Adult Bovine-Derived Small and Large Intestinal Organoids: In Vitro Development and Maintenance

. Recent progress in bovine intestinal organoid research has expanded opportunities for creating improved in vitro models to study intestinal physiology and pathology. However, the establishment of a culture condition capable of generating organoids from all segments of the cattle intestine has remained elusive. Although previous research has described the development of bovine jejunal, ileal, and colonic organoids, this study marks the frst report of successful bovine duodenal and rectal organoid development. Maintenance of these organoids through serial passages and cryopreservation was achieved, with higher success rates observed in large intestinal organoids compared to their small intestinal counterparts. A novel approach involving the use of biopsy forceps during initial tissue sampling streamlined the subsequent tissue processing, simplifying the procedure compared to previously established protocols in cattle. In addition, our study introduced a more cost-efective culture medium based on advanced DMEM/ F12, diverging from frequently used commercially available organoid culture media. Tis enhancement improves the accessibility to organoid technology by reducing culture costs. Crucially, the derived organoids from the jejunum, ileum, colon, and rectum faithfully preserved the structural, cellular, and genetic characteristics of the in vivo intestinal tissue. Tis research underscores the signifcant potential of adult bovine intestinal organoids as a physiologically and morphologically relevant in vitro model. Such organoids provide a renewable and sustainable resource for a broad spectrum of studies, encompassing investigations into normal intestinal physiology in cattle and the intricate host-pathogen interactions of clinically and economically signifcant enteric pathogens.


Introduction
Te feld of intestinal biology research has seen a transformative shift with the adoption of three-dimensional (3D) cultures of intestinal epithelial cells [1].Tese cultures, known as intestinal organoids, are derived from primary intestinal stem cells and faithfully replicate the structural complexity of the intestine.Comprising various cell lineages found in the intestinal epithelium, such as enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, these organoids ofer a unique opportunity to investigate intestinal physiology, nutrient absorption, and host-pathogen interactions [2,3].
Notably, the application of bovine intestinal organoids as an in vitro model for infectious diseases has demonstrated their immense potential [4][5][6][7].Tis is especially relevant in the context of the United States cattle industry, a signifcant contributor to the country's economy [8].Te health of cattle and their gut function directly impacts productivity, animal welfare, and production costs [9,10].In addition, cattle play a role in greenhouse gas emissions, particularly due to enteric methane, making the study of their gut health socially and economically signifcant [11].
Furthermore, cattle serve as a reservoir for pathogens with implications for human health [12].Understanding host-pathogen interactions for these zoonotic enteric pathogens requires consideration of the specifc gut segments they inhabit [13,14].Although intestinal organoid technology has been described for certain segments of the bovine gastrointestinal tract (i.e., the jejunum [5,15], ileum [16,17] and colon [18]), there remains a gap in our understanding of the duodenum and rectum.Addressing this gap is crucial for advancing our knowledge of pathophysiology and colonization processes.
To bridge these knowledge gaps and enhance our understanding of bovine gut physiology and pathogen interactions, we present a technique for culturing bovine intestinal organoids from fve diferent segments of the adult bovine gut.Tis method, distinct from previous fullthickness tissue sampling approaches [4,5,7,[15][16][17][18], involves mucosa extraction using biopsy forceps and subsequent organoid cultivation with an in-house organoid growth media supplemented with growth factors and inhibitors.Te resulting organoids are assessed for their fdelity to in vivo bovine gut traits through immunofuorescence and RT-qPCR analysis.

Animals and the Intestinal Tissue.
Intestinal tissue samples were collected from fve approximately 15-to 18month-old cattle at a local slaughterhouse.Signalments of donors are summarized in Supplementary Table 1.An overview of tissue sampling and subsequent processing techniques is illustrated in Figure 1(a).Five segments of the intestine, namely, the duodenum, jejunum, ileum, colon, and rectum, were identifed and incised longitudinally to expose the mucosal surface (Figure 1(b)).Approximately, 10-15 pieces of the fresh tissue were sampled from each segment of the intestine using biopsy forceps (Hildyard Post-Nasal Biopsy Forceps, Med-Plus) (Figure 1(c)) and immediately placed in a wash medium consisting of icecooled Dulbecco's phosphate-bufered saline (PBS) (Gibco) supplemented with 1x penicillin/streptomycin (Gibco) and 25 μg/mL gentamicin (Gibco).Te samples were kept on ice until further processing at the laboratory.

