Development and characterization of a 2D porcine colonic organoid model for studying intestinal physiology and barrier function

Masina Plenge; Nadine Schnepel; Mathias Müsken; Judith Rohde; Ralph Goethe; Gerhard Breves; Gemma Mazzuoli-Weber; Pascal Benz

doi:10.1371/journal.pone.0312989

. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989

Development and characterization of a 2D porcine colonic organoid model for studying intestinal physiology and barrier function

Masina Plenge ^1,^*, Nadine Schnepel ¹, Mathias Müsken ², Judith Rohde ³, Ralph Goethe ³, Gerhard Breves ¹, Gemma Mazzuoli-Weber ¹, Pascal Benz ¹

Editor: Mária A Deli⁴

PMCID: PMC12057940 PMID: 40333830

Abstract

The porcine colon epithelium plays a crucial role in nutrient absorption, ion transport, and barrier function. However ethical concerns necessitate the development of alternatives to animal models for its study. The objective of this study was to develop and characterize a two-dimensional (2D) in vitro model of porcine colonic organoids that closely mimics native colon tissue, thereby supporting in vitro research in gastrointestinal physiology, pathology, and pharmacology. Porcine colonic crypts were isolated and cultured in three-dimensional (3D) organoid systems, which were subsequently disaggregated to form 2D monolayers on transwell inserts. The integrity of the monolayers was evaluated through the measurement of transepithelial electrical resistance (TEER) and electron microscopy. The functional prerequisites of the model were evaluated through the measurement of the mRNA expression of key ion channels and transporters, using quantitative RT-PCR. Ussing chamber experiments were performed to verify physiological activity. The 2D monolayer displayed robust TEER values and retained structural characteristics, including microvilli and mucus-secreting goblet cells, comparable to those observed in native colon tissue. Gene expression analysis revealed no significant differences between the 2D organoid model and native tissue with regard to critical transporters. Ussing chamber experiments demonstrated physiological responses that were consistent with those observed in native colonic tissue. In conclusion, 2D porcine colonic organoid model can be recommended as an accurate representation of the physiological and functional attributes of the native colon epithelium. This model offers a valuable tool for investigating intestinal barrier properties, ion transport, and the pathophysiology of gastrointestinal diseases, while adhering to the 3R principles.

Introduction

The gastrointestinal tract (GIT) is vital for nutrient digestion and absorption, waste excretion, and metabolic homeostasis [1]. The GIT epithelium acts as a selective barrier, regulating the passage of nutrients, ions, and water. Tight junction proteins, such as occludin (OCLN), claudin (CLDN) and zonula occludens (ZO-1), are crucial in maintaining the integrity of the epithelial barrier by providing both a “fence” and a “gate” or barrier function [2,3]. The fence function serves to separate the apical from the basolateral sides of the epithelial cells, ensuring the proper distribution of membrane proteins and lipids. Moreover, the mucus layer, which is composed of mucins (MUC) and is secreted by goblet cells, serves as the primary defence against luminal pathogens and mechanical stress [4]. Various transporters, channels, and carriers facilitate the transport across the epithelium, such as the epithelial sodium channel (ENaC), sodium-hydrogen exchanger (NHE), calcium-dependent chloride channel (CaCC), and cystic fibrosis transmembrane conductance regulator (CFTR). Each of these regulates the transport of specific ions and molecules. The function of the GIT has historically been studied by animal experiments, with pigs being commonly used as models due to their relevance to both, pig-specific diseases and human intestinal conditions [5].

However, with the increasing relevance of ethical concerns and the principles of replacement, reduction, and refinement (3R) [6], alternative methodologies such as cell culture-based systems are gaining attraction. Cell lines have been instrumental in unravelling certain aspects of GIT physiology. Nevertheless, the usefulness of these models has often been limited by their focus on singular cell types. This limitation highlights the need for more comprehensive models that encompass the diverse cell populations found within the gastrointestinal epithelium. The colon hosts a variety of cell types, including enterocytes, goblet cells, enteroendocrine cells, tuft cells, and stem cells, each contributing uniquely to its function [7,8]. To overcome this limitation of a single cell type, the organoid model was introduced in 2009 [9], revolutionising our ability to model the complex architecture and functionality of the intestinal epithelium. Organoids are derived from stem cells, which impart two crucial properties to them. Firstly, these cells can differentiate into all cell types of the epithelium from which they were obtained. Secondly, organoids possess the ability to self-renewal [10]. Organoids cultivated in a 2D-monolayer system are highly valued as effective tools for studying barrier properties for drug discovery and development [11]. According to Hoffmann et al. [12], porcine jejunum organoid cultures are a useful model for investigating physiological transport properties of the epithelium. Nevertheless, a comprehensive model that takes into account the physiological attributes of the porcine colon is still lacking. Colon organoids offer unique advantages for investigating colon-specific disease mechanisms and treatments. They effectively model the thicker mucus layer that is essential for maintaining epithelial barrier integrity, and facilitate research into critical functions such as water and electrolyte absorption. Thus, this study aimed to delineate the physiological functionality of a 2D culture of porcine colonic organoids, emphasizing gene expression relative to native tissue and elucidating the transport properties of the organoid-derived epithelium.

Materials and methods

For this protocol, two healthy pigs were sacrificed by captive bolt shooting and exsanguination. According to the Animal Welfare Act (directive 2010/63 EU), this (slaughter and removal of tissues) is not classified as an animal experiment, but must be reported to the University Animal Welfare Officer (registration number TiHo-T-2017–22 and TiHo-T-2024–5).

Culture of 3D-organoids

To generate 3D organoids, intestinal crypts were isolated from the porcine colon. The cultivation of 3D organoids has already been described in detail by Hoffmann et al. [12]. In this instance, a 10 cm segment of the proximal porcine colon was employed for the isolation of crypts. Briefly, the organoids were cultured in Matrigel (Corning®, Kaiserslautern, Germany; Matrigel® Basement Membrane Matrix) droplets of 50 µl in a 24-well plate. Every 2–3 days, the organoid medium (S1 Table) was changed and at least once a week the droplet was resolved and the 3D organoids were passaged. The organoid medium was then aspirated, ice-cold PBS was added, and the Matrigel droplet dissolved by pipetting. The suspension was collected in a tube and was centrifuged at 500 x g for 10 minutes at 4 °C. The pellet was resuspended in medium and diluted 1:5 with Matrigel. The plate was incubated at 37 °C for at least 30 min and overlaid with 500 µl of organoid medium per well.

