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. 2020 Apr 17;15(4):e0231423. doi: 10.1371/journal.pone.0231423

Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions

Yoko M Ambrosini 1,2,#, Yejin Park 3,#, Albert E Jergens 4, Woojung Shin 1, Soyoun Min 1, Todd Atherly 4, Dana C Borcherding 2, Jinah Jang 3,5,6, Karin Allenspach 4, Jonathan P Mochel 2,*, Hyun Jung Kim 1,7,*
Editor: Mária A Deli8
PMCID: PMC7164685  PMID: 32302323

Abstract

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.

Introduction

Multiple chronic human disorders, including inflammatory bowel disease (IBD) and colorectal cancer (CRC), have been characterized in canine models based upon the spontaneous clinical analogs of gastrointestinal (GI) disorders [1,2]. For the investigation of human intestinal homeostasis, canine models are especially relevant to humans because their intestinal physiology and diet style have adapted to those of humans during domestication [3]. Due to this similarity, it is not surprising that dogs and humans share similar composition of the gut microbiota with ~60% taxonomic and functional overlap as compared to <20% for mice [4]. Therefore, dogs are considered a more predictable animal model for investigating environmental influences on human GI health and disease compared to conventional murine models [4].

There is currently a limited number of canine-specific primary cell lines to investigate intestinal physiology ex vivo or in vitro. Well-characterized immortalized cell lines including the Madin-Darby canine kidney (MDCK) cells do not accurately model intestinal epithelial interactions in the dog due to their origin from immature kidney cells [5,6]. Recently, isolated primary canine intestinal epithelial cells have been immortalized with a temperature-sensitive mutant of the Simian Virus 40 large tumor antigen (SV40 T-Ag) [7]. Although this cell line can be grown on a monolayer, the SV40 T-Ag may initiate pathways which could provide spurious, non-physiologic findings ex vivo given its tumorigenic cell line origin [8].

We have recently optimized the three-dimensional (3D) in vitro culture conditions of canine primary intestinal organoids and shown that isolated intestinal stem cells differentiate into organoids containing matured intestinal cell lineages within ~8 days of culture [9]. The 3D organoid culture technology not only offers a more physiological platform compared with conventional 2D cell lines [10], but also provides a “personalized” modeling to investigate the effect of environmental stimuli or dietary interventions on intestinal epithelium [11]. Altogether, the establishment of a robust canine organoid protocol allows for comparative biomedical initiatives in humans and dogs to be performed [2].

However, a notable limitation of the 3D intestinal organoid system has been identified. For instance, the 3D organoid body prevents the access to the lumen for studying the interactions with dietary constituents, microorganisms, drugs, or toxins transported through an epithelial layer [12]. While microinjection of a luminal component (e.g., living bacterial cells) into the lumen of an organoid has been feasible, the technique can be challenging due to the heterogeneity in organoid size, invasive injection, and the requirement of techniques and equipment [13]. Thus, cultures of a polarized intestinal cell monolayer are better suited for the standardized measurement of transepithelial permeability and epithelial-luminal interaction due to easier accessibility of the apical surface. Moreover, creating a canine-derived intestinal interface may be further improved by integrating the optimized protocol to the intestinal microphysiological systems [14–17].

In this study, we report an optimized method for generating an intact monolayer of the canine colonoid-derived epithelium. We characterized the formed epithelial monolayer that provides an accessible tissue interface, polarization, lineage-dependent differentiation, tight junction barrier, permeability, and the expression of key efflux pump using various imaging modalities. We envision that our optimized protocol and the robust culture of canine-derived epithelium may enable to develop an advanced in vitro model to demonstrate complex host-gut microbiome crosstalk and pharmacological assessment under various disease milieus.

Results

Recreating a canine colonoid-derived intestinal tissue interface

Canine colonic organoids derived from three independent canine donors were expanded in 3D geometry for up seven days in Matrigel (Fig 1A), allowing a long-term culture and storage of the primary intestinal epithelium [9]. A colonoid-derived monolayer was generated in a nanoporous insert of the Transwell pre-coated with the extracellular matrix (ECM) mix with Matrigel (100 μg/mL) and collagen I (30 μg/mL) by introducing the dissociated colonoid cells (Fig 1B). In terms of the colonoid dissociation, we employed an enzymatic dissociation method [15] to generate single-cell suspension to accomplish a confluent monolayer, which can be maintained for at least 13 days (Fig 1C).

Fig 1. Morphological analysis of the 3D colonoids and the 2D canine colonic monolayer.

Fig 1

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(A) A growth profile of the colonoid isolated from the canine colonic crypt. A small spherical colonoids progressively grows to form fully grown colonoids. Representative phase-contrast micrographs were taken at days 1, 3, and 5. The zoomed-in inset at each day shows the high-power magnification of a colonoid in the white dashed box. (B) A schematic displays the procedure of the formation of an epithelial monolayer derived from 3D canine colonoids. The fully-grown organoids are dissociated into single cells, then seeded into a nanoporous insert to form a monolayer. AP, apical; BL, basolateral. (C) Representative phase-contrast micrographs on day 3 and 13 are provided, respectively. Bars, 200 μm.

Apical microvilli formation in the canine colonic epithelial monolayer

The polarization of the colonic epithelium is critical to establish a biological tissue interface. Microvilli that illustrate the polarized apical membrane of the colonic epithelium were observed on the recreated monolayer using scanning electron microscopy (SEM; Fig 2A and 2B) and transmission electron microscopy (TEM; Fig 2C and 2D). A variation in microvilli frequency was observed in our dog colonoid-derived monolayer, which was also noted in other colonoid-derived studies [18,19]. The number of microvilli assessed by the SEM imaging was variable in the range from 9 to 18 microvilli/μm2, which was similar to the reports of human intestinal epithelial cell culture performed in vitro [20]. Glycocalyx, which provides a physical glycosylated barrier on the epithelial cells [21], was also well generated at the surface of the microvilli (Fig 2D).

Fig 2. Electron microscopic characterization of the apical surface and the tissue interface of the canine colonoid-derived monolayer.

Fig 2

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(A) A low magnification SEM image of the microvilli on the apical cell surface. (B) A high-power magnification of the microvilli from A indicated by a white dashed box. Bars, 5 μm. (C) A TEM image of the microvilli on the cell monolayer. MV, microvilli. Bar, 500 nm. (D) A high-power TEM image that shows the microvilli (MV) and the surrounding glycocalyx (GLX). Bar, 200 nm.

Lineage-dependent characterization of the differentiated canine colonic epithelial monolayer

RNA in situ hybridization (RNA-ISH), immunofluorescence (IF), and electron microscopic imaging were used to show the differentiated cell lineages in the canine colonoid-derived monolayer. The leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a seminal marker for adult intestinal stem cells [22], was detected sporadically in the 2D monolayer cultured for 14 days (Fig 3A). Also, the canine colonic epithelium retained a population of proliferative cells, as visualized by Ki67-positive signals for up to 2 weeks (Fig 3B). The differentiated absorptive enterocytes were visualized by the staining with intestinal alkaline phosphatase (ALPI) (Fig 3C) [23]. The enteroendocrine cells were highlighted using Neurogenin 3 (Neurog3; Fig 3D) and Chromogranin A markers (CgA; Fig 3E), respectively [24]. In the canine epithelial monolayer, we analyzed the appearance of each cell type based on the imaging results, where the Lgr5+ stem cells, Ki67+ proliferating cells, ALPI+ differentiated intestinal epithelium, Neurog3+ and CgA+ enteroendocrine cells were populated as 7.6±0.1, 38.4±2.4, 60.1±0.9, 41.2±10.3%, and 47.8±2.7%, respectively (Fig 3F).

Fig 3. The cell type-specific characterization of the canine colonoid-derived epithelial monolayer.

Fig 3

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The canine colonoid-derived monolayer on Day 13 was used to visualize the markers highlighting the cell lineages, proliferation, and mucus production. The population of stem cells (A; Lgr5+, Yellow), proliferative cells (B; Ki67, Red), absorptive enterocytes (C; ALPI, Magenta), and enteroendocrine cells (D; Neurog3, Red and E; CgA, Red) were visualized by using RNA in situ hybridization (for A, C, and D) or IF staining (for B and E). As a counterstaining, E-cadherin (Cyan for A, C, and D), F-actin (Green for B and Cyan for E), or nuclei (Grey for A, B, C, D, and E) were displayed. Bars, 20 μm. (F) Quantification of the population of the cells that show positive signals to the target markers normalized by the total numbers of nuclei. Three independent fields of view from two or more independent biological replicates were used. In each biological replicate, 2 technical replicates were performed. Error bars indicate SEM.

To investigate the presence of physiological mucus production in the monolayer, live-cell staining with Wheat Germ Agglutinin (WGA) was performed [17,25,26]. We found that the WGA-positive signals were detected across the entire monolayer, suggesting that the epithelial apical surface was covered by mucus-like molecules such as N-acetyl-D-glucosamine (Fig 4A). We also identified the mucin granule-containing goblet cells using TEM (Fig 4B, “MG”), where the goblet cell orifices (Fig 4D, “GO”) and fenestrated membranes (Fig 4D, “FM”) extending deep into the goblet cell were also confirmed using SEM, as shown in previous studies [27,28], demonstrating that the goblet cells were present in the canine colonoid-derived monolayer.

Fig 4. Visualization of mucus production and goblet cells in the canine colonoid-derived monolayer.