Crypt Isolation and Organoid
Generation.Te samples were processed as described previously with some modifcations to isolate intestinal crypts and obtain intestinal stem cells for organoid generation [19][20][21].In brief, the pieces of the tissue were frst washed with a sterile wash medium approximately 5 times or until the supernatant became clear by shaking vigorously and replacing the wash medium repeatedly.Once excess mucus and residual luminal contents were removed, the samples were resuspended into 20 mM EDTA solution and minced to small fragments in a standard tissue culture dish (Fisher Scientifc) using sharp-pointed dissecting scissors (Fisher Scientifc).Te fragmented tissue was then collected into a 15 mL conical tube and incubated for 15 (jejunum and ileum), 30 (colon), or 60 (duodenum and rectum) minutes at 10 rpm at 4 °C on a tube rotator (Fisher Scientifc) to isolate intestinal crypts (Figure 1(d)).
Following incubation, tissue fragments were allowed to settle to the bottom of the tube by gravity and the crypt-containing supernatant was collected in a new 15 mL conical tube.Te crypts were pelleted by centrifugation of the supernatant at 200 × g at 4 °C for 5 minutes.Te supernatant was discarded, and the pellet was resuspended in Matrigel (Corning).Te crypts were then seeded onto a 24-well plate in 30 μL per well.
Following the polymerization of Matrigel at 37 °C for 10-15 minutes, each well received 500 μL of the organoid expansion medium (OEM) consisting of the components outlined in Table 1.Te noggin and R-spondin-conditioned media were obtained by cultivating HEK293 cells engineered to secrete noggin (Baylor's College of Medicine) [22] and Cultrex HA-R-spondin1-Fc 293T cells (R&D Systems) as described previously [23].10 μM rho-associated kinase inhibitor (ROCKi) Y-27632 (STEMCELL Technologies) and 100 nM glycogen synthase kinase (GSK) 3β inhibitor CHIR99021 (Sigma-Aldrich) were added to the OEM for the frst 2-4 days of the culture and removed thereafter once organoids started growing in Matrigel (Figure 1(e)).Te plate was incubated at 37 °C with 5% CO 2 and the OEM was renewed every other day for growth and maintenance of organoids.Te growth of organoids was monitored daily, and images of organoids were taken using phase-contrast microscopy (DMi1, Leica).

2.3.
Organoid Subculture and Maintenance.Organoids were serially passaged and expanded every 6-8 days as they grew to form lumina and villi-like budding structures following initial culture.For passaging, Matrigel-containing organoids were frst dissociated by replacing the OEM with cold cell recovery solution (Corning) and incubating for 30 minutes at 4 °C.Organoids were recovered from Matrigel by centrifuging at 200 × g at 4 °C for 5 minutes and discarding the supernatant.Organoids were then disrupted by adding TrypLE Express (Gibco) and incubating in a 37 °C water bath for 1 minute.Enzymatic disruption was stopped by adding the basal medium (advanced DMEM/F12 with GlutaMAX, HEPES, and penicillin/streptomycin).Te dissociated organoids were pelleted down by centrifuging at 200 × g at 4 °C for 5 minutes.Te pellet was resuspended in Matrigel at an expansion ratio of approximately 1-8 wells and cultured as described above in 24-or 48-well plates with 500 μL or 300 μL of the culture medium, respectively.
Nuclei and F-actin were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 : 1000, Termo Scientifc) and Alexa Fluor 647-conjugated phalloidin (1 : 400, Invitrogen), respectively, according to the manufacturers' recommendation (Supplementary Table 2).EdU assay (Invitrogen) was also performed following the manufacturer's protocol to detect actively proliferating cells within organoids.Organoids were washed with PBS and mounted on glass bottom dishes (Matsunami) using the ProLong Gold Antifade reagent (Invitrogen).Fluorescence imaging was performed using a white-light point scanning confocal microscope (SP8-X, Leica) and images were processed using LAS X (Leica).Immunocytochemistry was performed in at least two technical replicates using organoids derived from four independent donors.
For quantitative analysis, the number of cells that stained positive for SOX9, EdU, and SNA was counted on randomly selected representative images.Te percent positive rate for each cell type was calculated by normalizing the positive cell number by the total number of nuclei.Tree to ten independent felds of view from four biological replicates were assessed.