Culture of 2D-organoids

2D organoids were generated by enzymatic and mechanical disintegration of 3D organoids as described in the following. The medium was aspirated and the Matrigel droplets were resolved with 1 ml ice cold PBS, at this stage 3–4 wells were pooled. The solution was transferred to a reaction tube with 10 ml ice cold PBS and then centrifuged at 250 x g for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended in 1 ml 0.05% Trypsin/ EDTA (Gibco™, Thermo Fisher) and incubated for 5 min at 37 °C. Afterwards, the solution was resuspended 20 times with a 1,000 µl tip and 15 times with a 200 µl tip, which was mounted on a 1,000 µl tip, all steps being performed on ice. 10 ml Advanced DMEM/F-12 (Thermo Fisher scientific, Waltham, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany; catalogue no.: F7524) were added to the cell suspension and centrifuged at 1,000 x g for 10 min at 4 °C. After removing the supernatant, the pellet was resuspended in 1 ml monolayer medium (S2 Table). Cells were counted and 1.5 ‧ 10⁵ cells were seeded on cell culture inserts (Corning®; Snapwells® or GREINER BIO ONE; ThinCert® Cell Culture Insert 12 Well; diameter: 12 mm; pore size: 0.4 μm) pre-coated with 1:40 (v/v) Matrigel in PBS. Every 2 days the monolayer medium was replaced and the transepithelial electrical resistance (TEER) was measured with a STX4 electrode (EVOM³; World Precision Instruments, Berlin, Germany). The differentiation was started at cultivation day 8 by changing the medium to differentiation medium (S3 Table). Differentiation medium was replaced daily and the TEER measured. Experiments were conducted on the 10^th day of cultivation.

RNA isolation and reverse transcription

On the 10^th day of cell cultivation, the cells were harvested for analysis. After aspirating the medium, cells grown on transwells were washed with ice-cold PBS and the cellular layer was detached from the membrane with a spatula. For the examination of tight junction proteins, cells were also collected on the 4^th, 7^th, and 9^th days of cultivation. Samples were centrifuged at 1,000 x g for 10 minutes at 4 °C, followed by swift freezing in liquid nitrogen and subsequent storage at -80 °C. For comparison, native tissues from the same breed, age and colon section as the organoids donors was used. These tissues were obtained from banked samples. The RNA extraction was performed with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of the isolated RNA was determined by spectrophotometric measurements using the NanoDrop™ One (Thermo Scientific™). The cDNA synthesis was performed via reverse transcription using TaqMan™ Reverse Transcription Reagents Kit (Applied Biosystems, Roche Molecular System, Darmstadt, Germany).

Quantitative real-time PCR

To determine mRNA expression of the target genes, quantitative realtime PCR was used. The gene expression of the genes in S4 Table was determined using the SYBR Green® PCR assay as previously described by Elfers et al. [13]. For the targeted gene MUC4 the annealing temperature was 63 °C and for ENaC alpha and NHE3 the annealing temperature was 65 °C for all other genes the annealing temperature was 60 °C. The gene expression of the target genes (S5 Table) were determined by TaqMan®. The reaction mixture contained TaqMan™ Gene Expression Master Mix (Applied Biosystems), 16 ng reverse transcribed cDNA, 300 nM of the specific primers and 100 nM of specific probe at a total volume of 20 µl. For each gene, a negative control and a standard series, which is a dilution series of the amplification product, were used to determine the primer efficiency. The amplification and detection was performed with the real-time PCR cycler Bio-Rad CFX96™ (BIO-RAD Laboratories, INC., Hercules, USA). The housekeeping genes of the 60S ribosomal protein L4 (RPL4) and the 40S ribosomal protein S18 (RPS18) were used to normalise the gene expression of the genes of interest. The relative gene expression was calculated with the Pfaffl [14] method and the mean of the two houskeeping genes. For the gene expression analysis, RNA was isolated from four independent 2D cultures of organoids, with each sample having two technical replicates to ensure the reliability and consistency of the data.

Ussing chamber experiments – transport physiology

In order to investigate epithelial transport characteristics, 2D monolayers were mounted in Ussing chambers at the end of differentiation as described by Hoffmann et al. [12]. After an initial period of 5 min, the tissues were set to short-circuit-conditions for another 15 min to allow equilibration. The short-circuit current (I_sc) and the epithelial membrane resistance (Rt) were monitored every 6 seconds for the whole experiment. The 2D culture was than challenged with one of the substances stated in Table 1. Each substance was tested individually in a chamber, followed by treatment with forskolin for the purpose of assessing viability. In the case of aldosterone, the 2D culture underwent a 3 h, 8 h and 48 h preincubation with aldosterone before Ussing chamber experiments were performed. The epithelia, which has been preincubated with aldosterone, and the control, which had not been preincubated with aldosterone, were subsequently treated in the Ussing chamber with 100 µ M amiloride. Except for forskolin and aldosterone dissolved in DMSO, all other substances are diluted in aqua destillata. Serosal addition of mannitol after the addition of glucose was carried out to ensure osmotic stability. For analysis, the basal values of I_sc determined in the Ussing chamber experiments were the mean calculated from the last ten values prior to the addition of an additive. The changes in short-circuit currents (ΔI_sc) are the difference between the maximum or minimum value after the addition of each substance and the the basal value. Each Ussing chamber setup was repeated in four independent experiments, with three technical replicates per condition, ensuring the reproducibility of the findings.

Table 1. Application for Ussing chamber experiments (all chemicals were obtained from Sigma-Aldrich, Darmstadt, Germany).

Substance	Concentration	application	Incubation time
DMSO	1:1000	serosal	15 min
glucose mannitol	10 mM 10 mM	mucosal serosal	15 min
carbachol	10 µM	serosal	15 min
forskolin	10 µM	serosal	15 min
amiloride	50 µM 100 µM 500 µM 1 mM	mucosal	15 min
aldosterone	3 nM, 1 nM, 0.3 nM	serosal	Preincubation for 3 h, 8 h or 48 h

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Scanning electron microscopy

Electron microscopy sample preparation was performed in a similar way as previously described [15]. In brief, cells on transwells were fixed in a 0.1 M EM Hepes buffer with 5% formaldehyde and 2% glutaraldehyde and washed twice to remove aldehyde residues. Dehydration was carried out in a gradient series of EtOH on ice (10%, 30%, 50%, 70%, 90%), each step for 10 min and two steps (100% EtOH) at room temperature. Ethanol was used to prevent damage of the transwell membrane. Afterwards, critical point drying (CPD300 from Leica) and sputter coating with gold palladium (SCD 500 from Bal-Tec) was performed. Samples were visualized with a field-emission scanning electron microscope Merlin (Zeiss) at an acceleration voltage of 5 kV and making use of both, an Everhart Thornley HESE2 and an inlens SE detector.

Data analysis and statistics

TEER values of the 8^th day of cultivation were set to 100% and the others calculated accordingly. The statistical analyses were performed using Prism version 9.0.0 (GraphPad, San Diego, USA). The normal distribution of all data was tested with the D’Agostino & Pearson test. Comparison between mean values of the gene expression on the 10^th cultivation day and native colon tissue was conducted using the unpaired t-test. The one-way ANOVA was employed to compare the gene expression on various cultivation times followed by Tukey’s test for post-hoc analysis to correct for multiple comparisons. The paired t-test was employed to compare the mean basal and maximum/minimum I_sc values in the presence of a normal distribution. When the data did not follow a normal distribution, the Wilcoxon test was used for the same purpose. A two-way ANOVA was used to evaluate the effects of different cultivation times and aldosterone concentrations. Basal and minimum I_sc values were compared as part of the analysis. Post-hoc analysis was performed using Dunnett’s test to account for multiple comparisons. Differences were considered statistically significant when p value <0.05. Within figure legends, the number of independent experiments is indicated by the symbol “n.”

