Crypt isolation and organoid culture

Crypt isolation from RCCHan™: WIST female rat duodenum and organoid culture were conducted according to the previous report²¹. Crypts were isolated using Gentle Cell Dissociation Reagent (STEMCELL Technologies) for 30 min on ice. The digested contents were filtrated through a 70 µm strainer and centrifuged at 300×g for 5 min at 4 °C. The pellets were resuspended with ice-cold Advanced DMEM/F-12 and centrifuged at 150×g for 2 min at 4 °C. The crypts fraction was embedded with 100% Matrigel (growth factor reduced, Corning, Cat# 356231) in the basal medium, consisting of Advanced DMEM/F-12, penicillin/streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 2 mM GlutaMAX-I (Thermo Fisher Scientific) supplemented with 1 × B-27 Supplement (Thermo Fisher Scientific), 1 mM N-acetyl-cysteine, 10 nM Gastrin (Sigma-Aldrich), 100 ng/ml recombinant mouse Noggin (Peprotech), 50 ng/ml recombinant mouse EGF (Thermo Fisher Scientific), 100 ng/ml recombinant human IGF-1 (BioLegend), 50 ng/ml recombinant human basic FGF (FGF-2) (Reprocell), 1 µg/ml recombinant human R-spondin 1 (FUJIFILM Wako Pure Chemical), 500 nM A83-01 and 10% Afamin/Wnt3a CM (JSR). Budding type organoids were picked up and passaged by mechanical disruption. 10 µM Y-27632 was supplemented for the first 2 days of culture until the first passage, and EGF was removed from the first passage. Rat organoids were maintained in ΔE medium, consisting of basal medium supplemented with 1 × B-27 Supplement, 1 mM N-acetyl-cysteine, 10 nM Gastrin, 100 ng/ml Noggin, 100 ng/ml IGF-1, 50 ng/ml heat stable recombinant human bFGF (Thermo Fisher Scientific), 1 or 0.5 μg/ml R-spondin 1, 500 nM A83-01 and 10% Afamin/Wnt3a CM. For the monolayer culture, rat organoids were dissociated with TrypLE Select Enzyme (10×) for 5 min in a 37 °C water bath and filtered through a 70 µm cell strainer. The filtrate was centrifuged at 300×g for 3 min at 4 °C and supernatant was discarded, and the single cells were resuspended with ΔE medium supplemented with 50 ng/ml EGF and 10 µM Y-27632. 100 µL of cell suspension (7.0 × 10⁴ cells/well) was added into cell culture insert (Corning, Cat#353095) coated with Matrigel diluted 30-fold with PBS for at least 2 h at 37 °C. Y-27632 was supplemented with culture medium only on the first day. Bright-field images were obtained with an inverted microscope (IX83, Olympus).

Development of cyst-enriched organoids

Rat organoids were dissociated with TrypLE Select Enzyme (10×) for 5 min in a 37 °C water bath and filtered through a 70 µm cell strainer. Single cells were resuspended with cyst-enriched medium consisting of basal medium supplemented with 1 × B-27 Supplement, 1 mM N-acetyl-cysteine, 10 nM Gastrin, 100 ng/ml Noggin, 50 ng/ml EGF, 100 ng/ml IGF-1, 50 ng/ml heat stable bFGF, 0.5 µg/ml R-spondin 1, 500 nM A83-01, 10 µM Y-27632 and 20% Afamin/Wnt3a CM. 3.0 × 10⁴ cells were embedded with 50 µl of Matrigel and cultured with cyst-enriched medium. The medium was replaced every 2 or 3 days, and cyst-enriched organoids were passaged every 3 or 4 days by mechanical dissociation.