Gene Expression Analysis. Expression of genes indicative of specifc intestinal cell types, namely,
LGR5 (stem cells), ChrA (enteroendocrine cells), LyzC (Paneth cells), Muc2 (goblet cells), and FABP2 (intestinal epithelial cells) [7,24,25], was determined and compared between organoids cultured in the OEM for 8 days and those treated with the organoid diferentiation medium (ODM) for 4 days following initial culture in OEM for 4 days.Te ODM was prepared by withdrawing Wnt3a, SB202190, and nicotinamide from OEM [1].Total RNA was extracted from the jejunal, ileal, colonic, and rectal organoids in the OEM and ODM using the RNeasy Plus Mini Kit (Qiagen) and concentrations were quantifed using NanoDrop One (Termo Scientifc).cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and was subsequently used to carry out RT-qPCR reactions using PowerUp SYBR Green Master Mix (Applied Biosystems).Primers used in this study are listed in Supplementary Table 3. Te primer sequence was amplifed at 60 °C for 40 cycles and generation of single product was confrmed using dissociation curves.Relative gene expression was calculated by applying the standard curve method and using GAPDH, RPL0, and ACTB as internal control [7,26,27].RT-qPCR reactions were carried out in triplicate from one (jejunum) or three (ileum, colon, and rectum) biological replicates.

Statistical Analyses.
Quantitative data were analyzed using R v.3.4.1 (the R foundation) and plotted using GraphPad Prism 9.5.1 (GraphPad Software).For immunocytochemistry, the percent positive rates were compared across the four intestinal segments using the Kruskal-Wallis test with a Bonferroni post-hoc test.For RT-qPCR analyses, data were compared between control (OEM-grown organoids) and ODM-treated organoids using Wilcoxon's signed rank test for independent samples.Results were presented as the mean ± standard error of the mean (SEM), with p values ≤0.05 being considered statistically signifcant.

Establishment of Bovine Intestinal Organoids. In vitro 3D
intestinal organoids were successfully generated from fresh tissue samples obtained via the biopsy technique from the duodenum, jejunum, ileum, colon, and rectum of fve adult cattle (Figure 2(a)).Organoids generally formed spherical or budding luminal structures consistent with previous reports [5,[15][16][17][18]28].Te success rate of initial organoid development using this biopsy technique was 100% in all the fve segments (Table 2).Serial in vitro passaging of these organoids demonstrated that organoids from the large intestine continuously and consistently grew and expanded for long term (>10 passages), yet those from the small intestine inconsistently retained their proliferating capability during early passages.Organoids which eventually lost their proliferating capacity were noted to disintegrate or cease to grow during early passages (i.e., passage 2-6; Supplementary Figure 1).Tese organoids were unable to expand in number or maintain culture thereafter; thus, we were unable to perform subsequent evaluations using immunocytochemistry and RT-qPCR.
Overall survival rates of organoids to passage 10 were 0% in the duodenum, 20% in the jejunum, 60% in the ileum, and 100% in both the colon and rectum (Table 2).Representative images of organoids following serial passages are shown in Figure 2(b).When organoids were cryopreserved in 10% DMSO in the FBS freezing medium and thawed after 8 months of storage, 100% of the organoid lines revived in all but duodenal organoids, where the resuscitation rate was 40% (Table 2, Figure 2(c)).

