{"paper_id":"306f59d5-3da2-4963-a283-5f67759dcef8","body_text":"Paediatric Intestinal Enteroids Derived from Very Early Onset Inflammatory Bowel Disease Reveal Differences in Growth, Morphology, and Barrier Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Paediatric Intestinal Enteroids Derived from Very Early Onset Inflammatory Bowel Disease Reveal Differences in Growth, Morphology, and Barrier Function Vijayalekshmi Balakrishnan, Bhuvaneswari Selvam, Sandya Rani B, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8034357/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract ​Paediatric inflammatory bowel disease (IBD) presents more aggressively than adult-onset disease; however,​ epithelial pathophysiology remains poorly understood due to limited access to patient tissue. Human intestinal enteroids (HIEs) are a powerful model for investigating disease phenotypes and heterogeneity. We characterized duodenal HIEs from two children with Crohn's disease(ED-71 and ED-81) and one control with intestinal obstruction(ED-19), all aged < 2years. Analyses included growth curves, epithelial cell heights, immunofluorescence, histology, electron microscopy, gene expression and intestinal permeability. All HIEs maintained consistent growth in vitro , with ED-81 displaying the steepest trajectory. Morphological examination revealed variations in epithelial cell height, with ED-71 and ED-81[median(95%CI) 10.2(7.76–11.4) and 12.5(11.5–15) µm] displaying a more flattened appearance than ED-19[18.7 (16.8–21.0) µm, p-value:<0.001]. HIE differentiation into intestinal cell types was confirmed by gene expression and microscopy. Intestinal permeability assays indicated compromised barrier integrity in IBD-derived monolayers, with ED-81 exhibiting the highest baseline permeability(5.27%) and EGTA-induced disruption (50.03%) compared to ED-19(3.88% and 36.82%). Pediatric intestinal enteroids in IBD demonstrate differences in epithelial growth, morphology, and barrier function. HIEs serve as a potential translational model for pediatric IBD, facilitating the study of epithelial pathophysiology and guiding precision therapy. Further studies with more pediatric HIEs are needed to confirm these findings. Health sciences/Diseases Health sciences/Gastroenterology Health sciences/Medical research Inflammatory Bowel Disease Patient-Derived Enteroids Intestinal Permeability Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and Ulcerative colitis (UC), presents significant challenges, affecting patients and burdening the healthcare system globally. Despite historically being reported more often in Europe and North America, the geographic distribution of IBD has since expanded, affecting populations in countries previously considered low-risk areas[ 1 – 4 ]. This rising trend in the incidence of IBD incidence has been attributed to multiple a range of factors such as increased awareness, improved diagnostics, and better access to specialized healthcare, and societal changes such as urbanization, adoption of westernized diet, and improved hygiene[ 5 ]., which This underscores the need for a deeper understanding of the pathogenesis and progression of IBD in these populations. This is even more important in pediatric IBD, where disease characteristics and treatment responses vary from those in adults[ 3 , 6 ]. Pediatric IBD is a distinct and often more aggressive subtype that is frequently associated with extensive intestinal involvement, growth failure, and complex therapeutic challenges[ 7 ]. The increasing prevalence of pediatric IBD is particularly significant among younger children, under the age of ten[ 8 ]. However, the study of pediatric IBD pathophysiology remains limited by the difficulty in obtaining sufficient patient-specific tissue, the absence of suitable animal models, and the inadequacy of conventional cell culture techniques to replicate the complexity of the intestinal epithelium. Recent advances in three-dimensional intestinal organoid culture systems from patient-derived intestinal crypts or pluripotent stem cells have provided unprecedented opportunities to model human intestinal physiology and pathology ex vivo [ 9 , 10 ]. Tissue-derived human intestinal enteroids (HIEs) closely mimic the cellular diversity, polarity, and functional characteristics of the native intestinal epithelium and can be expanded over time while maintaining donor-specific traits[ 11 – 13 ]. These have proven especially useful in the study of epithelial barrier function, host–microbe interactions, and responses to pharmacologic agents in IBD[ 14 – 17 ]. Yet, paediatric-derived HIEs are underutilised despite their potential to reveal age-specific disease mechanisms and inform targeted therapy. Additionally, there is poor representation of available HIE from low-and-middle income