Fig 4

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The mucus production (A; WGA) was visualized by live-cell imaging at the apical surface of the monolayer. Bar, 20 μm. (B) A representative TEM image shows the goblet cell with multiple mucin granules (MG) and mitochondria (M). Bar, 1 μm. (C) A low magnification SEM image of a goblet cell on the apical cell surface of the canine colonoid-derived monolayer. Bar, 5 μm. (D) A high magnification of a goblet cell orifice (GO), a fenestrated membrane (FM) extending deep into the cell, and microvilli (MV) from C indicated by a white dashed box. Bar, 1 μm.

In addition, we confirmed that the P-glycoprotein (P-gp) efflux transporters were diffusely expressed on the apical surface of the canine colonoid-derived monolayer (Fig 5A and 5B), which is consistent with the localization of the P-gp transporters in the canine colonic tissue [29]. Importantly, the IF assessment revealed that the polarized expression of P-gp was significantly (P < 0.0001) increased on Day 13 compared to the images acquired on Day 3 on the nanoporous insert, suggesting that the maturity of the colonoid-derived epithelial monolayer was achieved (Fig 5C).

Fig 5. Expression of the P-gp in the canine colonoid-derived monolayer.

Fig 5

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The expression of P-gp was visually characterized by IF staining. Angled (upper) and cross-sectional side views (lower) show the localization the P-gp proteins (Yellow) on the polarized colonoid-derived monolayer at days 3 (A) and 13 (B), respectively. Nuclei, Cyan. Dashed lines pinpoint the location of the basement membrane in the nanoporous insert. Bars, 50 μm. (C) Quantification of the P-gp expression at days 3 and 13, respectively. Total 10 randomly chosen fields of view were used to detect P-gp expression levels among 4 biological replicates. In each biological replicate, we performed 2 technical replicates. a.u., arbitrary unit. Error bars indicate SEM.*P<0.0001.

Assessment of the canine intestinal barrier integrity

The formation of tight-junction proteins was confirmed by IF staining for zonula occludens 1 (ZO-1) (Fig 6A) and E-cadherin (E-cad) expression (Fig 6B), where no significant difference of the expression at Day 3 and 13 was observed in both ZO-1 and E-cad (S2 Fig). After 4 days of cultures, the confluent colonoid monolayer showed stable transepithelial electrical resistance (TEER) values of approximately 1,000 Ω∙cm2 (Fig 6C). We observed that the TEER value was stably maintained for up to 14 days when the culture medium was replenished every other day for all 3 independent lines of canine colonoid-derived epithelium (S3 Fig).

Fig 6. Characterization of the junctional proteins and the barrier function in the canine colonoid-derived epithelial monolayer.

Fig 6

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Visualization of the spatial localization of the ZO-1 (A; Magenta) and E-cadherin (B; Cyan) on the same location of a canine colonoid-derived monolayer. Nuclei, Grey. Bar, 50 μm. (C) The profile of the epithelial barrier function was monitored by measuring TEER. The effect of culture medium on TEER was demonstrated by applying the regular proliferation medium in both the apical and basolateral side of the Transwell (Control, open circle) versus the differentiation/proliferation medium in the apical/basolateral compartments, respectively (Diff; closed circle). Both groups were cultured with the proliferation medium by Day 4 (a dashed line), then different culture media were applied (Diff vs. Control) for additional 4 days. Two biological replicates with 4 technical replicates were used in each condition. *P<0.01. (D) A TEM image of the intercellular junctional complex in the canine colonoid-derived monolayer and a zoom-in (E) that shows a high-power magnification of the white dashed area in D. MV, microvilli; M, mitochondria; and D, desmosome. Bars, 500 nm. (F) The profile of TEER (open circle) and corresponding apparent permeability (Papp) of fluorescein (closed square) on the days of 2, 3, 5, and 6 of the cultures. Each data point was prepared with 2 biological and 4 technical replicates. Error bars indicate SEM.

Next, we evaluated the effect of the complete medium with or without Wnt proteins on the growth of canine colonoid-derived monolayer to verify the effect of differentiated culture condition on the epithelial barrier function. Briefly, the overall profile of TEER cultured in both the differentiation (i.e., the Wnt-free and Wnt-containing medium in the apical and basolateral compartment, respectively; Fig 6C, “Diff”) and proliferation medium (i.e., Wnt-containing medium to both compartments; Fig 6C, “Control”) showed a similar decline as a function of time. However, the monolayer conditioned under the differentiation medium showed a temporal maintenance of the TEER for ~2 days compared to the control (P < 0.01). The effect of different culture medium on the TEER values became negligible over time by Day 7 (Fig 6C). This observation is consistent with the previous findings from our group where low Wnt3a-containing medium (i.e., differentiation medium) was not necessary for the development of mature canine tight junctions [9]. The TEM images revealed the presence of intercellular junctions as well as desmosomes at Day 13 (Fig 6D and 6E). Corresponding apparent paracellular permeability (Papp) to fluorescein sodium salt (Mw, 376.27 Da) was measured, and an inverted relationship of TEER and Papp values was observed (Fig 6F). Specifically, as TEER values significantly increased from Day 2 to Day 6 (P <0.0001), corresponding Papp values significantly decreased (P <0.0001), supporting that the TEER value may be used to predict the appropriate point to perform epithelial-luminal interactions.

Discussion

In this study, we report for the first time the development of an optimized method for the generation of an intact canine colonoid-derived monolayer from canine 3D colonoids. The enzymatic dissociation method can be applied to canine organoids as performed in other species [15,30] to generate single-cell suspension to accomplish a confluent monolayer. The multi-modal imaging techniques employed in this study confirmed the creation and stable maintenance of the 2D canine intestinal epithelial monolayer on a nanoporous insert up to two weeks with a physiological expression of structural tight-junctions and marker proteins.

Findings from TEM and SEM micrographs demonstrated the formation of a physiological brush border interface and the presence of glycocalyx on the microvilli, which is the characteristic of terminally differentiated canine intestinal epithelium [31]. We confirmed that the canine epithelium cultured on a nanoporous insert grew into multiple lineages of the differentiated intestinal epithelium including absorptive enterocytes, goblet cells, and enteroendocrine cells. Furthermore, our IF imaging data confirmed that P-gp efflux proteins were apically expressed similarly to canine colonic tissue in vivo [29], which disseminates a follow-up study in terms of the functional assessment of P-gp efflux pumps for pharmacological applications.

We confirmed that stable TEER values could be established by Day 4 of the monolayer culture, which is similar to the previous study using canine [8] or human cell lines [32]. The TEER values increased as a concurrent decrease in the apparent permeability of a paracellular marker similar to the previous study [8], suggesting that the ideal timeline to perform the barrier-associated experiments can be estimated once stable TEER values are achieved (here, after Day 4). In human intestinal organoid culture, Wnt protein-rich medium produced largely undifferentiated progenitors due to the central role that Wnt signaling plays in the maintenance of an undifferentiated crypt progenitor state [33,34]. We demonstrated minimal effect of low Wnt3a-containing medium (i.e., differentiation medium) for the development and maintenance of mature canine tight junctions as reported previously [9].

Moreover, we showed that the canine colonoid on the Transwell contain a stable population of the intestinal stem cells as well as other differentiated cells present in the intestinal tissue of origin [9]. Using RNA-ISH imaging technology, we were able to investigate the percentage of cells expressing multi-lineage cell differentiation RNA markers, including the Lgr5+ stem cells [9,35], ALPI+ differentiated intestinal epithelium [9,28], Neurog3+ enteroendocrine cells [36,37], which were all similar to what have been previously reported in human and dog in vitro intestinal systems. It is noted that the Ki67+ cells are not the population of lineage-dependent cells; however, we included in the same chart (Fig 3F) to provide a quantitative information. It is also critical to confirm the production of intestinal mucus and a glycocalyx on the engineered epithelial monolayer [19]. We demonstrated the presence of mucus with WGA staining and the presence of glycocalyx using TEM imaging. The presence of goblet cells was also demonstrated using TEM and SEM by detecting multiple mucin granules (MG) (Fig 4B) and goblet cell orifices (GO) as well as a fenestrated membrane (FM) (Fig 4D) as shown in previous studies [27,28].

A key advantage of the recreation of a 2D mucosal tissue interface is that this culture format will allow the access to the apical side of the epithelium for investigating the nutrient and drug absorption, host-microbe crosstalk, or drug metabolism and toxicity testing. The 2D mucosal tissue interface using primary 3D intestinal organoids could allow modeling of intestinal physiology ex vivo or in vitro compared to currently available canine-specific immortalized cell lines [5–7]. The measurement of the epithelial barrier function (e.g., TEER) is convenient when investigating the physiological responses of epithelial cells following exposure to toxins, therapeutic drugs, or nutrients [38,39]. On the contrary, the conventional 3D organoid culture method in Matrigel considerably prevents the access to the apical side of the epithelium [12], which hinders the aforementioned physiological reactions such as host-microbe crosstalk. Moreover, the Matrigel for the conventional culture of 3D organoid interferes with the compound exposure to the cells, and therefore, holds limited scalability for high-throughput pharmacological applications.

The physiological multi-lineage differentiation of a colonoid-derived monolayer suggests that patient-derived organoids on a 2D Transwell platform could potentially be used for translational researches targeting Precision Medicine purposes because the individual canine organoid lines obtained from various dog species can be established and utilized for pharmaceutical studies [10]. Comparative studies using various pharmacological agents or clinically approved drug compounds will bring translational value to bridge between experimental canine models to in vitro human models, then ultimately toward human in vivo. For instance, the Caco-2 human intestinal epithelial cell line, which has been predominantly used in the pharmaceutical industry [40], can be a good comparative model to interrogate the potential of canine organoid-derived epithelium for validating physiological and toxicological responses of the canine epithelium.