Results

TEER progression shows robust cellular monolayer integrity

Measurement of TEER provides an indication of the integrity of the cellular layer. Monolayers were confluent on day 8 of cultivation and the differentiation was started by changing the cultivation medium from monolayer medium to differentiation medium. Therefore, the electric resistance was set to 100% on this day, and subsequent TEER measurments were calculated relative to this baseline value. The progression of the TEER values showed an increase until the 8^th day of cultivation and formed a plateau during the differentiation period (Fig 1).

Fig 1 — The organoids were cultivated on transwell inserts (0.4 µm pore diameter). On the 8^th day of cultivation the mean TEER value was 187.4 ± 28.3 Ω ‧cm² (mean ± SEM). Displayed is the mean ± SD, n = 4.

Scanning electron microscopy reveals different cell types

Electron microscopy revealed an intact monolayer of 2D organoids at day 10 of culture. The monolayer was characterized by the development of well-defined microvilli at the apical surface. (Fig 2A, B). The goblet cells are indentified by multiple orifices, which arise from the fusion of mucin-containing granules (Fig 2A, B). This process has resulted in the formation of a mucus layer that covers the epithelium (Fig 2B).

Fig 2 — Green arrows: microvilli; blue arrows: mucin-containing granules; orange arrows: mucus.

RT-PCR demonstrates gene expression concordance between 2D organoids and native tissue

The gene expression of target genes was determined in 2D cultures of organoids and compared with gene expression in native porcine colon tissue. The focus was on key genes that are central to intestinal function, particularly those that control absorption and secretion and are critical for optimal colonic function. Genes involved in the transport of Na⁺, including ENaC and NHE, exhibited no differential expression in the organoid model when compared to native tissue. This pattern was similarly observed for genes for Cl^- secretion (CFTR and CaCC), and the gene encoding for the uptake of glucose (SGLT1). Moreover, the expression of the mucin genes (MUC) demonstrated no significant difference between the 2D organoid culture and native tissue (Fig 3).

Fig 3 — α, ENaC β, ENaC γ, ENaC δ), mucin 2, 4 and 5AC (MUC2, MUC4, MUC5AC), the sodium-potassium-exchanger (NHE1, NHE2, NHE3) and sodium/glucose cotransporter 1 (SGLT1) in 2D cultures of porcine colonic organoids in comparison to native porcine tissue. Ribosomal protein L4 (RPL4) and ribosomal protein S18 (RPS18) were used as reference genes and for normalization. Values shown: mean ± SD, n = 4, unpaired t-test was performed with no significant differences.

Gene expression of tight junction proteins during cultivation is constitutive

The development of tight junction protein gene expression was examined on 4^th, 7^th, 9^th and 10^th day of cultivation. CLDN3 showed no difference in gene expression during the cultivation period on 4^th, 7^th, 9^th and 10^th day. The same was true for CLDN2 with a higher variance on 4^th day. The other transmembrane tight junction protein OCLN also showed no significant differences over the cultivation period, but a slightly higher expression on the last day of cultivation (10^th day). The tight junction associated protein ZO-1 showed no differences in gene expression over the cultivation period and compared to native colonic tissue (Fig 4).

Fig 4 — The reference genes ribosomal protein L4 (RPL4) and ribosomal protein S18 (RPS18) were used to normalize the expression. Values shown: mean ± SD, n = 4, One-way ANOVA was performed as a statistical test with no significant differences.

Ussing chamber studies show physiological transport capacity of 2D-organoids

The addition of DMSO, glucose, forskolin and carbachol was used to alter the I_sc in Ussing chamber experiments (Fig 5). The addition of DMSO (0.1%), which was used as a solvent control, showed no effect to the I_sc. The same was observed after the addition of carbachol (10^-5 mol‧l^-1), which stimulates the Ca²⁺- dependent Cl^- secretion. A significant increase in I_sc was followed after the addition of glucose (10^-2 mol‧l^-1), which was added to test the functionality of the SGLT1, which cotransports Na⁺ with glucose. Forskolin (10^-5 mol‧l^-1) activates the cAMP-dependent Cl^- secretion through CFTR resulting in a significant increase in I_sc after serosal addition (Fig 5).

Amiloride influences the epithelial sodium channel in a dose-dependent manner leading to a significant decrease at 100 µ M and 500 µ M (Fig 6A). This effect could not be observed with 50 µ M or 1 mM amiloride. The addition of forskolin after amiloride led to a significant increase in I_sc, but showed no concentration-dependent effects (Fig 6B).

After the preincubation period with aldosterone, which activates apical Na⁺ channels, the monolayer was challenged with amiloride in the Ussing chamber. The basal I_sc did not undergo a notable alteration when 0.3 nM aldosterone was present. When the two higher concentration were introduced, the dispersion of the basal values increased, yet this did not result in a significant rise in basal I_sc (Fig 7). Notably, incubation with 1 nM aldosterone resulted in the smallest reduction in ΔI_sc. Preincubation showed significant concentration effects (Fig 8). Conversely, incubation with 0.3 nM aldosterone did not yield a significant change in ΔI_sc for the 3, 8, or 48-hour preincubation periods (Fig 8). Significant alterations, however, were observed for the concentration and incubation time.

Fig 7 — The ΔI_sc is the difference between of the aldosterone incubated basal I_sc and the control basal I_sc. Values shown: mean ± SD of four independent experiments, two-way ANOVA with no significant changes.

Fig 8 — ΔI_sc in 2D culture of colonic organoids, which were incubated with 0.3 nM, 1 nM or 3 nM aldosterone for 3 h, 8 h or 48 h, while the control (contr.) was not incubated with any aldosterone. The ΔI_sc were calculated from the basal I_sc and the minimal I_sc after the addition of 100 µ M amiloride. Values shown: mean ± SD, n = 4, two-way ANOVA. ns > 0.05, * p < 0.05, *** p < 0.001.

Discussion

The colonic epithelium serves as a critical barrier, regulating nutrient absorption and ion transport while protecting against pathogens and mechanical stress. This objective of this study was to develop a 2D organoid model that mimics the native porcine colon, therby facilitating detailed investigations of its functional properties. By assessing gene expression, tight junction formation, and transporter functionality, this model provides insights into epithelial barrier function and ion transport mechanisms. Furthermore, comparisons with native tissue revealed the model’s capabilities and limitations, establishing it as a valuable platform for investigating colonic physiology, pathophysiology, and pharmacological interventions. Additionally, the model supports the 3R principles by reducing reliance on animal studies.

