waters, Milford, MA, USA), an autosampler, and a Vacuubrand pump (Essex, CT, USA). For each sample, technical duplicates (10 μL) were injected. The instrument and processing methods have been previously described [26]. A Cortecs C18+ column held at 40 ◦C (4.6 × 150 mm and 2.7 μm particle size, Waters, Milford, MA, USA) was used to separate analytes and held at the temperature of 40 ◦C. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). The flow

rate was 1 mL/min. A linear gradient was used, specifically: 35–50% A over 30 min, a hold at 50% A for 1 min, an immediate transition to 65% A for 9 min, a gradual increase to 90% A over 2 min, and a hold at 90% A for 6 min. This was immediately followed by a washout with 90% A to 10% A for 6 min before returning to the initial 35% A at 54.1 min, which marked the end of each sample run. The column was allowed to equilibrate for 6 min in 35% A before the next injection. Pure compounds were used to produce standard curves for the quantification of BAs and CBD, as detailed in Table S1.

#### *2.6. Ileal Organoid Experiments*

Eight-month-old WT C57BL/6J female mice were euthanized by CO2 asphyxiation and ileal tissue was collected for cryp<sup>t</sup> isolation according to established methods [42]. Crypts were collected in 1× PBS (pH 7.4, Growcells, Irvine, CA, USA), counted manually, and the concentration of 300 crypts per μL was calculated. Culture was centrifuged at 200× *g* for 3 min, PBS was aspirated, Cultrex was added to obtain a density of 150 crypts per 25 μL volume, and 48-well plates were seeded with 25 μL Cultrex per well. The plate was incubated in a 37 ◦C, 5% CO2 incubator (Galaxy 170, Eppendorf Co., New Brunswick, NJ, USA) for 30 min to allow the polymerization of Cultrex then 250 μL of 1× complete growth medium (CGM) [43] was added per well. CGM was replaced every 2 days. Organoids were passaged every 7 days (1:3 ratio). Mature day 4 organoids were treated with 0, 100, 250, or 500 μM CBD in the presence or absence of Tnfα (100 ng/mL; STEMCELL, Vancouver, BC, Canada) + lipopolysaccharide (LPS; 100 μg/mL; Sigma-Alrich, Darmstadt, Germany) to induce inflammation. Six wells were pooled to create one biological sample (*n* = 1) and treatments were performed in triplicate. Organoids from passages 10–11 were used, and the experiment was performed twice.

CBD (1 mg/mL) was dissolved in 100% methanol and then calculated volumes of this stock were used to obtain 100, 250, or 500 μM CBD concentrations (in 250 μL/well) as well as these same CBD concentrations in combination with lipopolysaccharide (LPS 2 mg/mL 0.9% NaCl stock; 10 μg/mL in CGM; Cat#L6143 Sigma-Alrich) and TNFα (100 μg/mL sterile ddH2O stock; 100 ng/mL in CGM; Cat#78069, STEMCELL). TNFα and LPS alone served as a positive control for inflammation. Samples were dried in speed vacuum (CentriVap concentration system with cold trap, Labconco, Kansas City, MO, USA) and resuspended in CGM media the day of treatment. Organoids were treated for 24 h, CGM was removed, and 500 μL Cultrex organoid harvesting solution (Cat# 3700-100-01, R&D Systems, Minneapolis, MN, USA) was added per well. Organoids (6 wells/treatment) were collected into 15 mL conical tubes precoated with 1× PBS and left to incubate on ice for 1 h to dissolve Cultrex. Samples were centrifuged at 500× *g* for 5 min at 4 ◦C, washed with 2 mL of 1× PBS, supernatant was removed, 800 μL of Qiazol was added, and samples were transferred to 1.7 mL microfuge tubes with two 2.8 mm stainless steel beads and frozen at −80 ◦C until RNA extraction. Samples were thawed on ice followed by vortexing for 30 s, then RNA was extracted using RNeasy plus universal mini kit (Catalog#73404, QIAGEN, Germantown, MD, USA).

#### *2.7. MTT Analysis for Cell Viability*

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagen<sup>t</sup> (M6494, Thermofisher, Waltham, MA, USA) was diluted 5 mg/mL in sterile 1× PBS per manufacturer's instructions. Concurrently, treatments were performed on an additional 48-well plate of organoids for MTT assay to assess viability. Three wells were used per treatment, including 100% DMSO as a positive control for toxicity. At 24 h post-treatment, 27.5 μL of MTT solution was added to the 250 μL of 1× CGM in each well, then placed in an incubator (37 ◦C, 5% CO2) for 2 h. After media and MTT solutions were removed, viable organoids appeared purple/black. Then 50 μL of 2% sodium dodecyl sulfate (SDS) was added to each well and the plate was returned to the incubator (37 ◦C, 5% CO2) for 1 h. After incubation, 150 μL of pure dimethyl sulfoxide (DMSO) was mixed into each well and incubated for 4 h or overnight to solubilize the formazan crystals. Once solubilization was complete,

200 μL from each well was transferred to a microplate and absorbance was measured in a multimode plate reader (CLARIOStar, BMG Labtech, Cary, NC, USA) at 562 nm.

#### 2.7.1. qPCR of Ileal Organoids and Liver Tissue

RNA was extracted from liver tissue (10–20 mg of right median lobe) as previously described [43]. RNA extracted from organoids or liver samples was quantified by nanodrop and 5 mg was used to prepare cDNA followed by RT-qPCR (QuantStudio 3, Thermo) as previously described [26].

TaqMan™ assay primers (Life Technologies, Carlsbad, CA, USA) used were: *Nos2* (Mm00440502\_m1), *Tnfα* (Mm00443258\_m1), *Il6* (Mm00446190\_m1), and *Il1b* (Mm004342228 \_m1). *Hmbs* (Mm01143545\_m1) was used as the house keeping gene.
