Experimental design
The Bo-IOs was produced by separating the tissue with the crypt from the small intestine tissue, then 3D-cultured in Matrigel and re-cultured to produce the Apo-IOs (Fig. 4a). In this study, the expression of Apo-IOs genes and proteins was investigated, and functional analysis was performed by checking the absorption of amino and fatty acids using fluorescent substances (Fig. 4b). Additionally, we determined the LC50(Lethal Concentration 50) value of DON using the 4′,6-diamidino-2-phenylindole/propidium iodide (DAPI/PI) method. Afterwards, Apo-IOs were treated with DON alone, and a DON + Lactobacillus plantarum ATCC14917 culture supernatant (LCS) combination (DON + LCS). Then, FITC-4 kDa dextran was used to assess intestinal barrier function and the gene and protein expression levels were compared among the different treatments (Fig. 4c).
Experimental design. a Establishment of a bovine intestinal organoid with small intestine-derived stem cells. b Induction and characterization of a bovine apical-out intestinal organoid. c In vitro model for evaluating the toxicity of mycotoxins.
Establishment and treatment of bovine apical-out intestinal organoids
We isolated crypts from the bovine small intestine and generated organoids using a previously described method37. Briefly, the dome of the organoid was pipetted with 1 mL organoid harvest solution (Bio-techne, Minneapolis, MN, USA) and transferred to a 15 mL tube, whereafter 2 mL cold phosphate buffered saline (PBS) was added for centrifugation. The sample was left stationary at 4 °C for 1 h to remove the Matrigel. After centrifugation at 300 × g for 5 min at 4℃, intestinal human organoid medium was added to the resulting pellet. To produce organoids in the apical-out form, organoid pieces were cultured at 37 °C for two days by adding 1 mL intestinal human organoid medium to an ultralow-attachment plate54. The Apo-IOs were either untreated (control) or exposed to 25 µM DON, LCS, and 25 µM DON + LCS at 37 °C for 6 h to determine the effectiveness of mycotoxins and toxin reducers.
Preparation of bacteria
The L. plantarum ATCC14917 strain was cultured to the stationary phase (for approximately 24 h) in sterile Man–Rogosa–Sharpe medium at 37 °C in an anaerobic environment, then centrifuged at 5000 × g for 10 min at 4 °C. After optical density measurements (absorbance at 600 nm = 1.0 ± 0.1 corresponded to 1.0 × 108 CFU/mL), bacteria were harvested via centrifugation at 5000 × g for 10 min at 4℃. For cell culture assays, the LCS was reserved for subsequent treatment at a 10% (v/v) concentration.
RNA extraction and quantitative real-time PCR
TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract RNA from the Apo-IOs. The cDNA was synthesized via reverse transcription of RNA using the Superscript IV First-Strand Synthesis System (Invitrogen, Wilmington, DE, USA). The PCR reaction mixture was prepared using a combination of 1 µL cDNA, 1 µL of 10 pmol forward and reverse primers, 7 µL nuclease-free water, and 10 µL power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) to a final volume of 20 µL. Real-time PCR conditions were set as follows: 95 °C, 30 s; 40 amplification cycles (95 °C, 30 s; 60 °C, 30 s; 95 °C, 30 s); elongation step at 72 °C. Non-specific amplification was confirmed using the melting curve. The Ct value refers to the cycle number at which a fluorescent signal is expressed, and gene expression was quantified using the 2−ΔΔCt method. The qPCR primer used was for 18 S rRNA, and the target genes are shown in Table 1. The qPCR analysis was performed using a Step One Plus real-time PCR system (Applied Biosystems, Waltham, MA, USA).
Immunofluorescence staining of apical-out intestinal bovine organoids
Apo-IOs were washed three times with cold PBS for 5 min and treated with 4% cold paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The organoids were washed three times with PBS to remove the fixative and then treated with a permeabilization solution (Thermo Fisher Scientific) at room temperature for 15 min. After washing again with PBS three times, the organoids were treated with a blocking solution (Thermo Fisher Scientific) at room temperature for 1 h. Afterwards, anti-LGR5 (1:200; Cat. No. ab75732; Abcam, Cambridge, UK), anti-Ki67 (1:200; Cat. no. ab1667; Abcam), anti-E-cadherin (1:200; Cat. no. 61081; BD Biosciences, Franklin Lakes, NJ, USA), anti-F-actin (1:200; Cat. no. ab83746; Abcam), and anti-Mucin2 (1:200; Cat. no. Sc-515032; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were appropriately diluted in the blocking solution. The organoids were treated with these antibodies at 4 °C for 24 h, whereafter they were treated with Alexa Fluor-633 goat anti-mouse IgG (Cat. no. A21052; Thermo Fisher Scientific), Alexa Fluor-488 anti-mouse IgG (Cat. no. A11001; Thermo Fisher Scientific), and Alexa Fluor-488 anti-rabbit IgG (Cat. no. A21441; Thermo Fisher Scientific) antibodies at 4 °C for 24 h. Finally, the Apo-IOs were treated with DAPI (Cat. no. R37606; Thermo Fisher Scientific) for 20 min and then analyzed using a high-resolution confocal laser-scanning microscope (Nikon Instruments Inc., Tokyo, Japan).