The colonic epithelium maintains selective barrier function, regulating the passage of nutrients, ions and water while safeguarding against luminal pathogens and mechanical stress. Electron microscopy imaging revealed the presence of microvilli on the polarised monolayer and identified goblet cells secreting mucus by exocytosis. These features are consistent with those of the colonic epithelium [16,17]. Hoffmann et al. [12], identified two distinct cell types in porcine colonic organoids: goblet cells and enterocytes. These findings align with the current study, further validating the cellular composition of the model.

Mucins, the main structural components of mucus, were expressed in a manner consistent with the native colon. Specifically, MUC2 and MUC4 were observed, while MUC5AC, a mucin more common in the respiratory tract, was expressed at lower levels [18,19]. The similarity to native tissue underscores the physiological relevance of the model. Future studies could investigate mucin composition at the protein level to enhance the understanding of the functionalty and the role of mucus in diseases.

The barrier function is mediated by tight junction proteins, such as claudins (e.g., CLDN2, CLDN3), OCLN and ZO (e.g., ZO-1). The expression of claudins is organ-specific, and in the colon epithelium CLDN2, CLDN3, OCLN, and ZO-1 are expressed [2,20,21]. No significant changes in the gene expression of these proteins were observed during the cultivation period. The formation of tight junctions between the cells results in an increase in TEER values, which can be observed during the formation of the monolayer in two-dimensional (2D) culture. Since the gene expression of the observed tight junction proteins remains unchanged, we assume that the tight junction proteins are formed constitutively during cultivation and are assembled as a tight junction when the cells grow together to form a monolayer. In 2D cultured organoids, TEER values increased until day 8, after which they stabalised, maintiaing this plateau until the end of the cultivation period.

Ion and nutrient transport mechanisms are vital for colonic functions. The expression of key transporters and channels, including CaCC, CFTR, ENaC, and SGLT1, mirrored native tissue. SGLT1, known to be expressed to a major extent in the small intestine, has been shown to be expressed in the large intestine in various animal species [22–24]. Functional studies using Ussing chambers revelaed a response to glucose via SGLT1, in contrast to native tissue, where no glucose response is observed[25]. It is possible that the discrepancy between the organoids and native tissue was due to the former cultivation with glucose as the only carbon source. Yoshikawa et al. [24] observed an increase in SGLT1 expression in the colon of germ-free mice, where SCFA were absent as a carbon source. In contrast to the native colon, which primarily utilises SCFA derived from fermentation of fietary fibre for energy production [26], this experimental system relied exclusively on glucose. Future adaptations could incorporate SCFA in the culture medium to better reflect in vivo conditions.

The secretion of chloride through the CFTR, stimulated by forskolin, was observed to be consistent between the 2D organoids and the native tissue. Forskolin elevates intracellular cAMP levels, which subsequently activates CFTR, resulting inchloride secretion an and increased I_sc [27,28]. Carbachol typically elevates intracellular Ca²⁺ concentrations, which are known to stimulate the CaCC-mediated Cl^- secration [29,30]. However, the lack of response to carbachol indicates an absence of functional CaCC [27,31], potenially due to differences in protein expression. Although no significant difference in gene expression between 2D organoids and native colonic tissue has been detected, the discrepancy between gene expression and protein functionality may be attributable to post-translational modifications, improper protein localisation, or the detection method’s limited sensitivity. To elucidate this further, it would be beneficial to analyse protein expression in future studies using techniques such as Western blotting or immunohistology, which could provide insights into the presence and functionality of the CaCC protein.

However, the trend of lower responsiveness to carbachol is consistent with previous observations in porcine gastrointestinal tissues, which is also seen in porcine jejunum organoids [12,27].

Amiloride inhibition of the ENaC, which results in a reduction in I_sc when measured in Ussing chambers, was dose dependent [32–34]. Higher concentrations potentially target NHE instead of ENaC, which align with prevoius studies [33,35]. The NHE is an electroneutral exchanger, a characteristic that prevents its detection using the Ussing chamber technique. In particular, it has been demonstrated that 1 mM amiloride inhibits the activity of NHE [36,37]. A concentration of 1 mM amiloride did not result in a reduction in I_sc in the 2D culture of porcine colonic organoids, as the inhibition was not of the ENaC, but rather of the NHE. However, aldosterone-induced upregulation of ENaC, observed in native tissue, was not replicated [38–40]. Following the addition of the antagonist amiloride, a smaller decrease in I_sc was observed than in the control, which was not incubated with aldosterone and showed a greater decrease in I_sc. This suggests that the 2D culture may lack certain regulatory mechanisms or timeframes requiered for a aldosterone mediated ENaC modulation.

The developed model exhibited robust barrier function, transporter expression, and physiological relevance to the native porcine colon. However, it also demonstrated notable limitations. The absence of functional CaCC and aldosterone responsiveness highlights areas for further refinement. Additionally, while gene expression aligned with native tissue, protein-level analyses are needed to fully validate the model. The reliance on glucose as the carbon source underscores the need for culture conditions that better mimic native metabolic environments, such as incorporating SCFA.

A limitation of this study is the small number of biological donors. The 3D organoids were derived from two pigs and expanded through multiple passages, a method commonly used in immortalized cell lines, which allows reproducibility. This approach maintained consistency across experiments despite the limited biological donors. Similar procedure have been employed in previous studies, such as the work by van der Hee et al. [41], who developed a standardized 2D monolayer system from porcine intestinal organoids, also employing two donor pigs. Increasing the number of biological donors in future studies would strengthen the generalization of findings. Nevertheless, this approach provides valuable insights and a strong foundation for further research.

This two-dimensional organoid model offers a promising platform for the study of porcine colonic epithelium, exhibiting a high degree of fidelity in terms of gene expression and functional properties. The model is applicable to research in epithelial physiology, infection studies, and pharmacological research, in alignment with the 3R principles. While the model has certain limitations, it provides a foundation for future studies aimed at refining and expanding its applicability.

Supporting information

S1 Table. Compostion of organoid medium.

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pone.0312989.s001.docx^{(21.8KB, docx)}

S2 Table. Composition of monolayer medium.

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pone.0312989.s002.docx^{(20.5KB, docx)}

S3 Table. Composition of differentiation medium.

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pone.0312989.s003.docx^{(18.3KB, docx)}

S4 Table. SYBR Green primers for SYBR Green® assay.

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pone.0312989.s004.docx^{(22.9KB, docx)}

S5 Table. TaqMan primers for TaqMan® assay.