Another potential application that we envision is to leverage this proof-of-principle study of canine intestinal organoids towards the possible application of an advanced gut-on-a-chip microphysiological platform [14,41]. Over the past decade, numerous human organ- or tissue-on-a-chip models have been suggested and evaluated, whereas no significant animal-derived models have been developed. As for GI models, a couple of compelling gut-on-a-chip models have showcased that modeling physiologically relevant host-microbiome interactions is possible [16,19,41–44], as well as pathomimetic inflammatory disease modeling [16,17,45], or the co-culture of anaerobic gut bacteria in an anoxic-oxic interface [46]. Based on this newly established culture protocol, applications of the canine organoid-derived epithelial cells may help to develop a novel “Canine gut-on-a-chip” platform to demonstrate host-microbiome ecosystem and validate drug efficacy and toxicity.

One primary concern of the human biopharmaceutical industry lies in the fact that most preclinical studies fail to accurately predict the efficacy and safety of drug candidates in human clinical trials [47]. This concern is most likely due to the large translational gap between highly inbred or genetically-modified laboratory animals and humans who exhibit genetic variability influenced by environmental factors [1]. Therefore, an interdisciplinary collaboration between basic scientists, engineers, and clinician-scientists using companion animals with naturally occurring diseases is critical to bridge this gap and accelerate drug development [48]. Dogs are receiving more attention as a relevant translational in vivo model compared to rodents because they share similar genetic and environmental variations seen in humans [1]. For example, human intestinal disorders such as inflammatory bowel disease, ulcerative colitis, and colorectal cancer have been well characterized in clinical analogs of dogs [49–51]. Moreover, dogs and humans hold a strikingly similar composition of the gut microbiota; therefore, dogs are likely a more predictable model for studying microbial influences modulating human intestinal homeostasis [4]. The canine colonoid-derived monolayer that we report herein provides an intestinal tissue interface with multi-lineage cell differentiation, which is known to be expressed in primary tissues. This tool can also help reduce the number of dogs required to test intestinal physiology in health and disease, by the Three Rs Principle: Reduce, Replace, and Refine [52].

Although dogs are excellent animal models to study human diseases [1], dog studies are often limited by the number of commercially available reagents targeting major proteins shown to be relevant in mice [53,54]. The RNA-ISH technology provides an in situ analysis of biomarkers within the histopathological context of biological samples as they target the mRNA of select proteins [55]. RNA-ISH is a suitable alternative to IF in those cases where the detection of proteins lacks sensitivity or cellular resolution [55,56]. The customized probes for RNA-ISH can be engineered based on any RNA sequences, which allows investigators to overcome the lack of canine-specific reagents for the identification of intestinal stem cells and their lineage cells in dogs [9,55]. However, as RNA-ISH only detects mRNA expression, it provides no spatial information on actual protein expression or matured protein productive function in the cell. Regardless of the location of the positive signal, a positive signal is an indicative of the presence of the target gene(s) in that particular cell. This RNA-ISH technology has been successfully applied in dog organoids by our group [4] and similar findings (i.e., positive signals seem to be expressed in the nucleus) can be found in other studies [9,11] as well as our positive control provided in S1 Fig.

Stunted microvilli were observed in our system which could reflect the fact that colonic intestinal cells may not require longer microvilli due to minimal nutrient absorption in the colon [57]. Possibly, it could be due to the culture condition that is not completely adequate to promote longer microvilli [28]. As described before, Wnt-enriched medium produced largely undifferentiated progenitors comprising organoids in human intestinal organoid culture [33]. Our previous [9] and current work demonstrated that canine intestinal organoids are indeed capable of differentiating into functional epithelial cells even under Wnt-enriched condition; however, the effect of low Wnt-containing medium (i.e., differentiation medium) particularly on microvilli length would be beneficial to better understand the physiological demonstration and functions of the microvilli in the future study.

Our study demonstrates the methods to create the accessible apical surface of the intestinal epithelium generated from canine colonoids. In the future study, we will investigate epithelial-luminal interactions perturbed by microbial, metabolomic, and pharmacological stimulations that mediate GI health and disease. Moreover, the method developed herein can be applied to other segments of organoids (i.e., enteroids) as well as the organoids obtained from both diseased and other healthy dogs to enable segmental investigation of epithelial-luminal interactions.

Materials and methods

Creation of a biopsy-derived canine colonoid line

Intestinal biopsies were obtained via colonoscopy for intestinal stem cell isolation from healthy research colony dogs at the Iowa State University College of Veterinary Medicine. All animal procedures in this study were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC protocol: 9-17-8605-K). Colonic crypts containing primary adult intestinal stem cells were isolated and cultured, as previously described [9]. Briefly, endoscopic biopsy samples from colonoscopies were cut into small pieces, and intestinal crypt cells were released by incubating the samples with a complete chelating solution and EDTA (30 mM; Alfa Aesar) at 4°C for 60 min. After the crypt release, the crypt-containing pellet was suspended and seeded in 30 μL per well of Matrigel (Corning) and 500 μL per well of complete medium supplemented with intestinal stem cell (ISC) supporting factors including 10 μM rho-associated kinase inhibitor (ROCKi) Y-27632 (StemGent) and 2.5 μM glycogen synthase kinase 3β (GSK3β) inhibitor (StemGent) before the plate was incubated at 37°C [9]. The culture medium was changed to complete medium without any supplementation after 2 days of crypt isolation.

Colonoid culture

A basal medium containing 10 mM HEPES (Gibco), 1× GlutaMAX (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin in Advanced DMEM/F12 (Gibco) was first prepared. Conditioned medium was prepared by culturing Wnt3a-producing L cells (ATCC, CRL 2647), R-spondin1 (Rspo1) cells (Trevigen), and Noggin secreting cells (Baylor’s College of Medicine), as previously described [11]. In the complete medium, the volume ratio of basal and each conditioned medium is defined at 20/50/20/10% (v/v) and murine recombinant epidermal growth factor (EGF) (50 ng/mL; Peprotech), SB202190 (30 μM; Sigma Aldrich), A-8301 (500 nM; Sigma Aldrich), Gastrin (10 nM; Sigma Aldrich), N-acetylcysteine (1 mM; MP Biomedicals), nicotinamide (10 mM; Sigma Aldrich), N2 (1×; Gibco), and B27 (1×; Gibco) were also supplemented. The complete medium was changed every other day, and organoids were passaged once a week by mechanically breaking down the organoids, spinning down the fragmented organoids (100× g, 4°C, 5 min), resuspending centrifuged organoids with fresh Matrigel on ice, and then plating them in each well of a 24 well plate (Corning).

Culture of a colonoid-derived monolayer

The 3D colonoids were harvested from Matrigel after 7 days of culture by addition of EDTA solution (0.5 mM; Alfa Aesar) on ice, then transferred in 15 mL tubes and centrifuged (100× g, 4°C, 5 min). The organoid pellet was incubated in 1 mL TrypLE Express (Gibco) for 10 min while shaking at 37°C in a water bath. The centrifuged (100×g, 4°C, 5 min) organoid fragments were resuspended in complete medium [11] and further dissociated by repeated pipetting and subsequent filtering of the cell suspension through a cell strainer (cut-off size, 40 μm, Corning) to obtain a single-cell suspension. Transwell inserts (0.4 μm pores, Corning) were pre-coated with Matrigel (100 μg/mL; Corning) and collagen I (30 μg/mL; Fisher Scientific) in PBS or basal medium at 37°C for 1 h. Dissociated cells were counted manually using a cell counter (Hemocytometer; Hausser Scientific) and seeded at 106 cells/mL in pre-coated Transwell inserts. After 3 days of incubation in a humidified incubator at 37°C with 5% CO2, the cell monolayer was established. The morphology of a cell monolayer was intermittently monitored for up to two weeks by phase-contrast microscopy (Axiovert 40CFL, Zeiss).

Evaluation of the epithelial barrier integrity

The barrier function of the intestinal epithelial monolayer was measured by monitoring TEER. The TEER value was measured by using Ag/AgCl electrodes connected to an Ohm meter (Millicell ERS-2; Millipore). Normalization of TEER was performed following the equation as, TEER = (Ωt−Ωblank) × A, where Ωt is the resistance (in Ohms) at the measured time point since the start of the culture; Ωblank is the resistance of the blank, and A is the surface area cultured on the nanoporous insert in cm2. To investigate the reproducibility in TEER values from various canine colonoid-derived monolayers, TEER measurement was performed in 2 biological replicates with 4 technical replicates using 3 different canine colonoid lines (S3 Fig). To assess the effect of culture conditions on TEER, the colonoid-derived monolayer was cultured with proliferation medium (complete medium with Wnt3a proteins) or differentiation medium (a complete medium without Wnt3a) after forming a monolayer which was at Day 4. This study was performed in 2 biological replicates with 4 technical replicates in each condition (i.e., Diff vs. Control). The medium in the Transwell insert was changed to either differentiation medium or proliferation medium while the bottom wells were filled with proliferation medium.

To assess intestinal barrier permeability, fluorescein sodium salt (Mw, 376.27 Da; 0.05 μg/mL) was used as a paracellular marker. The concentration of fluorescein that transported through the cell monolayer (from apical to basolateral) was measured by SpectraMax microplate reader (Molecular Devices). The apparent permeability (Papp) was calculated using the following equation: Papp = (dQ/dt)/(C0 × A), where dQ/dt (μg/sec) is the steady-state flux, C0 (μg/mL) is the initial concentration of the fluorescein in the apical chamber, and A (cm2) is the surface area cultured on the nanoporous insert. This experiment was performed with 2 biological and 4 technical replicates.