Scanning electron microscopy
Apo-IOs were fixed in 2.5% buffered glutaraldehyde in 1× PBS for 2 h at 4 °C, followed by three washes with 0.1 M sodium cacodylate buffer, each wash lasting 10 min. Apo-IOs were post-fixed for 2 h at room temperature in 2% osmium tetroxide in 0.1 M cacodylate buffer, followed by two washes with 0.1 M cacodylate buffer for 10 min each and one wash with distilled water. Apo-IOs were then serially dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, and two 100% ethanol steps), with each step lasting 10 min. After the ethanol dehydration series, samples were further dehydrated in a 1:1 mixture of ethanol and hexamethyldisilazane (HMDS), followed by two additional 1-h incubations in 100% HMDS. After infiltration with pure HMDS, Ap-IOs were allowed to dry overnight in a fume hood at room temperature. Following drying, Apo-IOs were mounted onto carbon tape on an SEM stub. Apo-IOs were then coated with a Pt layer using an ion sputter coater (Hitachi E-1045, Hitachi Co.) with the following parameters: 15 mA for 60 s for the front face and 15 mA for 20 s for each lateral side. For SEM imaging, Apo-IOs were examined using the SE mode (ETD detector) on a TeneoVS SEM (FEI, USA). Images were captured at a resolution of 3072 pixels × 2048 pixels, with a working distance of approximately 10.0 mm, an acceleration voltage of 5 kV, and an emission current of 50 pA.
Fatty acid absorption of apical-out intestinal bovine organoids
After washing the Apo-IOs with cold PBS three times, BODIPY 500/510 C1, C12 (Invitrogen) and fatty acid-free BSA were combined to a final concentration of 5 µM/L, which was then added to the organoids. The organoids were then incubated at 37 °C in a CO2 incubator for 30 min. Fluorescent samples were seeded and mounted onto glass slides. Representative images were obtained using a confocal laser-scanning microscope (Nikon Instruments Inc., Tokyo, Japan).
Amino acid absorption of apical-out intestinal bovine organoids
The Apo-IOs were transferred to 15-mL conical tubes and centrifuged at 300 × g for 5 min. The supernatant was removed and the cells washed three times with prewarmed HBSS (Cat. no. 14175095; Thermo Fisher Scientific). A prewarmed BPA uptake solution (Cat. no. UP02; Dojindo, Kumamoto, Japan) was added to the Apo-IOs, which were then incubated at 37 °C for 5 min and centrifuged thereafter for 5 min at 300 × g. The supernatant was removed, and a prewarmed working solution (Cat. no. UP02; Dojindo) added to the organoids that were then incubated at 37 °C in a CO2 incubator for 5 min. Fluorescent samples were seeded and mounted onto glass slides. Representative images were obtained using a confocal laser-scanning microscope.
Epithelial barrier permeability of apical-out bovine organoids
Untreated Apo-IOs, as well as those treated with 25 µM DON, LCS, and 25 µM DON + LCS for 6 h, were exposed to FITC-4 kDa dextran (50 ng/mL; Sigma-Aldrich), and the penetration of FITC-4 kDa dextran into the Apo-IOs then confirmed using a confocal laser-scanning microscope.
Cell viability assay
The DAPI/PI assay was used to assess the cell viability of Apo-IOs in response to DON and LCS exposure. Untreated Apo-IOs, as well as those treated with 25 µM DON, LCS, and 25 µM DON + LCS for 6 h, were exposed to PI. After washing three times with cold PBS, 4% formaldehyde and DAPI were added for 15 min. Cell viability was then assessed using a confocal laser-scanning microscope.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (version 9; GraphPad Software, La Jolla, CA, USA). Statistical significance was determined using a t-test or one-way ANOVA, with corrections for multiple comparisons, as appropriate. Data are presented as the mean ± SD, and P-values < 0.05, < 0.01, < 0.001, and < 0.0001 were considered statistically significant. All experiments were performed at least three times.
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- Source: https://www.nature.com/articles/s41598-024-82928-0