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pone.0312989.s005.docx^{(19.8KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The Federal Ministry of Food and Agriculture (BLE #28N-2-071-00) funded this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989.r001

Author response to Decision Letter 0

22 Oct 2024

PLoS One. doi: 10.1371/journal.pone.0312989.r002

Decision Letter 0

Kevin Looi

26 Nov 2024

Dear Dr. Plenge,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments:

Editor's comments to authors:

1) The limited sample size (n=2 pigs) is a major concern and has significant implications on the interpretation of the data. The manuscript should explicitly acknowledge this limitation. Including individual data points in the figures, rather than bar charts, will also improve transparency and help readers better understand data variability. The Methods section, while thorough, contains redundancies that can be streamlined for readability. For instance, routine steps such as RNA extraction need not be described in detail if they follow standard protocols. Clarity is also needed regarding native tissue usage, specifically, whether the samples were derived from the same animals or archived. Additionally, ensure the number of technical replicates is clearly stated, and define all acronyms (e.g., Isc, Rt) upon their first use.

2) The Results section would benefit from adjustments to improve interpretability. For example, Figure 1 should display absolute TEER values rather than percentages to allow for meaningful comparisons with other studies. Similarly, all figures should include individual data points to reflect variability more effectively. Any missing details, such as error bars for ZO-1 gene expression on day 9 in Figure 4, should be added. Enhancing figure legends, such as clarifying aldosterone concentrations in Figure 7, will make the data presentation more complete and accessible. These are examples and the authors should refer to the individual Reviewer comments for further details.

3) A key point raised by the reviewers concerns the lack of response to carbachol in the Ussing chamber experiments, which contrasts with its reported effects in native tissue. If this discrepancy stems from methodological differences, dosing, or tissue-specific factors, these should be thoroughly discussed. While including native tissue in Ussing chamber experiments for direct functional comparison would have been ideal, if this was not feasible, the manuscript should acknowledge and explain this omission.

4) The Discussion section could benefit from a more concise and focused structure. Beginning with a brief summary of the main findings will provide a clear context for readers. Consider removing subheadings and ensuring smooth transitions between topics, such as mucin gene expression, TEER, and other findings. Expanding on functional differences and limitations, including the small sample size, lack of long-term data, and the absence of structural protein analysis will strengthen the discussion and provide valuable context for the study’s conclusions. If data on the long-term stability of the 2D cultures are not available, discussing potential implications and future directions for longer-term studies will still enhance the impact of your work.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I enjoyed reading the manuscript by Plenge and colleagues describing the establishment of an air-liquid interphase model of the porcine colonic epithelium. The introduction was well written and adequately presents the study rationale. The methods are appropriate for the study aim. The results are justified throughout the discussion, though the discussion may benefit from some refinement and focus. Overall this is an interesting study and presents a novel approach to allow future studies to experimentally assess the porcine colon without the need for harvesting fresh tissue.

Generalised comments:

1. Perhaps this is naivety as I work primarily with airway epithelium, but it is not clear why stem cells were utilised to construct 3D organoids prior to the 2D culture. Could the stem cells not have been directly differentiated in the ALI culture? The authors may wish to expand the rationale behind this step, as my understanding is that barrier function could still be assessed if proceeding directly to ALI culture.

2. The methods section overall is rather wordy. I think this could be significantly reduced to aid in readability – for example, the RNA extraction paragraph states the kit was performed according to manufacturer's instructions but then proceeds to describe each step.

3. The correct gene notation needs to be utilised throughout the manuscript.

4. I am concerned about the small sample size used for many of the results (typically n=4). I do not think this necessary precludes publication, but I would strongly advocate for the graphs to be updated to include the individual data points so readers can better understand any variation present. The authors should also consider adding a line addressing that this study is a ‘proof of concept’ rather than a detailed comparison against native tissue.

Specific comments:

5. Can the authors please provide additional information regarding the Animal Welfare Act that provides the ethics exemption (year and country).

6. Very minimal information is provided in regard to the native tissue that was utilised. A brief sentence should be included in the methods regarding the RNA collection of native tissue – was it from the same animals or was this banked sample?

7. Lines 76 to 79 repeat the same information provided in lines 70-73.

8. In Figure 1 it would be preferred if the actual TEER values were reported, rather than reporting a percentage. It is difficult to objectively determine whether a robust barrier was formed and compare to other studies when the results have been normalised to a previous timepoint.

9. Similarly, as mentioned earlier, figures 3-8 would significantly benefit from showing the individual data points on the graphs. Some of the error bars appear quite large and given the small number of samples it would help interpretation if individual data points are shown.

10. The arrows in Figure 2 are a bit difficult to see. Could the authors consider enlarging the arrows and perhaps making them less transparent/brighter?

11. There is a minor typo in line 138 – should be epithelial.

12. In line 140 and 141 the abbreviations Isc and Rt are used but a full definition is not provided.

13. The reference or references used in lines 188 and 189 appear to be corrupted/missing.

14. On line 223, the title should be shortened to ‘Ussing chamber studies show physiological transport capacity of 2D-organoids’ as the results do not present any native tissue to assess similarity.

15. The discussion would benefit from a generalised introductory paragraph summarising the main findings. The authors may also wish to remove the subheadings from the discussion, as it could be argued that everything they assessed is considered barrier function.

16. The ‘Barrier Function’ component of the discussion could be streamlined – I can understand what the authors are trying to say and agree with the findings but it jumps between topics with little consistency. For example, Line 272 states “To gain further insight and utilise the model for the study of pathophysiology, an analysis of gene expression was conducted.” Which comes directly after a discussion on mucin gene expression and is immediately followed by discussion of TEER results. Similarly, a strength of the study is the comparison to native tissue, but the mucin similarity is not discussed. Yet it does discuss the comparative expression of the mucins, which one could argue is not particularly relevant if the mucin expression is the same as native tissue.

17. Why do the authors speculate the Ussing chamber is not sensitive to carbachol induced changes when numerous other studies have used Ussing chambers to assess the effects of carbachol? Are they referring to the effect of this tissue specifically? Could it be a dosing issue? I am not familiar with the referenced papers assessing the carbachol response in native tissue, but were there experimental differences that could explain this?

Reviewer #2: The work conducted by Plenge et al in this manuscript characterises a 2D porcine colonic organoid model for studying the physiology and barrier function of the gut. The manuscript is overall well-written, and the techniques used are appropriate. The authors were able to generate a 2D organoid model within 10 days of seeding that recapitulates many of the morphological and transcriptional characteristics observed in the native colonic epithelium, albeit with slight functional differences shown via Ussing chambers. This in vitro model could be utilised for translational research in gastrointestinal physiology, as well as testing intestinal-specific responses to therapeutic applications.

However, my major concern about the study pertains to the extremely limited sample size – the authors only had a biological replicate of two pigs. It is unclear whether the n numbers referenced in the figure legends are technical replicates per animal or the combined total over two animals, or whether multiple samples were taken from each pig. Nevertheless, such a small sample size does not provide the reader with any confidence that the mean (SD) is accurate. Primary cell cultures are known to display larger variations in their morphology and physiology compared to immortalised cell lines. Hence the authors should conduct additional studies to ensure their observations are indeed real and do not occur by chance.