Immunofluorescence imaging

For IF microscopic analysis, a confluent cell monolayer grown on a nanoporous insert was fixed with 4% (w/v) paraformaldehyde (Electron Microscopy Science) for 15 min at room temperature. Samples were then permeabilized with 0.3% (v/v) Triton X-100 (Sigma) and blocked with 2% (w/v) bovine serum albumin (BSA; Sigma) followed by PBS (Ca2+and Ma2+free; Gibco) washing. The monolayer was incubated at room temperature for 1 h with primary antibodies against ZO-1 (Invitrogen), P-gp (Thermo Fisher Scientific), CgA (Abcam), and Ki67 (Abcam) diluted in 2% (w/v) BSA in PBS. Alexa Fluor 488 conjugated E-cadherin (BD Biosciences) was applied in a same procedure. Secondary antibodies of Alexa Fluor 555-conjugated goat polyclonal anti-rabbit IgG (Abcam) for ZO-1, P-gp, CgA, and Ki67 diluted in 2% (w/v) BSA in PBS were applied under light protected conditions at room temperature for 1 h. For the counterstaining, samples were incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 μg/mL; Fisher Scientific) and Alexa Fluor 647-conjugated phalloidin (7.5 units; Thermo Fisher) for nuclei and F-actin visualization, respectively. To detect the mucus production on the monolayer, samples were directly stained with Alexa Fluor 488-conjugated WGA (5.0 μg/mL; Thermo Fisher). The monolayer was imaged using a differential interference contrast (DIC) or laser-scanning confocal microscopy (DMi8; Leica). Acquired images were processed using LAS X (Leica) or ImageJ v1.52q [58]. The percentage of cell numbers (Ki67 and CgA) or fluorescence intensity (P-gp, ZO-1, and E-cad) was assessed using ImageJ to the randomly selected images that show representative characteristics. The number of cells that show positive signals was manually counted (ImageJ), then the number was normalized by the total number of nuclei to calculate the % population. For this quantification, 3 independent fields of view from 4 independent biological replicates were used, while at least two technical replicates were performed (Fig 3F). For the quantification of the P-gp expression, total 10 randomly chosen fields of view to detect P-gp expression levels among 4 biological replicates, while at least two technical replicates were performed (Fig 5C). For the quantitative assessment of ZO-1 and E-cadherin, total 10 and 6 randomly chosen fields of view for ZO-1 and E-cadherin, respectively, to quantify the relative intensity of fluorescence among 4 biological replicates of IF staining experiment. We also applied two technical replicates to the individual biological replicate. (S2 Fig).

In situ hybridization of mRNA

We employed RNA-ISH using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostic, Newark, CA) [55] on a canine colonoid-derived monolayer to characterize the multi-lineage differentiation. In brief, a colonoid-derived monolayer was fixed and underwent dehydration/hydration, permeabilization, and protease treatment. Samples were hybridized in the ACD HybEZ II Hybridization System (110v) oven at 40°C while placed in light protected humidified trey as instructed by the manufacture [55]. The samples were then stained for specific oligonucleotide probes for visualizing intestinal stem cells (CL-Lgr5-C2; Advanced Cell Diagnostic), differentiated intestinal epithelial cells (Cl-ALPI; Advanced Cell Diagnostic), and secretory enteroendocrine cells (Cl-NEUROG3-C3; Advanced Cell Diagnostic), respectively. Next, amplification and visualization using Opal 520 (FP1487001KT), Opal 570 (FP1488001KT), and Opal 650 (FP1496001KT) were performed. Sections were imaged using a confocal microscope (DMi8; Leica). Acquired images were processed using LAS X (Leica) or ImageJ. The number of cells staining positive for mRNA detection for each RNAscope probe was manually counted at random positions. Specifically, the number of cells staining positive was manually counted, then normalized by the total number of nuclei. Quantification of the positive cells to individual RNA markers was performed with 3 independent fields of view from 2 independent biological replicates (Fig 3F). Probes against RNA Polymerase II Subunit A (POLR2A) and Ubiquitin C (UBC) were applied and the same amplification and visualization steps were performed to prepare the positive control (S1 Fig).

Transmission and scanning electron microscopy

After 13 days of culture, the culture medium was gently removed from the apical and basal chambers of the Transwell, and cells were fixed with 2% (v/v) glutaraldehyde (Electron Microscopy Sciences) in 0.1 M cacodylate buffer (Electron Microscopy Sciences) for 1 hr at room temperature, and washed in 0.1 M cacodylate buffer. Samples were then fixed and stained with 1% (v/v) osmium tetroxide (Electron Microscopy Sciences) and 1% (v/v) ferrocyanide in cacodylate buffer, and then stained with 2% (v/v) uranyl acetate for a negative contrast. Samples were finally dehydrated through serial dehydration in ethanol from 50% to 100% (v/v) and then infiltrated with resin (Electron Microscopy Sciences) to be polymerized at 60°C and sectioned for TEM. Ultrathin (50–100 nm) sections were cut by a microtome with a diamond blade, then collected on copper grids and observed under the Transmission Electron Microscope (FEI Tecnai) using an accelerating voltage of 80 kV. SEM samples were fixed in 2.5% (v/v) glutaraldehyde (Electron Microscopy Sciences), treated with 1% (v/v) osmium tetroxide (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 30 min at room temperature. Samples were dehydrated through serial dehydration in ethanol from 50% to 100%, and hexamethyldisilazane (HDMS) method. Samples were coated with a thin (12 nm) layer of Pt/Pd using a sputter coater (Cressington 208 Benchtop Sputter) prior to imaging using an SEM (Zeiss Supra 40V SEM) with an accelerating voltage of 5 kV. The average frequencies of microvilli in less frequent and frequent areas were performed at 4 random independent positions from 3 different SEM images.

Statistical analysis

All results are expressed as mean±standard error (SEM). Shapiro-Wilk tests were used to assess the normality of the data. Mann–Whitney U test (for non-parametric data) or student’s t-tests (for parametric data) were used to compare the expression levels of proteins between two different time points (Day 3 vs. Day 13), TEER and Papp values on different culture time points (Day 2 vs. Day 6), or TEER values in different culture conditions (proliferation medium vs. differentiation medium) at each culture time point. All statistical analyses were performed using Prism 8.2.1 (GraphPad Software, San Diego, CA). P values < 0.05 were considered statistically significant.

Supporting information

S1 Fig. Verification of the RNA in situ hybridization on the canine colonoid-derived epithelial monolayer.

A 3-Plex Positive Control Probe (Advanced Cell Diagnostics) was applied to the canine monolayer cultured for 13 days to confirm the functionality of the kit applied. A low (RNA Polymerase II Subunit A (POLR2A), Opal 650; S1A) and a high expressor RNA (Ubiquitin C (UBC), Opal 520; S1B) confirmed the functionality of the probes applied in the canine epithelial monolayer. An overlaid image is displayed in S1C. Nuclei, blue. Bars, 50 μm.

(TIF)

Click here for additional data file. (1.2MB, tif)
S2 Fig. Expression of the epithelial junctional proteins in the canine colonoid-derived epithelial monolayer.

Quantification of the expression level of ZO-1 and E-cadherin at days 3 and 13 was performed using total 10 and 6 randomly chosen fields of view for ZO-1 and E-cadherin, respectively, among 4 biological replicates of IF staining experiment. We also applied two technical replicates to individual biological replicates. a.u., arbitrary unit. NS, not significant.

(TIF)

Click here for additional data file. (116.8KB, tif)
S3 Fig. Reproducibility of the barrier function of colonoid-derived epithelial monolayers derived from three different canine colonoid lines.

Three independent lines of canine colonoids show similar profile of epithelial barrier function when those three lines were used to form a monolayer on a nanoporous insert. The result was produced with 2 biological replicates, where each biological replicate was performed with 4 technical replicates. Error bars indicate SEM.

(TIF)

Click here for additional data file. (314KB, tif)

Acknowledgments

We thank Mrs. Michelle A. Mikesh (Center for Biomedical Research Support Microscopy & Imaging Core Facility, UT Austin) and Dr. Sonali Jog (Advanced Cell Diagnostics) for their technical support.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported in part by the Burroughs Wellcome Fund Collaborative Research Travel Grant (BWF 1019990.01 to Y.M.A.), the American College of Veterinary Internal Medicine Advance Research Fellowships (K.A. and Y.M.A.), the Ministry of Science and ICT Korea under the ICT Consilience Creative program supervised by the Institute for Information & communications Technology Planning & Evaluation (IITP) (IITP-2019-2011-1-00783 to Y.P. and J.J.), Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2015R1A6A3A04059015 to Y.P. and J.J.), Iowa State University (Faculty startups, College of Veterinary Medicine Seed Grant and Miller Award), Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science and ICT (2018M3A9H3025030 to H.J.K.), Technology Impact Award of the Cancer Research Institute (UTA18-000889 to H.J.K.), the National Cancer Institute of the National Institutes of Health, IMAT program (R21CA236690 to H.J.K.), the Leona M. & Harry B. Helmsley Charitable Trust (Grant #1912-03604 to H.J.K.), and F99/K00 Predoctoral to Postdoctoral Transition Award (F99CA245801 to W.S.), and Asan Foundation Biomedical Science Scholarship (W.S.). 3D Health Solutions provided support in the form of equity interest for J.P.M., A.E.J., K.A., and H.J.K. and in the form of a salary for T.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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Decision Letter 0

Mária A Deli

23 Jan 2020

PONE-D-20-00243

Recreation of an Accessible Interface of the Biopsy-Derived Canine Intestinal Organoids to Study Epithelial-Luminal Interactions

PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #2: Yes

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Reviewer #1: Ambrosini et al present a method to generate epithelial monolayers from canine intestinal organoids. Although this is the first study of this kind generating canine intestinal monolayers, the methods used to generate these monolayers are not novel, asides from slight variations in the dissociation and plating protocols/media, having been demonstrated by multiple groups in both mouse and human systems. They suggest that these monolayers form epithelial cell layers with multiple differentiated cell types, including enteroendocrine cells, intestinal stem cells and polarised enterocytes. Although some of the data supports this conclusion, such as the TEM images of polarised cells and the results from the TEER experiments, there is a lack of data to show fully that these cells are properly differentiated into functioning intestinal epithelium. Some of the markers used are not appropriate and it is not clear from the select number of TEM images presented whether goblet cells are present and whether there are true microvilli on the apical surface of these cells as has been observed in human and murine organoid culture. Furthermore, it is not clear in the manuscript how many canine lines were used in these experiments. The authors suggest in their discussion that this protocol “could potentially be used for translational Precision Medicine purposes because the individual canine organoid lines obtained from various dog species can be established and utilized for pharmaceutical studies”. However, it is not clear that multiple organoid lines were used in this study, limiting the potential applicability for the future uses of the system the authors suggest. Overall, although I agree with the authors that canine organoids would provide a useful system for various areas of research, I don’t believe that the data presented here is enough to demonstrate successful monolayer development.