Characterization of Bovine Intestinal Organoids.
Immunofuorescence staining of organoids demonstrated their basal-out structural characteristics with cellular polarization, heterogeneity of epithelial lineages, and retention of stemness and proliferative capacity in the jejunum, ileum, colon, and rectum (Figure 3(a)).More specifcally, staining against E-cadherin confrmed the formation of basolateral epithelial adherence junctions.Factin and nuclei stains confrmed cellular polarity characterized with apical brush border and basal nuclei, each of which was observed on luminal surfaces and towards the periphery of organoids, indicating basal-out cellular organization in organoids.
Te epithelial nature of the organoid cells was confrmed by positive staining for EpCAM.Te presence of SOX9positive cells within organoids, as noted with distinct nuclei staining, indicated retention of stem cell characteristics which typify in vivo intestinal crypts.Te EdU assay confrmed the maintenance of organoid proliferative capacity by depicting actively proliferating cells.In addition, fuorescein-labeled SNA, a sialic acid-specifc lectin, was used to detect mucin which is produced by goblet cells.SNApositive cells were noted with distinct cytoplasmic staining, where mucin is normally stored in vivo.Some SNA-positive cells appeared with mucus excreting into the organoid lumen at the apical surface of those cells, adding support to goblet cell diferentiation.Te mean percentages of positively stained cells for SOX9, EdU, and SNA across the four intestinal segments were 67.8 ± 22.3% (range: 58.8-78.8),49.5 ± 20.9% (36.1-66.2),and 19.6 ± 12.1% (10.9-27.2),respectively (Figures 3(b)-3(d)).No signifcant diference was noted for all markers between the segments.

Relative Gene Expression of OEM-Grown vs. ODM-Treated
Organoids.RT-qPCR of organoids cultured in the OEM and ODM revealed variable expressions of genes indicative of specifc cell types found in the intestine in vivo.Te stem cell marker LGR5 gene was expressed at signifcantly higher levels in ileal, colonic, and rectal organoids grown in the OEM compared with those treated with the ODM (p < 0.01; Figure 4(a)).Te expression of LGR5 was suppressed in ODM-treated jejunal organoids compared to those in OEM-grown organoids, although a diference was not noted between the groups (p � 0.25).In fact, LGR5 expression was suppressed to barely detectable levels in ODM-treated jejunal, ileal, and colonic organoids.Te expression of enteroendocrine cell marker ChrA gene was upregulated in ODM-treated colonic and rectal organoids (p < 0.01), whereas no diference was observed in jejunal and ileal organoids (p � 0.75 and 0.65, respectively; Figure 4(b)).Te Paneth cell marker LyzC gene was expressed at higher levels in OEM-grown jejunal organoids (p � 0.25; Figure 4(c)).LyzC expression was slightly increased in ODM-treated organoids in the ileum, colon, and rectum, although no diference between the groups was noted (p � 0.50, 0.50, and 0.20, respectively).ODM treatment upregulated the expression of goblet cell marker Muc2 and enterocyte marker FABP2 genes in organoids of all the four segments (Figures 4(d) and 4(e)).Signifcant upregulation was noted in colonic organoids for Muc2 (p � 0.02) and in the ileal, colonic, and rectal organoids for FABP2 (p � 0.004 for all).