There are a few grammatical and typographical errors that should be amended before acceptance in the journal. The authors are referred to the uploaded feedback where a number of these errors (but not exhaustive) are highlighted. Other general comments are noted below:

1. Lines 75-79 are repeats of lines 70-73. The latter should be deleted.

2. Line 80: Although the procedure has been described in Hoffman et al, the authors should still state which part of the intestine the samples were derived from. Is it also the jejunum? Additional description would be helpful.

3. Line 82: what medium? The reference to S1 table should be made here. Clarify as 'organoid medium' (S1 Table).

4. Line 86: Again, authors are advised to specify which medium - organoid media

5. Line 125-6: genes should be italicised

6. Line 138: unclear how many technical replicates were performed for the Ussing chambers? Is it at least 3?

7. Line 140: First time the acronym is used - should be spelled out here. Short-circuit current (Isc) and resistance (Rt)

8. Line 142: Is Table 1 also the order that the substances were tested?

9. Line 142-3: If all cultures had aldosteroine pre-incubation, the highlighted sentence should immediately follow the first sentence after reference to Hoffman et al.

10. Technically, it would have been nice to see the native tissue also mounted on the Ussing chamber as a true control to the 2D organoids to compare functional physiology.

11. Line 166: what mean values are being compared? Also the same for line 168 – what is the measured variable being compared?

12. Line 172: I disagree with the statement “the number of biological experiments”. Please refer to comments above.

13. Lines 176-178: arguably these sentences could be in the methodology section as it provides background information on the methodological approach.

14. Lines 188-9: Please fix Error Reference source not found

15. Line 190: Why not consider a simple H&E cross-section to complement the data? It would enable the visualization of the different cell types too. Although it is understandable to make assumptions working with monolayers.

16. Do the cultures continue to differentiate past day 10? What happens to the culture if you continue to maintain it?

17. Line 268: please italicise genes.

18. Line 327: Agree with the authors comments. Perhaps this reasoning can be expanded to the junctional proteins too -, it may be useful to observe with immunofluorescence whether the structural orientation of the proteins is the same as in native colon tissue. But limitations are well acknowledged.

FIGURES

ALL figures: please show individual points instead of bar charts for transparency

1. Figure 1: authors are recommended to keep the y-axis origin as 0

2. Figure 3: please show individual points instead of bar charts for transparency. Native is missing an ‘e’ at the end of the legend.

3. Figure 4: as above please show individual points. Missing error bars for day 9 of ZO-1 gene.

4. Figure 7: include aldosterone name on the x-axis for ease of understanding

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: PONE-D-24-46719_reviewer.pdf

pone.0312989.s006.pdf^{(2.2MB, pdf)}

PLoS One. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989.r003

Author response to Decision Letter 1

7 Jan 2025

Additional Editor Comments:

Editor's comments to authors:

� The authors thank the editors for their efforts in summarizing the reviewers' feedback on our manuscript. We have addressed all individual points directly in our responses to the reviewers' comments below.

� We acknowledge that the use of organoids from two pigs is a limitation and have explicitly addressed this in the manuscript. Each biological replicate corresponds to a separate passage of 3D organoids used to create independent 2D cultures. For gene expression, RNA was isolated from four independent 2D cultures, each with two technical replicates, while transport studies involved four independent experiments with three technical replicates each. These details, along with updated figure legends showing individual data points, provide greater transparency and help readers assess variability. We have streamlined the Methods section by removing redundant details (e.g., RNA extraction protocols) and clarified that native tissue samples were derived from four different animals. Acronyms (e.g., Isc, Rt) are now defined upon first use.

� Thank you for your suggestions to enhance the interpretability of the Results section. We have updated all figures to include individual data points to better reflect variability. Regarding Figure 1, we chose to report relative TEER values normalized to day 8 (100%) to emphasize relative changes in barrier integrity across time points. To provide additional clarity, the mean absolute TEER value on day 8 has been included in the figure legend, facilitating comparisons with other studies.

� Thank you for your comment regarding the carbachol response. We acknowledge that the lack of response in our study is not due to Ussing chamber sensitivity, as similar carbachol concentrations have been used successfully in other studies. We believe the discrepancy may be due to post-translational modifications affecting transporter functionality, despite comparable gene expression to in vivo conditions.

Although including native tissue for direct comparison would have been ideal, it was not included in this study. The functional physiology of native tissue in Ussing chambers has been well-established (e.g. . Leonhard-Marek et al. (1), Klinger et al. (3), Bridges et al. (4), Inagaki et al. (5)), so a direct comparison was not necessary for the scope of this work. We have revised the manuscript to clarify these points.

� Thank you for your constructive feedback. We have revised the Discussion section to make it more concise and focused, starting with a brief summary of the main findings to provide clear context for readers. We have also removed subheadings and ensured smoother transitions between topics, such as mucin gene expression, TEER, and other findings. We hope these revisions improve the clarity and impact of the Discussion section.

Reviewers' comments:

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Generalised comments:

� Thank you for your insightful question. We utilized stem cells to construct the 3D organoids prior to transitioning to the 2D culture. This approach was adopted because 3D organoids contain a higher proportion of stem cells, which are essential for generating the monolayer in the subsequent 2D culture. While direct differentiation of stem cells in the 3D culture is a viable option, it is not be suitable for the transport studies we intend to investigate. Additionally, the use of an air-liquid interface (ALI) culture was not employed in this study, as our focus was specifically on using a submerged 2D culture system to facilitate functional assays, such as electrophysiological measurements in Ussing chambers. ALI cultures are more commonly used to study airway epithelium. Therefore, we have opted to utilise the 2D culture. An alternative approach would be to use an undifferentiated 2D monolayer to form a new 2D monolayer. However, this would be less efficient in terms of both time and cost. The use of 3D organoids provides a more reliable and consistent source of stem cells, which is essential for the formation and differentiation of the monolayer.

� Thank you for your comments on the methods section. We have conducted a comprehensive rearranging of the section and agree that certain details could be streamlined to enhance readability. Therefore, the methods section has been significantly shortened to enhance readability (line 112).

3. The correct gene notation needs to be utilised throughout the manuscript.

� The gene notation has been verified throughout the manuscript and updated as required.

� We are grateful for your considered response. We are aware of the issue of the small sample size and of the necessity to ensure that readers have a comprehensive understanding of the variability in our results. For the gene expression analysis, RNA was isolated from four independent 2D cultures of organoids, with each sample having two technical replicates to ensure the reliability and consistency of the data. Similarly, for the transport characteristics, four independent experiments were conducted, each with three technical replicates, thus facilitating an assessment of the reproducibility of the findings. This has also been included to the manuscript. It is also important to note that the organoids used in this study were derived from two pigs and the 3D organoid cultures were treated with high similarity to an immortalised cell line, thereby facilitating continuous passage and reproducibility due to their high amounts of stem cells. In this context, the passage of the 3D organoids used to generate the 2D cultures can be considered a biological replicate, thereby further reinforcing the robustness of the study. In accordance with your recommendation, we will incorporate individual data points into all the graphs, thus facilitating a more detailed visual representation of the data and any inherent variations.

