Canine Biopsy Derived Colonoid Culture
In this study, intestinal biopsies were derived from healthy donors anesthetized for dental cleaning procedures. The donor population of one- to twelve-year-old dogs with no history of chronic diseases affecting the gastrointestinal tract, heart, kidney, and liver was recruited. These dogs were comprehensively screened with physical and blood examinations as pre-anesthetic evaluations and deemed healthy other than dental diseases. This study was conducted with the approval of the Washington State University Institutional Animal Care and Use Committee (IACUC Approval: ASAF#6993). Colonoids were established and maintained as previously described [16]. Briefly, colonoids were embedded in 30 µL of Matrigel (Corning, New York, USA) on 48-well plates (Corning), incubated at 37°C for 10 minutes, and added with 300 µL of expansion medium. The basal medium consisted of Advanced Dulbecco's Modified Eagle Medium (DMEM) /F12, 2mM of Glutamax-I, 10 mM of N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid, 1x Penicillin/streptomycin. The composition of the expansion medium was as follows; basal medium, 1x B27 supplement (provided from Gibco, Thermofisher Scientific, Massachusetts, USA), 100 ng/mL of Noggin Conditioned Medium, 200 ng/mL of R-Spondin-1 Conditioned Medium (made in the laboratory by culturing HEK293 cells), 100 ng/mL of Recombinant Murine Wnt-3a, 50 ng/ml of murine Epidermal Growth Factor, 100 µg/mL of Primocin (derived from PeproTech, Thermofisher Scientific, Massachusetts, USA), 10nM of gastrin I human, 500 nM of A 83 − 01, 10 µM of SB202190, 10 mM of Nicotinamide (Sigma Aldrich, Missouri, USA), 1 mM of N-Acetyl-L-Cysteine (MP Biomedicals, Southern California, USA), 100 ng/mL of Recombinant Murine Wnt-3a, 1x N2 Max Media supplement (R&D Systems, Minnesota, USA). For the first two days after passaging, 10 µM of Y-27632 and 2.5 µM of CHIR 99021 (Stem Cell Technologies, Vancouver, Canada) were added to the medium.
Polarity Reversal and Differentiation of Colonoids
Apical-out canine colonoids were established according to the previous reports that established apical-out organoids in humans [17]. Briefly, the colonoids were harvested four days after passage, suspended and cultured in 5 mM of ethylenediaminetetraacetic acid (EDTA)-phosphate-buffered saline (PBS) for an hour on a rotating platform, centrifuged and removed EDTA, washed with the basal medium, and resuspended to the 400 µL of desired medium to a 24-well ultra-low-attachment plate (Corning, New York, USA). One well of Matrigel-embedded colonoids was suspended to three wells of floating colonoids. The number of colonoids was counted and confirmed as not exceeding 500 colonoids/well. Twelve hours after resuspension, floating colonoids were dislodged by pipetting to prevent sticking to each other. To clarify the effect of the different culture conditions on gene expression, apical-out colonoids were cultured either in the expansion medium (EM) or differentiation medium (DM) (Fig. 1A). According to the previous report, DM was prepared as an EM without Wnt-3a, nicotinamide, and SB202190 [13]. The medium was changed every other day.
Immunofluorescent Staining
Colonoids were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and washed with PBS. The sample was permeabilized with 0.3% Triton™ X-100 (Sigma Aldrich, Missouri, USA) in PBS for ten minutes at room temperature and washed with PBS. The sample was incubated with 2% bovine serum albumin (BSA) for an hour to prevent unspecific binding of antibodies. When indicated, colonoids were incubated with 1:50 anti-chromogranin A antibody (ab45179, Abcam, Cambridge, United Kingdom) for enteroendocrine cells and 1:100 Sambucus nigra lectin; SNA (Vector Laboratories, California, USA) for mucus overnight at 4°C, washed with PBS, and then treated with 1:1000 Alexa Fluor 555-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (Abcam) for an hour at room temperature, followed by another wash with PBS. Colonoids were incubated with 1:400 phalloidin (Alexa Fluor™ 647 Phalloidin) for actin filaments and 1:1000 diamidino-2-phenylindole (DAPI) for nuclear staining (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA) for 30 minutes at room temperature and washed. Colonoids were suspended in ProLong™ Gold Antifade Mountant (Invitrogen) onto the Glass Bottom Culture Dishes (Matsunami Glass, Osaka, Japan) and imaged using TCS SP8 X White Light Laser Confocal Microscope (Leica, Wetzlar, Germany). Images were processed with LAS X software (Leica). The number of polarity reversed cells was counted under the confocal microscope and considered apical-out if F-actin was aligned outside and mixed if F-actin alignment was observed both outside and inside. This experiment was conducted using three biological replicates.