Major comments

1. In my opinion, the authors do not convincingly show that there are multiple fully differentiated cell types present in their canine epithelial model system. % positive cells presented in figure 2F is not showing distinct cell types because if it was the total % would equal 100, whereas the number shown is much greater than this. One reason may be the assumption that differentiated enteroendocrine cells can be identified by expression of Neurog3, whereas Neurog3 is expressed in progenitor cells, which become mature enteroendocrine cells but also other cell types (see Gehart et al., 2019. Cell: 176(5)). To identify mature enteroendocrine cells it is more common to use markers such as ChgA and Reg4 to define mature cells of this subset. Although the limitations of using Neurog3 are discussed by the authors in their discussion, it is not clear why the authors not used one of these more common enteroendocrine markers? The authors have used transmission electron microscopy to nicely show the presence of tight junctions between epithelial cells in Figure 3, therefore if there are fully differentiated epithelial cell subsets such as goblet cells and enteroendocrine cells these should be identifiable based on distinct morphology in comparison to absorptive enterocytes by TEM (see Figure S1, Forbester et al., 2018. PNAS: 115(40)). As the authors point out, the availability of canine-specific antibodies is a limitation of this study, however TEM would allow them to convincingly show varying morphology between different cell types, without requiring antibodies. The reliance on the results from the FISH experiments, which are difficult to interpret, is not convincing enough data. Therefore, the statement in the discussion: “Canine epithelium cultured on a Transwell insert differentiated into multiple lineages of the differentiated intestinal epithelium (e.g., intestinal stem cells, absorptive enterocytes, and enteroendocrine cells)” is not validated by enough convincing data within the manuscript.

2. The authors imply that one advance of their study is that they use enzymatic rather than mechanical disruption to disrupt the organoid ultrastructure to single cells. This is not a novel method for generating monolayers, and has been used to dissociate and grow monolayers in both and mouse and human intestinal organoid systems (see Altay et al., 2019. Scientific reports; 9(10140) and Thorne et al., 2018. Developmental Cell; 44(5)). This needs to be clarified in text, because at the moment in my opinion the novelty is overstated. However, in mouse and human systems extensive mechanical disruption is needed in conjunction to TrypLE treatment to ensure dissociation to single cells. Can the authors explain why dissociation of canine intestinal organoids is much easier in comparison to mouse and human intestinal organoids?

3. The microvilli in Figure 3A and Figure 4E look disrupted, or more similar to the structures seen on the surface of M-cells? Can the authors explain why this is? Why is only a single ‘normal length’ villus shown in Figure 3C? See Llanos-Chea et al., 2019. J Pediatr Gastroenterol Nutr; 68(4) for sample microvilli on human intestinal epithelial organoids.

Minor comments

1. None of the figures specify number of replicates/number of canine intestinal organoid lines used. Are these figures representative for lines from multiple canine donors?

2. Description in figure legend for Figure 4D is confusing, needs to be clearer what the control and the experimental samples are, the sentence doesn’t make sense

3. Figure 2F – not clear what N=3 is, 3 fields of view of the same sample; 3 replicate monolayers and staining experiments from the same canine organoid line; 3 replicate monolayers and staining experiments from different canine organoid lines?

4. Figure 3F- As above what is N equivalent to? Multiple experiments, multiple replicates, multiple organoid lines?

5. Figure 4C, D & G – as above, what does N represent in terms of replicates?

Reviewer #2: In this manuscript, Ambrosini and colleagues describe the culture of canine cells derived from 3D canine colonic organoids as monolayers. Various measures of cellular differentiation and monolayer integrity are described. The study is meant as follow up of previously published work (ref 9 in this manuscript) and in part overlapping statements in introduction and discussion are used to justify the need for development of canine culture models. Also TrypLE express is commonly used for dissociation of colonic 3d and 2d intestinal cultures and cited in multiple papers (Thorne CA, Chen IW, Sanman LE, Cobb MH, Wu LF, Altschuler SJ. Developmental cell. 2018;44(5):624-33.e4, VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Gut. 2015;64(6):911-20…), therefore attributing it as a novel enzymatic approach (line 9,18, 67 and so on…) is simply overstating. Except for “optimization” of cell numbers needed to seed one type/brand of transwell, this study does not significantly contribute to the “reproducible method” either as it is not clear if the 2D lines used are derived from different animals(line 67).

Other Concerns:

Title: Please consider changing “Recreation of accessible interface….” to “ Establishment of accessible interface or Recapitulation of accessible interface….”

Line 60-63 Consider changing the sentence: “Thus, cultures of polarized…..” into: Thus cultures of polarized intestinal cell monolayers are better suited for standardized measure of transepithelial permeability and epithelial-luminal interaction due to easier accessibility of the apical surface.

Methodologic issues:

1. Throughout the paper N is mentioned for each experiment, but not described properly. Does N means 3 biopsies from the same dog or 1 biopsy of three different dogs. In the opinion of the referee in order to contribute to the statement of “standardized protocol” the authors should use different dogs as N. Currently this is not clear Throughout the manuscript. For example: figure 2F, are 3 biopsies from the same animal of are 3 different animals used? Figure 3 D and E , what is N precisely, 10 different donors or 10 different biopsies from the same donor?

2. figure 2: authors performed WGA staining for mucus layer. Which cells are producing the mucus, are there Goblet cells present in canine colonic monolayers?

3. figure 2: it would be useful to show dapi staining. Right now it is difficult to evaluate these staining, for example ALPI seems to be expressed in the nucleus. Besides, magnification insets are too small to provide more detail.

4. In support to figure 4A-D authors should measure permeation by performing FD4 permeation rate and really show the tight barrier already at day 3. This is beneficial to those wishing to use this model in order to decide the right time for an intervention study. The idea for authors is to elucidate it and provide a simple timeline of potential intervention study.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Apr 17;15(4):e0231423. doi: 10.1371/journal.pone.0231423.r002

Author response to Decision Letter 0

9 Mar 2020

Responses to Reviewers (PONE-D-20-00243)

Reviewer 1:

1. Ambrosini et al present a method to generate epithelial monolayers from canine intestinal organoids. Although this is the first study of this kind generating canine intestinal monolayers, the methods used to generate these monolayers are not novel, asides from slight variations in the dissociation and plating protocols/media, having been demonstrated by multiple groups in both mouse and human systems. They suggest that these monolayers form epithelial cell layers with multiple differentiated cell types, including enteroendocrine cells, intestinal stem cells and polarised enterocytes. Although some of the data supports this conclusion, such as the TEM images of polarised cells and the results from the TEER experiments, there is a lack of data to show fully that these cells are properly differentiated into functioning intestinal epithelium. Some of the markers used are not appropriate and it is not clear from the select number of TEM images presented whether goblet cells are present and whether there are true microvilli on the apical surface of these cells as has been observed in human and murine organoid culture. Furthermore, it is not clear in the manuscript how many canine lines were used in these experiments. The authors suggest in their discussion that this protocol “could potentially be used for translational Precision Medicine purposes because the individual canine organoid lines obtained from various dog species can be established and utilized for pharmaceutical studies”. However, it is not clear that multiple organoid lines were used in this study, limiting the potential applicability for the future uses of the system the authors suggest. Overall, although I agree with the authors that canine organoids would provide a useful system for various areas of research, I don’t believe that the data presented here is enough to demonstrate successful monolayer development:

We appreciate the Reviewer’s positive comment on the promising perspectives of our canine colonoid- derived monolayer system for various areas of research applications. In this research article, we have claimed the value of outcomes in terms of the optimization of the protocol of dissociation, seeding, and culture of canine colonoid-derived epithelium on the standpoint of an enduser for assessing the morphological characteristics, polarization of apical brush border, basic barrier function and permeability, and expression of lineage-dependent or functional markers.

We agree with the Reviewer’s critique in terms of the insufficient novelty of the dissociation method that we adapted in this study to recreate a tissue interface, which has been applied and optimized in mouse, pig, or human intestinal organoids by multiple groups (1,2). Thus, we toned down the overstatement of the “novel” or “standardized” enzymatic dissociation approach to “an optimized protocol” in the revised manuscript in lines of 10, 65, 69, and 157. Instead, we sufficiently claimed the specific “usefulness” and “functionality” of our canine colonoid-derived epithelial monolayer reconstituted on the nanoporous membrane for the experimental assessment including barrier integrity and permeability, localization of the key structural markers, spatial visualization of the stem cells and other colonic cells with lineage-dependent cytodifferentions, and the stability and reproducibility of the formation and maintenance of a primary colonic epithelial monolayer with required statistics.