countries (LMIC). In this study, we utilised an established HIE biobank to characterise duodenal enteroids from paediatric IBD cases and a non-IBD age-matched control in southern India. We employed morphological and intestinal permeability assays to compare epithelial cell properties across IBD and control HIE lines. By integrating these methods, we aim to advance the use of paediatric enteroids as a translational platform for intestinal disease biology in IBD, evaluate therapeutic responses and guide precision therapy. Results Clinical History, Tissue Histopathology and Other Laboratory Parameters of IBD cases and control Three paediatric subjects who were enrolled for duodenal tissue-derived HIE generation were included and comprised two IBD cases (derived HIEs ED-71 and ED-81) and one non-IBD control (ED-19). ​ED-71 was a 1-year-old female who underwent endoscopy for complaints of chronic blood mixed diarrhoea and failure to thrive. Colonoscopy showed multiple ulcers in the entire colon except the descending colon, and biopsy changes indicated Crohn’s disease. Duodenal biopsy showed minimal chronic duodenitis and increased intraepithelial lymphocytes. There was no stricture or fistula, and he improved with immunomodulator therapy(azathioprine and mesalazine). ED-81 was a 2-year-old male who presented with a history of fever, diarrhoea with occasional blood in the stool and failure to thrive. Colonoscopy showed multiple colonic ulcers from caecum to sigmoid colon and histopathology showed features of Crohn’s disease. Duodenal biopsy showed moderate chronic duodenitis. He also had no strictures or fistula and was treated with immunomodulators(methotrexate and mesalazine)​. Both ED-71 and ED-81 cases are currently 9 and 10 years old, and at the last follow-up (2025 and 2024, respectively) were continuing treatment for Crohn’s disease. The control, ED-19, was an 11-month-old male who underwent intestinal surgery for neonatal intestinal obstruction and was diagnosed postoperatively with duodenal atresia requiring duodenoplasty. Duodenal mucosal biopsy did not show chronic inflammation. On the last follow-up at the age of 9 in 2025, he was healthy, and laboratory parameters were normal. Laboratory parameters for the two cases and the control are shown in Table 1 . Table 1 Clinical and Biomarker Profile of Study Participants ED-71 ED-81 ED-19 Age 1yr 2 yr 11months Sex Female Male Male Diagnosis Crohn’s disease Crohn’s disease Intestinal Obstruction due to duodenal web Biopsy reports Duodenum: Minimal chronic duodenitis with focal increase in intraepithelial lymphocytes with minimal activity (~ 30/100 enterocytes). Ileum: Mild chronic active ileitis with mild villous atrophy and increased intraepithelial lymphocytes Cecum to rectum: Mild to patchy moderate colitis with mild activity Duodenum: Mild to moderate chronic active duodenitis with patchy regenerative change. Ileum: No significant lesion Cecum to rectum: Patchy chronic active colitis with chronic ulceration and fibrosis. Consistent with duodenal web with normal mucosa and submucosa (excision biopsy) Fecal Calprotectin > 2000µg/g > 2000µg/g 60.62µg/g Myeloperoxidase (MPO) 8962.06ng/mL 101381.33ng/mL Below detection limit Growth Curve Analysis To evaluate the proliferative capacity of the paediatric duodenal tissue-derived intact HIEs, we monitored growth over a 7-day culture period. As shown in Fig. 1 , all HIE lines exhibited time-dependent increases in diameter of the enteroids, indicating sustained growth. ED-81 HIEs showed the steepest growth trajectory, particularly between Days 4 and 6. In contrast, ED-71 HIEs showed moderate growth, while ED-19 HIEs displayed the slowest growth trajectory. These differences suggest inherent variability in growth potential among donor-derived lines. Differentiation of HIE To evaluate epithelial lineage differentiation in the pediatric HIEs, we performed immunofluorescence staining and confocal imaging of lineage-specific markers on HIEs (Fig. 2 ). Enterocytes of all three HIE lines identified by sucrase-isomaltase (SI) staining localized prominently to the apical brush border of the epithelium, confirming mature absorptive cell differentiation. Goblet cells detected using mucin-2 (MUC2) staining showed cytoplasmic staining and localization towards the luminal aspect of the HIEs, indicative of mucin production. Paneth cells visualized by lysozyme (LYZ) staining, showed a discrete punctate red signal. Enteroendocrine cells, marked by chromogranin A (CHGA) staining, were sparsely distributed but positive within the epithelial layer. To investigate epithelial alterations in pediatric IBD, we assessed gene expression markers representing epithelial lineages and stemness compared to controls. SI expression, an enterocyte differentiation marker, was significantly reduced