Discussion
Te present study demonstrates that bovine intestinal organoids can be generated at a high success rate (100%) from the tissue obtained from the duodenum, jejunum, ileum, colon, and rectum using the biopsy technique previously described in dogs with some modifcations [19][20][21].Although the development of bovine jejunal, ileal, and colonic organoids has been described previously [5,[15][16][17][18], this is the frst study which reports the successful development of bovine duodenal and rectal organoids.In addition, the use of biopsy forceps during initial tissue sampling is novel in cattle and was able to simplify subsequent tissue processing compared to the protocols previously described in cattle.Furthermore, the present study established a more cost-efective culture medium based on advanced DMEM/ F12, which was similar but not identical to the media described to grow porcine jejunal and ileal organoids previously [29,30].Tis is in contrast to often employed commercially available organoid culture media used in Journal of Tissue Engineering and Regenerative Medicine   , and intestinal epithelial cell (FABP2, (e)) marker genes using organoids cultured either in the OEM for 8 days or in the OEM for 4 days followed by the ODM for 4 days.Te results are from samples derived from one (jejunum) or three (ileum, colon, and rectum) independent animals, with three technical replicates.Te gene expression levels of each of the target genes were calculated relative to that of GAPDH, RPL0, and ACTB as internal control.Te results are presented as the mean ± standard error of the mean (SEM).Statistical analysis was performed with Wilcoxon's signed rank test for independent samples.* p < 0.05, * * p < 0.01.
previous studies, thus improving accessibility to the organoid technology through reduced culture costs.
In the present study, multiple small pieces of the partial thickness intestinal tissue were sampled using biopsy forceps.Tis contrasts with previously described techniques which usually sampled a portion of the full-thickness intestinal tissue in the feld for transportation back to the laboratory for washing and further tissue processing [15][16][17].Advantages of using biopsy forceps include the fact that samples can be collected with minimal debris or cleansed easily on site through wash media replacements in the case that large amount of ingesta or debris is noted at the time of sampling.Collecting cleaner samples is expected to minimize possible cellular damage resulting from bacterial exposure, potentially preserving cellular viability and improving the organoid generation rate.Although the importance of the minimizing gross contamination of intestinal specimens at the time of sampling has not been emphasized in previous studies, possible organoid development failure due to autolysis during transportation was reported in the gall bladder [31].Terefore, it is likely a good practice to adopt cleaner sampling techniques when a delay in sample processing is expected (e.g., transporting to a distant laboratory or sampling multiple animals consecutively).Moreover, the present sampling technique was simpler as it did not require scraping of the mucosal layer and villi using a glass slide before mincing samples into small fragments [15][16][17].No diference in crypt isolation and subsequent organoid generation efciencies was noted between the samples collected using biopsy forceps before or after scraping with a glass slide (data not shown).Potential disadvantages of using biopsy forceps might include a reduction in the amount of the tissue that is available for further processing as compared to previous techniques; hence, the number of crypts isolated from the samples also would be reduced.However, the current study demonstrated that sampling of 10-15 pieces of biopsy tissues from each segment of the intestine resulted in the isolation of a sufcient number of crypts to generate organoids successfully and consistently during the initial cultivation.
Organoids derived from all the fve segments of the intestine were successfully resuscitated following cryopreservation and all, but duodenal organoids were maintained and expanded stably for a long term through serial passages without losing their proliferating capacity.Te present study observed the maintenance of stable culture in all but duodenal organoids for greater than 15 passages at the time of manuscript preparation, which was similar to or greater than previous observations [5,15,16].Cryopreservation and resuscitation of viable bovine intestinal organoids was only reported previously with jejunal and ileal organoids [4,5,7,[15][16][17].Te commercially available cryopreservation medium has been used most commonly, with two studies preparing their own freezing medium consisting of 10-20% DMSO in the culture medium.Te freezing medium used in the present study was adopted from the work reported with canine intestinal organoids and was not tested previously in bovine organoids [20].Terefore, the present results demonstrate that rectal organoids can be maintained for long term through serial passages similar to jejunal, ileal, and colonic organoids.Furthermore, our results demonstrate that duodenal, colonic, and rectal organoids as well as jejunal and ileal organoids can be cryopreserved for later use and the freezing medium previously reported in canine intestinal organoids is applicable to bovine intestinal organoids.
Success rates for cryopreservation and long-term cultivation varied across the organoids derived from diferent bovine intestinal segments.High success rates were generally observed in colonic and rectal organoids, suggesting that our OEM composition provide a more favorable in vitro environment for the growth of large intestinal organoids.In contrast, duodenal organoids from all the fve donors lost proliferating capacity following several passages and failed to expand to provide enough organoids for subsequent characterization with immunocytochemistry and RT-qPCR.Although exact reasons for this failure remain unknown, it is possible that bovine duodenal organoids require additional support for long-term survival.For example, this might take the form of continuous ROCKi supplementation to the OEM as was the case in rabbits in contrast to other species [32] or the addition of prostaglandin E2 to a culture medium as was described in the human duodenal organoid culture [33].Tese treatments were helpful in reducing dissociationassociated apoptosis in human embryonic stem cell cultures [34,35] and in promoting survival and growth of chicken embryo intestinal organoids [36].Further work is necessary to optimize culture conditions in order to achieve long-term cultivation and expansion of bovine small intestinal organoids, especially duodenal organoids.However, the ability to maintain organoids in vitro and revitalize them at a later time through cryopreservation has important implications for livestock research.For instance, researchers can establish a Biobank of valuable samples, share resources among institutions regardless of their geographical distance, expand organoids for use in multiple independent research projects, or apply their use to various investigations simultaneously, all while limiting the number of animals required for different studies.
Characterization of the jejunal, ileal, colonic, and rectal organoids by means of immunocytochemistry confrmed the presence of a multilineage cellular population including intestinal epithelial stem cells, intestinal epithelial cells, and goblet cells.It also confrmed the presence of cellular polarity, adherens junction formations, and cellular proliferative capacity as previously reported both in vivo and in vitro of bovine and other species [5,17,18].Furthermore, RT-qPCR of jejunal, ileal, colonic, and rectal organoids demonstrated expression of genes which are specifc to diferentiated cell types such as enterocytes (FABP2), enteroendocrine cells (ChrA), Paneth cells (LyzC), and goblet cells (Muc2), as well as a the stem cell marker gene (LGR5) in all the four segments.Tese results suggest that the bovine intestinal organoids generated in this study can serve as an in vitro bovine intestinal model as they retained the regenerative capacity of crypts and possessed structural and physiological similarity to the in vivo intestinal tissue.
Te percentages of cells retaining stemness, displaying active proliferative capacity, and diferentiating into mucus-Journal of Tissue Engineering and Regenerative Medicine producing goblet cells were found to be comparable to or even higher than previously reported observations in both the human and porcine in vivo intestinal as well as in vitro intestinal systems [37,38].Moreover, the distribution of stem cells and actively proliferating cells across the four segments aligned with the patterns observed in the in vivo intestine, where crypt niches and mitotic rates of intestinal stem cells are consistent between the small and large intestines [39].In contrast, the relative abundance of mucussecreting goblet cells between the small and large intestines, as observed in our study, deviated from the typical in vivo trend, where the number of goblet cells generally increases from the proximal to the distal intestine [40,41].Interestingly, a similar trend to our fndings has been documented in a study involving human small and large intestinal organoids [42].While further investigations are warranted to elucidate the precise reasons behind this discrepancy between in vivo and in vitro models, their observation of low ATOH1 expression in large intestinal organoids suggests the potential existence of an alternative pathway, distinct from the well-known Wnt and Notch pathways that regulates goblet cell diferentiation in the large intestine.
Expression of LGR5 and LyzC genes decreased or remained unchanged when organoids were treated with the ODM as compared to being OEM-grown.Tese results indicated that our ODM does not support maintenance of stem cells nor diferentiation of stem cells into Paneth cells, which is somewhat expected as Wnt signaling plays a pivotal role in these functions [1,43].On the other hand, treatment with the ODM promoted the expression of FABP2, ChrA, and Muc2 genes, indicating that ODM-treated organoids are a superior model of the in vivo intestine than OEM-grown organoids due to the exhibition of a greater heterogeneity of cellular populations related to intestinal epithelial cell lineages.
As a limitation, the present study did not evaluate the direct gene expression of the donor tissue; thus, it is not possible to compare and discuss similarities or diferences in genetic properties between the organoids and their originating tissues.However, previous studies reported an overlapping gene expression pattern between bovine intestinal organoids and the in vivo tissue [15,16,44].Tis characteristic has also been reported for other types of organoids such as the human and murine liver [45,46].Te highest level of FABP2 expression was in ileal organoids followed by jejunal and large intestinal organoids in this study, which is in accordance with a previous report that evaluated gene expression in various locations of intestinal tissues collected from Jersey calves [47].Indistinguishable levels of Muc2 gene expression across jejunal, ileal, colonic, and rectal organoids was also in agreement with a previous study using tissues collected from 4-year-old cows [48].Furthermore, a high resemblance of organoids and the in vivo tissue as compared to a cell line was documented by applying a transcriptome analysis on the porcine jejunum and human duodenum [49,50].Our bovine intestinal organoids treated with the ODM ofer an in vitro model that aligns more closely with physiological relevance compared to conventional cell culture methods or organoids grown in the OEM.