Monolayer culture

Conventional organoids/cyst-enriched organoids were dissociated into single cells using TrypLE Select Enzyme (10×). After a filtration by 70 µm cell strainer and centrifugation, the cell pellet was resuspended in cyst-enriched medium and adjusted to 3.5 × 10⁵ cells/mL. 200 µL of cell suspension was seeded on Transwell (Corning, Cat#3378 or 3470) coated with Matrigel diluted 50-fold with cold-PBS at 37 °C for 2 h, and medium was added to the basal compartment. On day 2 or 3, the medium was changed to the differentiation medium which consisted of basal medium containing 1 × B-27 Supplement, 1 mM N-acetyl-cysteine, 10 nM Gastrin, 100 ng/ml Noggin, 0.5 µg/ml R-spondin 1, 500 nM A83-01, 10 µM Y-27632, 3 mM VPA (Abcam). The medium was replaced every 2 or 3 days. For the induction assay, differentiation medium containing 50 µM β-naphtoflavone (Sigma), 5 µM 3-methylcholanthrene (Sigma), or 100 nM 1α, 25-dihydroxyvitamin D3 (Sigma) was exposed for 48 h. CYP1A activity and CYP3A activity were analyzed by P450-Glo Assays according the manufacturer’s protocol (luciferin-CEE for CYP1A, luciferin-IPA for CYP3A, Promega). The monolayer integrity was assessed by TEER. The TEER value was measured using Millicell ERS-2 system (Merck).

Histological examination

A paraffin block of rat organoids were prepared by the method described previously⁴⁷. Briefly, rat organoids were fixed with 4% Glutaraldehyde for 1 h at room temperature. Matrigel dome was disrupted by pipetting and collected, then embedded in paraffin by the AMeX method⁴⁸. HE slides were prepared and an AB-PAS was performed.

Whole mount immunostaining for organoids and monolayer

After withdrawing the culture medium, Matrigel dome was fixed by 2% paraformaldehyde (PFA) for 30 min at room temperature. After PBS washing, PBS containing 0.5% Triton X-100 was added and permeabilized at 4 °C overnight. Blocking was performed with BlockAid Blocking Solution (Thermo Fisher Scientific) containing 0.5% Triton X-100 for 6 h at room temperature. Primary antibodies [Alexa Fluor 555-labeled anti-Ki-67 antibody (Clone B56, BD Biosciences), anti-Sox9 antibody (Clone D8G8H, Cell Signaling Technology), anti-CK20 antibody (Clone SA35-03, Thermo Fisher Scientific), anti-P-gp antibody (Clone F4, Thermo Fisher Scientific), anti-ZO-1 antibody (Clone ZO1-1A12, Thermo Fisher Scientific), and anti-phospho-Ezrin antibody (Clone 48G2, Cell Signaling Technology)] in blocking solution containing 0.5% Triton X-100 were added and incubated for 3 days at 4 °C. After that, the sample were washed with PBS containing 0.5% Triton X-100 three times, incubated for 1 or 2 days at 4 °C with secondary antibody [Alexa Fluor 488-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific), Alexa Fluor Plus 555-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific), or Alexa Fluor 555-labeled anti-mouse IgG1 antibody (Thermo Fisher Scientific)] in blocking solution containing 0.5% Triton X-100. After washing with PBS containing 0.5% Triton X-100 three times, the wells were then incubated with SeeDB2G Solution 1 [1/3 × Omnipaque350 with 2% saponin] for 3 h at room temperature, Solution 2 (1/2 × Omnipaque350 with 2% saponin) containing Phalloidin-DyLight 650 and 1 µg/mL DAPI overnight at 4 °C, Solution 3 (1 × Omnipaque350) for 3 h at room temperature to clear the organoids⁴⁹, which were observed with a confocal fluorescence microscope (A1, Nikon).

For the monolayer culture, after withdrawing the culture medium, the monolayer was fixed by 4% PFA for 15 min at room temperature. After PBS washing, blocking solution containing 0.5% Triton X-100 was added and incubated for 1 h at room temperature. The monolayer was stained with primary antibodies [anti-P-gp antibody, anti-ZO-1 antibody, anti-phospho-Ezrin antibody, anti-E cadherin (Thermo Fisher Scientific, PA5-32178), anti-Villin-1 antibody (Clone R814, Cell Signaling Technology)] in blocking solution containing 0.3% Triton X-100 at 4 °C for 2 days, then washed with PBS three times, incubated overnight at 4 °C with secondary antibody in blocking solution containing 0.3% Triton X-100.

Transmission electron microscopy for organoids

Organoids were fixed with half Karnovsky solution at 4 °C for 24 h. Fixed organoids with Matrigel were minced into pieces of approximately 1 mm³ with a razor blade and washed with 0.1 M cacodylate buffer. Further fixation was performed using 1% osmium tetroxide in 0.1 M cacodylate buffer at 4 °C for 1.5 h. The fixed organoids were dehydrated while gradually increasing the ethanol concentration from 50 to 100%, replaced with propylene oxide, and embedded in epoxy resin (Quetol812, Nisshin EM). Resin was heat polymerized; ultrathin (60–70 nm) sections were prepared using a ultramicrotome (Leica EM UC7, Leica Microsystems), double-stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (HT7700, Hitachi High-Technologies).