Reverse Transcription-Quantitative Polymerase Chain Reaction
After 96 hours of culture in EM or DM, colonoids were collected and washed with PBS. A time point of 96 hours was chosen based on previous reports [14, 18]. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA) with the C1000 Touch Thermal Cycler (Bio-Rad Laboratories, California, USA). The quantitative polymerase chain reaction (qPCR) was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems) with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Gene expression levels of the following marker genes were evaluated: leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), intestinal alkaline phosphatase (ALPI), mucin 2 (MUC2), and chromogranin A (CHGA). Hydroxymethyl-bilane synthase (HMBS) and Succinate dehydrogenase complex subunit A (SDHA) were selected as internal references [19]. The relative quantity of each gene was calculated using the standard curve method and normalized with the internal reference genes as previously described [20]. The primers used in this study are listed in Supplementary Table 1 [14, 19, 21, 22]. This experiment was conducted using three biological replicates and three technological replicates, and each sample was examined in duplicate for qPCR, and the mean values were used for analysis.
Dextran Diffusion Barrier Integrity Assay
The barrier integrity was assessed in accordance with the previous report [17]. After 96 hours of polarity reversal and subsequent culture in DM, colonoids were collected and pelleted at 300g for 1 minute. To make the barrier-disrupted model a positive control, apical-out colonoids were treated with 5 mM EDTA for 15 minutes on ice. After treatment, colonoids were washed with basal medium and resuspended to 100 µL of 0.5 mg/mL 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich). After five minutes, 90 µL of FITC-dextran supernatant was removed, and 10 µL of the remaining bottom FITC-dextran was transferred to a well marked using a PAP pen on a glass microscope slide. To avoid crushing colonoids when putting the coverslip, grease spots were loaded on the slide in a way consistent with the four corners of the coverslip. After preparation, confocal images were immediately obtained using the EVOS FL Fluorescence Microscope (Advanced Microscopy Group, Washington, USA). FITC intensity of colonoids was measured by tracing its outline and normalized divided by intensity outside (three data points averaged; [23]) using ImageJ version 2.14.0. Therefore, the low FITC permeability ratio means low permeability. This experiment was conducted using three biological replicates.
Escherichia coli Infection
A wild strain of Enterohemorrhagic Escherichia coli O157:H7 (EHEC) derived from bovine was used as clinical isolates of canine were unavailable [24]. A nonpathogenic strain of Escherichia coli (E. coli) tagged with GFP, MC4100, was used as an E. coli infection control. E.coli were incubated overnight in Luria-Bertani broth at 37°C on shaking at 200 rpm, then diluted 1:10 and subcultured for 1.5 hours. Bacteria were harvested, washed with PBS, and resuspended to 1.0×108 CFU/mL in DM without antibiotics. After 96 hours of polarity reversal and subsequent culture in DM, colonoids were harvested and washed with basal medium without antibiotics, resuspended to the bacteria-containing DM, and incubated at 37°C for 4h. This experiment was conducted using two biological replicates.
Statistical Analysis
The gene expression levels between apical-out colonoids and Matrigel-embedded basal-out colonoids and within apical-out colonoids cultured in different mediums (EM or DM) were compared using the Willcoxon rank-sum test, followed by Bonferroni correction. Results were shown as mean ± standard error of the mean (SEM). The FITC dextran assay data was also compared using the Willcoxon rank-sum test, followed by Bonferroni correction when comparing three groups. p < .05 were considered statistically significant differences. Graphs were produced using Prism (10.2.1) (GraphPad Software, San Diego, California, USA), and statistical analysis was conducted using R version 4.0.2.