To improve our argument based on the Reviewer’s comments, we additionally performed morphological characterization using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to demonstrate the presence of goblet cells in our canine colonoid-derived monolayer. We summarized the new results that specifically support the expression of goblet cells in Figure 4 in the revised manuscript. Here, we identified the mucin granule-containing goblet cells using TEM (revised Fig 4B, “MG”), goblet cell orifices (revised Fig 4D, “GO”), and fenestrated membranes (revised Fig 4D, “FM”) extending deep into the goblet cell surface using SEM. This new finding shows a good agreement with the previous studies (3,4), demonstrating that the goblet cells were present in the canine colonoid-derived monolayer.

Based on the Reviewer’s comments, we re-evaluated the stem and differentiated cells populated on the caine colonoid-derived epithelial monolayer in the revised manuscript using various imaging modalities including additional antibody-based immunofluorescence (IF) staining as well as RNA in situ hybridization. This precision imaging technology with high specificity reveals that our marker staining results show the presence of stem- and differentiated cells that are pertinent in the colonic functions and cytodifferentiation with strong in vivo relevance. In terms of the quantification of epithelial populations, the % positive cell numbers for the Lgr5+ stem cells (5,6), ALPI+ differentiated intestinal epithelium (4,6), Neurog3+ and CgA+ enteroendocrine cells (7,8) were detected as 7.6±0.1, 60.1±0.9, 41.2±10.3%, and 47.8±2.7%, respectively, which are all strongly supported by the previous reports published in both human and dog in vitro studies. Our statistical analysis also supports the reproducibility and robustness of our experimental outcomes in the revised manuscript (see Statistical analysis).

We used three independent lines of canine colonoids in this study. We additionally inserted a new Supplementary Figure 3 in the revised manuscript to show the reproducibility of the functional outcome in terms of the barrier function using the TEER measurement of the used 3 indepenent colonoid lines with 2 biological replicates and 4 technical replicates. We provided this updated information in both Materials and Methods (lines 131-136) and Discussion (lines 343-346) sections. As a proof-of-principle study, we applied reasonable sets of biological and technical replications (see details in the revised manuscript, especially in the Legend) to claim the potential contribution to the translational researches between canine and human studies. Our manuscript clearly demonstrates the optimized protocol to recreate the mucosal tissue interface using primary canine colonic epithelium for the “future applicability”. The epithelial barrier functions and colon-specific characteristics support that our technical advance may contribute to validate our system toward inter-species variation in barrier permeability, transport, absorption, efflux, or metabolism of the administered drug compounds. We currently perofrm these aspects as follow-up studies; however, we do not include them in this manuscript as they are beyond the scope.

2. In my opinion, the authors do not convincingly show that there are multiple fully differentiated cell types present in their canine epithelial model system. % positive cells presented in figure 2F is not showing distinct cell types because if it was the total % would equal 100, whereas the number shown is much greater than this. One reason may be the assumption that differentiated enteroendocrine cells can be identified by expression of Neurog3, whereas Neurog3 is expressed in progenitor cells, which become mature enteroendocrine cells but also other cell types (see Gehart et al., 2019. Cell: 176(5)).

In terms of the interpretation of our data, the Reviewer’s argument of “if it was the total % would equal 100, whereas the number shown is much greater than this” is incorrect because Ki67+ cells are not the population of lineage-dependent cells. Thus, we do not expect that the add-up of the provided chart would be 100%. In the revised manuscript and figures, we provide additional marker staining results (Chromogranin A, CgA) as well as the quantification (revised Fig 3). In terms of the quantification of epithelial populations, the % positive cell numbers for the Lgr5+ stem cells (5,6), ALPI+ differentiated intestinal epithelium (4,6), Neurog3+ and CgA+ enteroendocrine cells (7,8) were detected as 7.6±0.1, 60.1±0.9, 41.2±10.3%, and 47.8±2.7%, respectively, which are all strongly supported by the previous reports published in both human and dog in vitro studies. Our statistical analysis also supports the reproducibility and robustness of our experimental outcomes in the revised manuscript (see Statistical analysis).

In order to demonstrate the mature enteroendocrine cell differentiation and its % positive cell number, we performed an IF staining against canine Chromogranin A, which actually showed similar % expression level as Neurog3 as well as a reference from a human large intestinal tissue (7). For this quantification, three independent fields of view from two or more independent biological replicates were used. We also applied at least two technical replicates (revised Fig 3F). We appreciate the suggestion of references by the Reviewer.

3. To identify mature enteroendocrine cells it is more common to use markers such as ChgA and Reg4 to define mature cells of this subset. Although the limitations of using Neurog3 are discussed by the authors in their discussion, it is not clear why the authors not used one of these more common enteroendocrine markers?

As suggested, we performed IF staining against canine Chromogranin A (CgA) in the revised manuscript and shown in Fig 3E. We updated the Results (lines 105-111), and Materials and Methods (lines 366-381) sections accordingly.

4. The authors have used transmission electron microscopy to nicely show the presence of tight junctions between epithelial cells in Figure 3, therefore if there are fully differentiated epithelial cell subsets such as goblet cells and enteroendocrine cells these should be identifiable based on distinct morphology in comparison to absorptive enterocytes by TEM (see Figure S1, Forbester et al., 2018. PNAS: 115(40)). As the authors point out, the availability of canine-specific antibodies is a limitation of this study, however TEM would allow them to convincingly show varying morphology between different cell types, without requiring antibodies.

In response to the comment for the verification of goblet cells, we performed SEM and additional TEM image acquisitions to demonstrate the presence of goblet cells in our canine organoid-derived monolayer. Our new findings are now summarized in Fig 4 in the revised manuscript. Briefly, we identified the mucin granule-containing goblet cells using TEM (revised Fig 4B, “MG”). In addition, we leveraged the SEM iamge results to identify the goblet cells, where goblet cell orifices (revised Fig 4D, “GO”) and fenestrated membranes (revised Fig 4D, “FM”) were clearly indicated. This observation shows a strong agreement with the previous studies (3,4), demonstrating that the goblet cells were present in the canine colonoid-derived monolayer. In response to the comment for the verification of enteroendocrine cells, we performed IF staining against canine Chromogranin A in addition to the visualization using RNA in situ hybridization of Neurog3+ cells and summarized in Fig 3E and 3F in the revised manuscript. We updated the Results (lines 105-111) and Materials and Methods (lines 366-381) sections accordingly. We appreciate the suggestion of references by the Reviewer.

5. The reliance on the results from the FISH experiments, which are difficult to interpret, is not convincing enough data.

We understand the Reviewer’s concern in terms of the obviousness and easiness of the data interpretation of the RNA in situ hybridization results. However, we believe that our RNA in situ hybridization data successfully provide a localized marker-positive cell that highlights the expression of mRNA. Our argument is supported by the following reasons. First, RNA in situ hybridization specifically show the presence and its location of the target RNA, not protein, by which the marker-positive cell show the “on” signal via fluorophore. Thus, the signal may be somewhat weak with a form of “dots” (revised Figs 3A, 3C, 3D, and S1) rather than the positioned “space” in fluorescence (revised Fig 3B and 3E). The displayed signal of collected “dots” may position on the nucleus or the cytoplasm, but it does not compromise the evidence that the cell shows the targeted fluorescence signal. Second, although RNA in situ hybridization is a relatively new technology, the validation and reproducibility of this technology have been sufficiently established and proved by multiple studies with in vitro intestinal models (9–13). Third, we previously provided the successful demonstration of the RNA in situ hybridization on the canine intestinal 3D organoid cultures, where we specifically visualized stem cells (Lgr5, Sox9, and Ephb2), paneth-like cells (Fzd5 and Cath), absorptive enterocytes (ALPI), and enteroendocrine cells (Neurog3) (6). Finally, we provided positive control results in Supplementary Fig 1 to confirm the appropriate performance of the RNA in situ hybridization in the canine colonoid-derived monolayer, verifying that the positive and negative staining provide the precision and accuracy of the RNA in situ hybridization that targets the genes that are express in a lower (revised Fig S1A) and higher degree (revised Fig S1B) in the cells.

6. The authors imply that one advance of their study is that they use enzymatic rather than mechanical disruption to disrupt the organoid ultrastructure to single cells. This This is not a novel method for generating monolayers, and has been used to dissociate and grow monolayers in both and mouse and human intestinal organoid systems (see Altay et al., 2019. Scientific reports; 9(10140) and Thorne et al., 2018. Developmental Cell; 44(5)). This needs to be clarified in text, because at the moment in my opinion the novelty is overstated. However, in mouse and human systems extensive mechanical disruption is needed in conjunction to TrypLE treatment to ensure dissociation to single cells. Can the authors explain why dissociation of canine intestinal organoids is much easier in comparison to mouse and human intestinal organoids?