Conclusion
Te present study demonstrates that bovine intestinal organoids can be generated from tissues of the duodenum, jejunum, ileum, colon, and rectum using a biopsy sample technique and OEM prepared in our laboratory, flling in a knowledge gap in bovine organoid technology.Maintenance of organoids through serial passages and cryopreservation was feasible in all fve segments, with more consistent and higher success rates observed in large intestinal organoids as compared to small intestinal organoids.Organoids from the jejunum, ileum, colon, and rectum retained structural, cellular, and genetic resemblance to the in vivo intestinal tissue.Tese results suggest that adult bovine intestinal organoids ofer a morphologically and physiologically relevant in vitro system that can be used as a long-term renewable resource for various studies including the investigation of normal intestinal physiology in cattle and host-pathogen interactions of clinically and economically important enteric pathogens with public health signifcance.dilution, and manufacturers of each antibody/probe are listed.Supplementary Table 3: primers used to evaluate the gene expression of bovine intestinal organoids.Gene name, types/markers, and forward and reverse sequences are listed.Supplementary Figure 1: representative images of disintegrating bovine intestinal organoids taken with phasecontrast brightfeld microscopy.Duodenal, jejunal, and ileal organoids derived from some individuals started to disintegrate following routine passaging during early passages.Tese organoids eventually lost their proliferating capacity and were unable to maintain in long term through serial passages.(Supplementary Materials)