Organoid formation assay

Conventional organoids and cyst-enriched organoids were dissociated into single cells in the same way as the monolayer culture. These cells were embedded with Matrigel and cultured in expansion medium supplemented with 10 μM Y-27632 for 4 days. Organoid growth was measured using CellTiter-Glo^® 3D Cell Viability Assay (Promega) after solubilizing Matrigel with dispase (Dispase I, FUJIFILM Wako Pure Chemical). For the Imaging analysis was performed with cellSens (OLYMPUS) according the manufacturer. The formation of an organoid was defined to be when the major axis of its structure was measured to be over 100 μm in the bright-field image.

EdU uptake assay

EdU staining was performed according the manufacturer’s protocol (Click-iT Plus EdU Cell Proliferation Kit for imaging, Alexa Fluor 555 dye, Thermo Fisher Scientific). Nuclei and EdU positive cells were counted using NIS elements (Nikon) according to the manufacturer’s instructions.

Quantitative RT-PCR

Total RNA was extracted from monolayer culture using TRIzol. cDNA was synthesized with the Superscript III First-strand synthesis system for RT-PCR (Thermo Fisher Scientific). Quantitative RT-PCR was conducted in duplicate for each gene on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green (Thermo Fisher Scientific). β-Actin was used as the housekeeping gene. The sequences of the primers for RT-PCR are shown in Supplementary Table S1.

Compound permeability assay

For the lucifer yellow permeability assay, a transporter buffer consisting of 10 mM HEPES and 1% (w/v) BSA in HBSS (pH 7.4) with 200 μM lucifer yellow was added to the apical side. HBSS was purchased from Thermo Fisher Scientific (Cat#14025092). The transporter buffer without lucifer yellow was also added to the basal side and cultured by shaking. After 2 h, the basal buffer was sampled for analysis. Lucifer yellow concentration was determined by a fluorescent signal measured with EnSpire (PerkinElmer) using 428 nm excitation and 536 nm emission filters. For the digoxin permeability assay, a transporter buffer containing 200 μM lucifer yellow and 10 μM digoxin in the presence or absence of 5 μM Zosuqudar was added to the apical or basal side, respectively. After 2 h, the basal or apical buffer was sampled for analysis. For the midazolam metabolic assay, HBSS containing 200 μM lucifer yellow, 5 μM midazolam, 10 mM MES, and 1% (w/v) BSA (pH 6.5) in the presence or absence of 1 mM ABT was added to the apical side of the differentiated monolayer. Transporter buffer consisting of 10 mM HEPES and 4% (w/v) BSA in HBSS (pH 7.4) was added to basal side of the monolayer. After 2 h, the basal buffer was sampled for analysis. The monolayer which was cultured for 3 days in expansion medium and for 4 days in differentiated medium with 100 nM VD3 induction for 48 h was used for the midazolam assay. The concentration of digoxin, and 1-hydroxymidazolam were determined by LC–MS/MS analysis. The apparent permeability coefficient (Papp) was calculated according to following equation:

where dQ/dt represent compound transported to the basal or apical side per unit time, A is the surface area of the cell culture insert, and C₀ is the initial concentration of compound.

LC–MS/MS analysis

The collected buffer samples were mixed with methanol/acetonitrile (1/1, v/v) containing an internal standard (d3-digoxin for digoxin analysis, d4-1-hydroxymidazolam for 1-hydroxymidazolam). After centrifugation or filtration, supernatant was analyzed using the Nexera X3 system (Shimadzu), QTRAP6500 system (SCIEX), and the ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm, Waters) for digoxin, or the XBridge BEH C18 Column (3.5 μm, 2.1 mm × 100 mm, Waters) for 1-hydroxymidazolam.

Statistical analysis

Statistical analyses were performed using the GraphPad Prism 7.04 (GraphPad Software) by Student t-test and Dunnett’s multiple comparison test. A p-value of < 0.05 was considered statistically significant.

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• Source: https://www.nature.com/articles/s41598-023-39425-7