We agree with the Reviewer’s critique in terms of the insufficient novelty of the dissociation method that we adapted in this study to recreate a tissue interface, which has been applied and optimized in mouse or human intestinal organoids by multiple groups (1,2). Thus, we toned down the overstatement of the “novel” or “standardized” enzymatic dissociation approach to “an optimized protocol” in the revised manuscript in lines of 10, 65, 69, and 157. Instead, we sufficiently claimed the specific “usefulness” and “functionality” of our canine colonoid-derived epithelial monolayer reconstituted on the nanoporous membrane for the experimental assessment including barrier integrity and permeability, localization of the key structural markers, spatial visualization of the stem cells and other colonic cells with lineage-dependent cytodifferentions, and the stability and reproducibility of the formation and maintenance of a primary colonic epithelial monolayer with required statistics. It is noted that we did not claim “dissociation of canine intestinal organoids is much easier in comparison to mouse and human intestinal organoids”. For the Reviewer’s information, we have applied the same dissociation protocol to both human and canine intestinal organoids, where we have not observed a significant difference in the dissociation yield. We discussed this point in the revised manuscript (lines 158-160). We appreciate the suggestion of references by the Reviewer.

7. The microvilli in Figure 3A and Figure 4E look disrupted, or more similar to the structures seen on the surface of M-cells? Can the authors explain why this is? Why is only a single ‘normal length’ villus shown in Figure 3C? See Llanos-Chea et al., 2019. J Pediatr Gastroenterol Nutr; 68(4) for sample microvilli on human intestinal epithelial organoids?

In the revised manuscript, we provide new data sets performed with the SEM imaging. Briefly, SEM images demonstrate the overall patterns of the recreated microvilli on the apical surface of our canine colonoid-derived monolayer, confirming the frequency and density of the microvilli (revised Fig 2). The variation in microvilli frequency was observed in our dog colonoid-derived monolayer (revised Fig 2B), which was also noted in other colonoid-derived studies (14,15). The number of microvilli assessed by the SEM imaging was variable in the range from 9 to 18 microvilli/µm2, which was similar to the report of human intestinal epithelial organoid culture performed in vitro organ-on-a-chip (16).

In terms of a “single normal length (micro)villus”, we removed that image from the figure because we confirm that it is not representative based on the investigation of SEM images. Stunted microvilli were observed in our system which could reflect the fact that colonic intestinal cells may not require longer microvilli due to minimal nutrient absorption in the colon (17). Possibly, it could be due to the culture condition that is not completely adequate to promote longer microvilli (4). Although assessing the effect of different culture conditions on dog colonoid-derived monolayer, particularly to the length of microvilli, is beyond our scope in this manuscript, such investigation would be beneficial to better understand the physiological demonstration and functions of the microvilli in the future study. We addressed this point in the revised manuscript (lines 272-282). We appreciate the suggestion of references by the Reviewer.

Minor comments

8. None of the figures specify number of replicates/number of canine intestinal organoid lines used. Are these figures representative for lines from multiple canine donors?

The number of replicates and the number of canine intestinal organoid lines were specifically delineated in each figure lengend as well as in the Materials and Methods section (lines 383-391, lines 408-412) in the revised manuscript. As previously mentioned, we used three independent lines of canine colonoids derived from three independent canine donor biopsies. Based on the provided biological and technical replicates, we confirm that the figures are representative for lines from multiple canine donors.

9. Description in figure legend for Figure 4D is confusing, needs to be clearer what the control and the experimental samples are, the sentence doesn’t make sense

We provided a new legend for Fig 4D (revised Fig 6C) in the revised manuscript as suggested with improved clarity, accuracy, and brevity.

10. Figure 2F – not clear what N=3 is, 3 fields of view of the same sample; 3 replicate monolayers and staining experiments from the same canine organoid line; 3 replicate monolayers and staining experiments from different canine organoid lines?

The N number indicates the number of a field of view. In Fig 2F (revised Fig 3F), we chose three independent fields of view from two or more independent biological replicates. We also applied at least two technical replicates to all the experimental setup. The image was randomly chosen for the analysis. The described information is now specifically provided in the figure legend in the revised manuscript.

11. Figure 3F- As above what is N equivalent to? Multiple experiments, multiple replicates, multiple organoid lines?

In Figure 3F (revised Figure 5C), we used total 10 randomly chosen fields of view to detect P-gp expression levels among 4 biological replicates. In each biological replicate, we performed 2 technical replicates. The described information is now specifically provided in the figure legend in the revised manuscript.

12. Figure 4C, D & G – as above, what does N represent in terms of replicates?

In both Fig 4C and 4D (revised Supplementaary Fig 2), we used total 10 and 6 randomly chosen fields of view for Fig 4C and 4D, respectively, to quantify the relative inteinsity of fluorescence among 4 biological replicates of IF staining experiment. We also applied two technical replicates to individual biological replicates. In Fig 4G (revised Fig 6C), TEER values were caculated from total eight replicates (N=8), where 2 biological replicates with 4 technical replicates in each condition were applied for the TEER assessment in each data point.

Reviewer 2:

1. The study is meant as follow up of previously published work (ref 9 in this manuscript) and in part overlapping statements in introduction and discussion are used to justify the need for development of canine culture models. Also, TrypLE express is commonly used for dissociation of colonic 3d and 2d intestinal cultures and cited in multiple papers (Thorne CA, Chen IW, Sanman LE, Cobb MH, Wu LF, Altschuler SJ. Developmental cell. 2018;44(5):624-33.e4, VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Gut. 2015;64(6):911-20…), therefore attributing it as a novel enzymatic approach (line 9,18, 67 and so on…) is simply overstating. Except for “optimization” of cell numbers needed to seed one type/brand of transwell, this study does not significantly contribute to the “reproducible method” either as it is not clear if the 2D lines used are derived from different animals (line 67).

We agree with the Reviewer’s critique in terms of the insufficient novelty of the dissicoation method that we adapted in this study to recreate a tissue interface, which has been applied and optimized in mouse or human intestinal organoids by multiple groups (1,2). Thus, we toned down the overstatement of the “novel” or “standardized” enzymatic dissociation approach to “an optimized protocol” in the revised manuscript in lines of 10, 65, 69, and 157. Instead, we sufficiently claimed the specific “usefulness” and “functionality” of our canine colonoid-derived epithelial monolayer reconstituted on the nanoporous membrane for the experimental assessment including barrier integrity and permeability, localization of the key structural markers, spatial visualization of the stem cells and other colonic cells with lineage-dependent cytodifferentions, and the stability and reproducibility of the formation and maintenance of a primary colonic epithelial monolayer with required statistics. In terms of the argument of “reproducible method”, we provided repeated, consistent methods of statistics to all our provided data set. In terms of the reproducibility of the efficiency of monolayer generation, it can be influenced by the quality of colonoid (e.g., original viability), yield of dissociation into single cells, applied protocols (mechanical vs. enzymatic), duration of time performed, and incubation condition, except for “optimization” of cell numbers needed to seed one type/brand of Transwell. We used three independent lines of canine colonoids derived from the biopsies of three different canine donors. In terms of the data analysis, we used at least two independent biological replicates with at least two technical replicates, where the imaging data were acquired from at least three different locations. The person-to-person variation of the yield of culture performance as well as the variability of results between batches were also carefully considered to implement the “reproducible method” in this study. Now, we all specifically restate in the revised manuscript in the main text as well as in the Methods section.

2. Title: Please consider changing “Recreation of accessible interface….” to “Establishment of accessible interface or Recapitulation of accessible interface….”

Line 60-63 Consider changing the sentence: “Thus, cultures of polarized…..” into: Thus cultures of polarized intestinal cell monolayers are better suited for standardized measure of transepithelial permeability and epithelial-luminal interaction due to easier accessibility of the apical surface.

Based on the Reviewer’s comment, the title was changed to “Recapitulation of the Accessible Interface of Biopsy-Derived Canine Intestinal Organoids to Study Epithelial-Luminal Interactions” in the revised manuscript. The sentence that the Reviewer pinpointed was changed as suggested in the revised manuscript as follows; “Thus, cultures of a polarized intestinal cell monolayer are better suited for the standardized measurement of transepithelial permeability and epithelial-luminal interaction due to easier accessibility of the apical surface.” (revised Lines 61-64).

Methodologic issues:

3. Throughout the paper N is mentioned for each experiment, but not described properly. Does N means 3 biopsies from the same dog or 1 biopsy of three different dogs. In the opinion of the referee in order to contribute to the statement of “standardized protocol” the authors should use different dogs as N. Currently this is not clear Throughout the manuscript. For example: figure 2F, are 3 biopsies from the same animal of are 3 different animals used? Figure 3 D and E , what is N precisely, 10 different donors or 10 different biopsies from the same donor?

The N number indicates the number of a field of view. In Fig 2F (revised Fig 3F), we chose three independent fields of view from two or more independent biological replicates. We also applied at least two technical replicates to all the experimental setup. The image was randomly chosen for the analysis. The described information is now specifically provided in the figure legend in the revised manuscript. As previously mentioned, we used three independent lines of canine colonoids that were derived from different canine donors.

For Figure 3D and 3D (revised Figure 5A and 5B) were selected from 4 biological replicates of experiments. We also applied at least two technical replicates to all the experimental setup. In Figure 3F (revised Figure 5C), we used total 10 randomly chosen fields of view to detect P-gp expression levels among 4 biological replicates. In each biological replicate, we performed 2 technical replicates. The described information is now specifically provided in the figure legend in the revised manuscript.

As stated, we used three independent lines of canine colonoids, where scientifically rigorous multiple biological and technical replicates were applied in this study to increase the statistical significance. However, we agree with the Reviewer’s point in terms of the limited size of the canine cohort, thus, we toned down the overstatement of the “standardized protocol” to “an optimized protocol” in the revised manuscript.

4. figure 2: authors performed WGA staining for mucus layer. Which cells are producing the mucus, are there Goblet cells present in canine colonic monolayers?