In regard to the scope of the study, we acknowledge the necessity of explicitly delineating the context within which it is situated. Although this is an early-stage investigation, we have chosen not to frame the work as a proof of concept, as we believe this term can be somewhat limiting and often underestimates the value of studies that explore novel approaches. Instead, our intention is to position this work as a significant contribution towards a deeper understanding of the characteristics of organoid models, with the objective of stimulating further research in this area. We are committed to providing transparency regarding the experimental design, the limitations of the study, and the necessity for further validation in future studies. It is our intention that this approach will provide readers with a clear understanding of the study's intent and its position within the broader context of ongoing research in this field.

Specific comments:

5. Can the authors please provide additional information regarding the Animal Welfare Act that provides the ethics exemption (year and country).

� Thank you for your comment. We have included the information regarding the Animal Welfare Act in the document (line 71).

� Thank you for your feedback. The missing information has been included in the method section (line 109-110). The native tissue was sourced from different pigs from banked samples. The pigs were of the same breed and comparable age as the pigs which were used to isolated the crypts.

7. Lines 76 to 79 repeat the same information provided in lines 70-73.

� Thank you for bringing this to our attention, the repeated lines have been removed.

� We appreciate your feedback regarding the reporting of TEER values. In the figure, we chose to report the relative TEER values as a percentage normalised to the day 8 as 100 %, as this approach allows for easier comparison of the relative changes in barrier integrity across time points. However, we understand that presenting the actual TEER values could offer additional clarity. To address this, we have included the mean TEER value on day 8 along with the standard deviation in the figure description. We hope this additional information will assist in evaluating the robustness of the barrier formation and facilitates comparisons with other studies.

� Thank you for your comment. The figures 3-8 have been changed into discrete data points representing the results of the independent experiments.

10. The arrows in Figure 2 are a bit difficult to see. Could the authors consider enlarging the arrows and perhaps making them less transparent/brighter?

� Thank you for bringing this to our attention. The arrowheads have been modified to larger and, hopefully, brighter arrows to improve the visibility to the reader.

11. There is a minor typo in line 138 – should be epithelial.

� The typo has been fixed.

12. In line 140 and 141 the abbreviations Isc and Rt are used but a full definition is not provided.

13. The reference or references used in lines 188 and 189 appear to be corrupted/missing.

� Thank you for your feedback; we have made the requested changes by providing full definitions for Isc and Rt, correcting the references on lines 137.

� We are grateful for your valuable input. In response, we have incorporated a comprehensive introductory paragraph in the discussion section, summarising the primary findings (line 267 – 275). Additionally, we have eliminated the subheadings, aligning with your observation that all assessed elements pertain to the subject of barrier function.

� We have ref

Attachment

Submitted filename: 20241220_Response to Reviewers.docx

pone.0312989.s008.docx^{(44.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312989.r004

Decision Letter 1

Kevin Looi

21 Feb 2025

Dear Dr. Plenge,

Please submit your revised manuscript by Apr 07 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Kevin Looi, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The authors have done well to address the initial reviewer comments. I am satisfied with the science but have some minor comments to improve the manuscript:

The description of the native tissue has been added as requested; however, it is described without any prior introduction. For reader clarity, this should be slightly modified to make clear that native tissue was used for a comparator before they give the details of where the tissue was collected.

The discussion would benefit from addressing the small sample size – it was rationalized in the reviewer responses but is not specifically addressed in the manuscript. The inclusion of specific sample sizes was an important and welcome addition. However, it is still a small sample and that does limit the study findings. Given many readers will be critical of the work because of the limited sample number, the authors should pre-emptively address these concerns.

Line 283: Space missing between mucins and were

Line 314: Typographical error in ‘expression’.

Line 330: Typographical error in ‘replicated’.

Reviewer #2: Thank you to the authors for addressing many of the comments from the first review. The manuscript has improved clarity and transparency, with apt acknowledgment of model limitations and future investigations. This novel culture model will enable investigations into the pathophysiology of intestinal diseases, which are increasingly becoming prevalent.

Minor comments - Thank you for changing the figures to represent individual data points. However, error bars are missing from figures 3-8 (despite the figure legend indicating mean+- SD). While the individual datapoints allow readers to deduce the spread of the data, it would still be nice to have the T error bars to indicate the calculated SD. This can be added behind the datapoints.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989.r005

Author response to Decision Letter 2

25 Feb 2025

Review Comments to the Author

Reviewer #1: The authors have done well to address the initial reviewer comments. I am satisfied with the science but have some minor comments to improve the manuscript:

Thank you for your constructive comments on our manuscript. We have revised the methods section to introduce the native tissue as a comparator, providing an introduction.

Thank you for pointing this out. We have now addressed the small sample size directly in the manuscript. This should address concern from readers regarding this limitation.

Line 283: Space missing between mucins and were

Line 314: Typographical error in ‘expression’.

Line 330: Typographical error in ‘replicated’.

The typographical errors have been corrected.

Thank you for your positive feedback. We also appreciated your suggestion regarding the error bars. They have been added to the figures 3-8.

Attachment

Submitted filename: rebuttal letter.docx

pone.0312989.s009.docx^{(15.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312989.r006

Decision Letter 2

Kevin Looi

14 Mar 2025

Dear Dr. Plenge,

Please submit your clarification and comments by Apr 28 2025 11:59PM. If you will need more time to supply your clarifications and comments, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Kevin Looi, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments :

Dear Dr Plenge,

We are currently reviewing your manuscript, titled “Development and Characterization of a 2D Porcine Colonic Organoid Model for Studying Intestinal Physiology and Barrier Function”.

During our assessment, we identified a highly similar manuscript published in Journal of Visualized Experiments titled “Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut, DOI: 10.3791/67666.” The significant overlap in content between the two manuscripts raises concerns regarding potential duplication of publication.

To ensure transparency and adherence to ethical publishing standards, we kindly request clarification on the following points:

1) The relationship between the two manuscripts (e.g., whether they represent distinct studies or overlapping analyses).

2) Whether the submission to Journal of Visualized Experiments was made with the understanding that the content had already been accepted/published elsewhere.

3) Any additional context or justification for the similarities between the manuscripts.

We take issues of publication ethics very seriously and aim to resolve this matter promptly. Your response will help us better understand the situation and determine the appropriate next steps.

Please provide your clarification at your earliest convenience. If you have any questions or require further assistance, do not hesitate to reach out to the PLOS One team.

Thank you for your attention to this matter, and we look forward to your response.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: A final minor correction - I noticed that in Figure 3, 2nd panel (relative gene expression of MUC genes) the y-axis changed to -1 (compared to Revision 1). The y-axis origin should be 0, consistent with other panels. Please update figure 3. Otherwise, the authors have addressed all concerns.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989.r007

Author response to Decision Letter 3

19 Mar 2025

Dear Editor,

Thank you for your detailed feedback regarding the perceived overlap between our revised manuscript and our previously published work in the Journal of Visualized Experiments (DOI: 10.3791/67666). We appreciate the opportunity to clarify the relationship between these two studies.