Figure 1 :
Figure 1: Development of bovine intestinal organoids.(a) A schematic demonstrating the fow of the technique used to generate organoids from the bovine intestine created with https://www.biorender.com/.(b-e) Gross and microscopic images showing tissue sampling (b), collected tissue samples after washing with the wash medium (c), isolated crypts (d), and a developing organoid 24 hours after seeding in Matrigel (e).

Figure 2 :
Figure 2: Representative images of bovine intestinal organoids taken with phase-contrast brightfeld microscopy.(a) Organoids were generated from the freshly sampled tissue of the duodenum, jejunum, ileum, colon, and rectum of adult cattle.(b) Organoids from all the fve segments were maintained over serial passages, with the highest passage numbers of P6 in the duodenum and >P10 in the jejunum, ileum, colon, and rectum.(c) Organoids from all the fve intestinal segments were successfully resuscitated after cryopreservation for over 8 months.Images were captured between Day 2 and 12 either at P1-3 (a), P11 except for the duodenum which was imaged at P6 (b), or P4-5 (c), respectively.

Figure 3 :Figure 4 :
Figure 3: Immunocytochemical characterization of bovine intestinal organoids.(a) Confocal microscopy images of bovine intestinal organoids demonstrated the formation of basolateral epithelial adherens junctions (E-cadherin, green) and cellular polarity characterized with apical brush border (F-actin, red) and basal nuclei (DAPI, blue) and the presence of mixed cell populations including epithelial cells (EpCAM, yellow), stem cells (SOX9, yellow), actively proliferating cells (EdU, cyan), and mucin-producing goblet cells (SNA, green).(b-d) Te bar graphs show quantifcation of the cells that stained positive for SOX9, EdU, and SNA normalized by the total numbers of nuclei.Tree to ten independent felds of view from two technical and four biological replicates were assessed.Te results are presented as the mean ± standard error of the mean (SEM).

Table 1 :
Composition of the organoid expansion medium (OEM) used to generate and maintain bovine intestinal organoids.

Table 2 :
Summary of development, maintenance, and cryopreservation of organoids.