For the verification of goblet cells, we performed SEM and additional TEM image acquisitions to demonstrate the presence of goblet cells in our canine organoid-derived monolayer. All this points are now summarized in Fig 4 in the revised manuscript. Briefly, we identified the mucin granule-containing goblet cells using TEM (revised Fig 4B, “MG”). In addition, we leveraged the SEM iamge results to identify the goblet cells, where goblet cell orifices (revised Fig 4D, “GO”) and fenestrated membranes (revised Fig 4D, “FM”) were clearly indicated in the revised manuscript. This observation shows a strong agreement with the previous studies (3,4), demonstrating that the goblet cells were present in the canine colonoid-derived monolayer. In addition to the verification of goblet cells using electron microscopy, we also performed immunofluorescence confocal microscopy to confirm the actual mucus production on the colonoid-derived epithelial monolayer. As the WGA staining showed, the mucus-like molecules such as N-acetyl-D-glucosamine is present on the apical side of the canine colonoid-derived monolayer (revised Fig 4). Unfortunately, there are no commercially available antibodies against goblet cell marker (e.g., canine MUC2). Thus, we used the WGA live-cell staining to visually characterize the mucus production as we previously used this marker at various studies (18,19). We revised the Results (lines 112-116), Discussion (lines 196-197), Materials and Methods (lines 375-3768), and Figure 4 Legend sections accordingly.

5. figure 2: it would be useful to show dapi staining. Right now it is difficult to evaluate these staining, for example ALPI seems to be expressed in the nucleus. Besides, magnification insets are too small to provide more detail.

The ALPI staining provided in previously Figure 2C (revised Figure 3C) had the visualized nuclei stained with DAPI and pseudo-colored to grey. We provided this information in figure legend. In terms of the localization of the fluorescence signal of ALPI, RNA in situ hybridization specifically stains RNAs, which commonly exist in both nucleus and cytoplasm. Thus, the positive signals observed in the nucleus may be the overlay of the RNA signal dots above the nucleus or truly the positive signal within the nucleus. Regardless of the location of the positive signal, that is what the probe is detecting and should be interpreted as the way that the positive signal (either a single dot or a collection of dots) is indicative of the presence of the target gene(s) in that particular cell. This RNA in situ hybridization technology has been successfully applied in dog organoids by our group (6) and similar findings (i.e., positive signals seems to be expressed in the nucleus) can be found in other studies (11,13) as well as our positive control provided in Supplementary Fig 1. We increased the magnification insets to avoid any difficulty in data interpretation. We reflected all the discussion here in the revised manuscript.

6. In support to figure 4A-D authors should measure permeation by performing FD4 permeation rate and really show the tight barrier already at day 3. This is beneficial to those wishing to use this model in order to decide the right time for an intervention study. The idea for authors is to elucidate it and provide a simple timeline of potential intervention study.

In response to this comment, we performed a transport assay using fluorescein sodium salt (MW, 376.27 Da) as a small molecule paracellular fluorescent marker for estimating the apparent permeability (Papp) value. The new results are shown in Figure 6F in the revised manuscript and each data point was prepared with 2 biological and 4 technical replicates. We used fluorescein instead of FD4 because this molecule will be more specific to quantitatively compared the Papp profile as the TEER value showed pretty high level (>1,000 Ohm cm2) after day 4 (not day 3), suggesting that any medium- or large-sized fluorescent paracellular markers may not penetrate through the formed monolayer. Based on the TEER profile, we anticipated that the permeability barrier integrity will be matured and stabilized after day 4 (revised Fig 6C and 6F), whereas morphologically the monolayer showed reasonable tight junctions before day 4 (revised Figs 1C and 5A). Thus, we further confirmed if the day 4 may be a good temporal indicator to recognize the maturity of a colonoid-derived monolayer applied in our study for the functional assessment. We confirmed by applying the fluorescein transport assay, where the inverse correlation was observed compared to the TEER profile (revised Fig 6F) with statistical significance (P <0.0001). We revised our manuscript based on the discussion provided here.

References:

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2. Shin W, Hinojosa CD, Ingber DE, Kim HJ. Human Intestinal Morphogenesis Controlled by Transepithelial Morphogen Gradient and Flow-Dependent Physical Cues in a Microengineered Gut-on-a-Chip. iScience. 2019 May;15:391–406.

3. Marsh MN, Swift JA. A study of the small intestinal mucosa using the scanning electron microscope. Gut. 1969 Nov 1;10(11):940–9.

4. Wang Y, Kim R, Gunasekara DB, Reed MI, DiSalvo M, Nguyen DL, et al. Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer. Cell Mol Gastroenterol Hepatol. 2018;5(2):113–30.

5. Zhong X-Y, Yu T, Zhong W, Li J-Y, Xia Z-S, Yuan Y-H, et al. Lgr5 positive stem cells sorted from small intestines of diabetic mice differentiate into higher proportion of absorptive cells and Paneth cells in vitro. Dev Growth Differ. 2015;57(6):453–65.

6. Chandra L, Borcherding DC, Kingsbury D, Atherly T, Ambrosini YM, Bourgois-Mochel A, et al. Derivation of adult canine intestinal organoids for translational research in gastroenterology. BMC Biol. 2019 Apr 11;17(1):33.

7. Cristina ML, Lehy T, Zeitoun P, Dufougeray F. Fine structural classification and comparative distribution of endocrine cells in normal human large intestine. Gastroenterology. 1978 Jul;75(1):20–8.

8. Gunawardene AR, Corfe BM, Staton CA. Classification and functions of enteroendocrine cells of the lower gastrointestinal tract: Classification and functions of colorectal enteroendocrine cells. Int J Exp Pathol. 2011 Aug;92(4):219–31.

9. Workman MJ, Gleeson JP, Troisi EJ, Estrada HQ, Kerns SJ, Hinojosa CD, et al. Enhanced Utilization of Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids Using Microengineered Chips. Cell Mol Gastroenterol Hepatol. 2018;5(4):669-677.e2.

10. Sáez de Guinoa J, Jimeno R, Gaya M, Kipling D, Garzón MJ, Dunn-Walters D, et al. CD1d-mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis. EMBO J. 2018 Mar 1;37(5):e97537.

11. Bullman S, Pedamallu CS, Sicinska E, Clancy TE, Zhang X, Cai D, et al. Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science. 2017 15;358(6369):1443–8.

12. Hughes KR, Harnisch LC, Alcon-Giner C, Mitra S, Wright CJ, Ketskemety J, et al. Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner. Open Biol. 7(1):160155.

13. Yamakawa T, Tomita K, Sawai J. Characteristics of Biofilms Formed by Co-Culture of Listeria monocytogenes with Pseudomonas aeruginosa at Low Temperatures and Their Sensitivity to Antibacterial Substances. Biocontrol Sci. 2018;23(3):107–19.

14. In J, Foulke-Abel J, Zachos NC, Hansen A-M, Kaper JB, Bernstein HD, et al. Enterohemorrhagic Escherichia coli Reduces Mucus and Intermicrovillar Bridges in Human Stem Cell-Derived Colonoids. Cell Mol Gastroenterol Hepatol. 2016 Jan 1;2(1):48-62.e3.

15. Sontheimer-Phelps A, Chou DB, Tovaglieri A, Ferrante TC, Duckworth T, Fadel C, et al. Human Colon-on-a-Chip Enables Continuous In Vitro Analysis of Colon Mucus Layer Accumulation and Physiology. Cell Mol Gastroenterol Hepatol. 2019 Nov 26;

16. Kasendra M, Luc R, Yin J, Manatakis DV, Apostolou A, Sunuwar L, et al. Organoid-derived Duodenum Intestine-Chip for preclinical drug assessment in a human relevant system. bioRxiv. 2019 Aug 5;723015.

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19. Macierzanka A, Mackie AR, Krupa L. Permeability of the small intestinal mucus for physiologically relevant studies: Impact of mucus location and ex vivo treatment. Sci Rep. 2019 Nov 26;9(1):1–12.

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Mária A Deli

24 Mar 2020

Recapitulation of the Accessible Interface of Biopsy-Derived Canine Intestinal Organoids to Study Epithelial-Luminal Interactions

PONE-D-20-00243R1

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Acceptance letter

Mária A Deli

6 Apr 2020

PONE-D-20-00243R1

Recapitulation of the Accessible Interface of Biopsy-Derived Canine Intestinal Organoids to Study Epithelial-Luminal Interactions

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Verification of the RNA in situ hybridization on the canine colonoid-derived epithelial monolayer.

    A 3-Plex Positive Control Probe (Advanced Cell Diagnostics) was applied to the canine monolayer cultured for 13 days to confirm the functionality of the kit applied. A low (RNA Polymerase II Subunit A (POLR2A), Opal 650; S1A) and a high expressor RNA (Ubiquitin C (UBC), Opal 520; S1B) confirmed the functionality of the probes applied in the canine epithelial monolayer. An overlaid image is displayed in S1C. Nuclei, blue. Bars, 50 μm.

    (TIF)

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    S2 Fig. Expression of the epithelial junctional proteins in the canine colonoid-derived epithelial monolayer.

    Quantification of the expression level of ZO-1 and E-cadherin at days 3 and 13 was performed using total 10 and 6 randomly chosen fields of view for ZO-1 and E-cadherin, respectively, among 4 biological replicates of IF staining experiment. We also applied two technical replicates to individual biological replicates. a.u., arbitrary unit. NS, not significant.

    (TIF)

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    S3 Fig. Reproducibility of the barrier function of colonoid-derived epithelial monolayers derived from three different canine colonoid lines.

    Three independent lines of canine colonoids show similar profile of epithelial barrier function when those three lines were used to form a monolayer on a nanoporous insert. The result was produced with 2 biological replicates, where each biological replicate was performed with 4 technical replicates. Error bars indicate SEM.

    (TIF)

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    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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