1. Independence of the studies

Although both manuscripts use the two-dimensional porcine intestinal organoid model, they are based on independent experiments with different scientific objectives. The JOVE paper focuses primarily on establishing and visually demonstrating the protocol for generating 2D organoid cultures, thus providing a validated methodological framework. In contrast, our revised manuscript presents a detailed functional and molecular characterisation of the model, with particular emphasis on intestinal barrier integrity, ion transport and gene expression.

2. Experimental use of different donor animals and timeline

The two studies were performed using organoids derived from different donor animals, as reflected by the different animal registration numbers reported in the respective manuscripts (e.g. JOVE: TiHo-T-2023-15; revised manuscript: TiHo-T-2017-22 and TiHo-T-2024-5). In addition, the experiments were carried out in different time periods, further confirming their independence. Despite these differences, the high similarity of the results obtained in both studies underlines the robustness and reproducibility of the 2D porcine organoid model.

3. Justification for Methodological Overlap

As both studies use the same well-established organoid system, it is inevitable that certain parts of the materials and methods will be similar. However, the research questions, experimental approaches and data analyses are distinctly different between the two manuscripts. The JOVE study serves as a methodological reference, while the revised manuscript extends this work by exploring unique aspects of the model that are critical for understanding its physiological and pathophysiological properties.

In summary, although both manuscripts share a common experimental platform, they represent independent investigations with complementary but non-duplicative aims. We hope that this clarification reinforces our commitment to transparency and adherence to ethical publishing standards.

Sincerely,

Masina Plenge

Attachment

Submitted filename: Response to Editor.docx

pone.0312989.s010.docx^{(17.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312989.r008

Decision Letter 3

Mária A Deli

4 Apr 2025

Development and Characterization of a 2D Porcine Colonic Organoid Model for Studying Intestinal Physiology and Barrier Function

PONE-D-24-46719R3

Dear Dr. Plenge,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: Thanks to the authors for clarifying. I recognise that the two studies were completed during an independent time period and do not share the same animal registration numbers. The JoVe article describes the methodologic aspects of monolayer formation whereas the current revised manuscript explores the physiological characteristics.

line 348 - missing a space between "strong" and "foundation". This can be edited during typesetting.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0312989.r009

Acceptance letter

Mária A Deli

PONE-D-24-46719R3

PLOS ONE

Dear Dr. Plenge,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Mária A. Deli

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Table. Compostion of organoid medium.

(DOCX)

pone.0312989.s001.docx^{(21.8KB, docx)}

S2 Table. Composition of monolayer medium.

(DOCX)

pone.0312989.s002.docx^{(20.5KB, docx)}

S3 Table. Composition of differentiation medium.

(DOCX)

pone.0312989.s003.docx^{(18.3KB, docx)}

S4 Table. SYBR Green primers for SYBR Green® assay.

(DOCX)

pone.0312989.s004.docx^{(22.9KB, docx)}

S5 Table. TaqMan primers for TaqMan® assay.

(DOCX)

pone.0312989.s005.docx^{(19.8KB, docx)}

Attachment

Submitted filename: PONE-D-24-46719_reviewer.pdf

pone.0312989.s006.pdf^{(2.2MB, pdf)}

Attachment

Submitted filename: 20241220_Response to Reviewers.docx

pone.0312989.s008.docx^{(44.2KB, docx)}

Attachment

Submitted filename: rebuttal letter.docx

pone.0312989.s009.docx^{(15.4KB, docx)}

Attachment

Submitted filename: Response to Editor.docx

pone.0312989.s010.docx^{(17.1KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Development and characterization of a 2D porcine colonic organoid model for studying intestinal physiology and barrier function

Masina Plenge

Nadine Schnepel

Mathias Müsken

Judith Rohde

Ralph Goethe

Gerhard Breves

Gemma Mazzuoli-Weber

Pascal Benz

Roles

Abstract

Introduction

Materials and methods

Culture of 3D-organoids

Culture of 2D-organoids

RNA isolation and reverse transcription

Quantitative real-time PCR

Ussing chamber experiments – transport physiology

Table 1. Application for Ussing chamber experiments (all chemicals were obtained from Sigma-Aldrich, Darmstadt, Germany).

Scanning electron microscopy

Data analysis and statistics

Results

TEER progression shows robust cellular monolayer integrity

Fig 1. Change of the transepithelial electrical resistance (TEER) during the 2D cultivation of the porcine colonic organoids.

Scanning electron microscopy reveals different cell types

Fig 2. Visualization of microvilli, goblet cells and mucus in the 2D culture of porcine colonic organoids.

RT-PCR demonstrates gene expression concordance between 2D organoids and native tissue

Fig 3. Gene expression of calcium dependent chloride channel (CaCC), cystic fibrosis transmembrane conductance regulator (CFTR), the domains of epithelial sodium channel (ENaC.

Gene expression of tight junction proteins during cultivation is constitutive

Fig 4. Relative gene expression of the tight junction proteins claudin 2 (CLDN 2), claudin 3 (CLDN 3), occludin (OCLN) and zonula occludens 1 (ZO-1) in 2D-culture of porcine colonic organoids on different cultivation days (day 4, day 7, day 9 and day 10) and native porcine colon.

Ussing chamber studies show physiological transport capacity of 2D-organoids

Fig 5. Basal and maximal Isc values of 2D culture of organoids after addition of DMSO, glucose, forskolin and carbachol using Ussing chamber experiments.

Fig 6. Comparison of the Isc in 2D culture of colonic organoids with different concentrations of amiloride.

Fig 7. Comparison of the basal Isc in 2D culture of colonic organoids, which were incubated with 0.3 nM, 1 nM or 3 nM aldosterone for 3 h, 8 h or 48 h, while the control was not incubated with any aldosterone.

Fig 8. Comparison of the.

Discussion

Supporting information

Data Availability

Funding Statement

References

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Decision Letter 0

Kevin Looi

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Author response to Decision Letter 1

Decision Letter 1

Kevin Looi

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Author response to Decision Letter 2

Decision Letter 2

Kevin Looi

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Author response to Decision Letter 3

Decision Letter 3

Mária A Deli

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Acceptance letter

Mária A Deli

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Fig 5. Basal and maximal I_sc values of 2D culture of organoids after addition of DMSO, glucose, forskolin and carbachol using Ussing chamber experiments.

Fig 6. Comparison of the I_sc in 2D culture of colonic organoids with different concentrations of amiloride.

Fig 7. Comparison of the basal I_sc in 2D culture of colonic organoids, which were incubated with 0.3 nM, 1 nM or 3 nM aldosterone for 3 h, 8 h or 48 h, while the control was not incubated with any aldosterone.