in both ED71 and ED81 IBD cases (p < 0.0001) compared to the ED-19 control, indicating impaired epithelial maturation in IBD HIEs. The Paneth cell marker LYZ and the goblet cell marker MUC2 were significantly elevated in ED81 (p < 0.0001) while CHGA, an enteroendocrine marker, was downregulated in both ED71 and ED81 (p < 0.0001). LGR5 which is a stem cell marker showed increased expression in ED81 and decreased expression in ED71 compared to the ED-19 control. KI67, a marker of proliferation, was significantly elevated in ED71 and 81 compared to ED19 (p < 0.001), indicating hyperproliferative responses in IBD HIE (Fig. 3 ). TLR4, TLR3, CXCL-8, and IFN-β gene expression was significantly upregulated in ED81 (p < 0.01 to p < 0.0001), whereas they were downregulated in ED71. These variations could be attributed to donor-specific differences in innate immune gene expression or polymorphisms or potentially to the disease stage of IBD. Barrier function and structural integrity Cell height measurements revealed significant variability among the pediatric tissue-derived HIE monolayers (Fig. 4 A). Quantitative analysis demonstrated that ED19 monolayers had the highest mean cell height, followed by ED81, while ED71 exhibited the lowest mean height. Statistical analysis indicated that the cell height of ED71 was significantly reduced compared to both ED19 ( p < 0.05) and ED81 ( p < 0.05). These findings are supported by hematoxylin and eosin (H&E) staining in intact HIEs (not shown), which showed taller and more columnar epithelial morphology in ED19. The variation in cell height may reflect differences in epithelial differentiation and structural integrity among the ED lines, potentially correlating with disease severity or barrier function. Trans-epithelial electrical resistance (TEER) and FITC-dextran flux assays were performed on differentiated HIE monolayers (grown in transwells) derived from ED19, ED71, and ED81 to evaluate epithelial barrier integrity. At baseline (0hrs) all three HIE lines exhibited high TEER values, indicating intact monolayers. After 2hrs, TEER values decreased significantly on EGTA treatment in all three HIE lines, suggesting a potential compromise in barrier function (Fig. 4 B). FITC-dextran flux revealed significant differences in permeability upon treatment with EGTA. Under untreated conditions, all three lines maintained low permeability; however, baseline FITC-dextran flux was slightly higher in ED81 than ED19 and ED71, suggesting inherently weaker barrier properties in the former. Upon exposure to treatment with EGTA, all HIEs exhibited a marked increase in FITC-dextran flux (Fig. 4 C). Notably, ED81 showed the highest permeability, followed by ED71 and ED19. These results indicate that the ED81-derived monolayer is the most susceptible to barrier disruption, with elevated baseline permeability and heightened sensitivity to EGTA treatment. Paradoxically, an upregulation in the expression of genes associated with tight junctions, specifically Claudin-1 and ZO-1, was observed in ED81 (Fig. 4 D). Ultrastructure of HIE Electron microscopy of all three HIEs showed cells arranged in rings and nests. Some of the rings were lined by flattened to cuboidal epithelium with rudimentary microvilli on the inner luminal surface. Others were lined by tall columnar epithelium with more prominent microvilli. Some of the columnar cells had mucin vacuoles in the apical cytoplasm, suggesting differentiation towards a goblet cell phenotype. (Fig. 5 A, C, D). Apart from rings, ‘nests’ of epithelial cells were also seen. Some of the nests had small lumina between the tall columnar cells, the luminal surface of which showed prominent microvilli (Fig. 5 B). All epithelial cells in both rings and nests showed apical intercellular tight junctions and desmosomes were seen between lateral cell margins. ED71 HIEs showed rings and nests with focal prominent clumping of nuclear chromatin, consistent with pyknosis, an early change of apoptosis. These cells also showed increased size and number of lysosomes. Many shrunken, dark cells with nuclear fragments containing condensed chromatin and degenerate organelles suggestive of apoptosis were also seen in ED71, some being extruded into the lumen of rings or smaller lumina of nests (Fig. 5 E, F). Discussion In this study, we characterised paediatric patient-derived HIEs from two cases with IBD and one non-IBD control, generating a comparative platform to assess characteristics of the intestinal epithelium pertinent to paediatric IBD. By combining morphological, molecular, and permeability assays, we offer a thorough examination of HIE characteristics in both IBD and non-IBD conditions, highlighting the value of paediatric HIEs for translational research in IBD. Reports from LMICs remain limited, and pediatric inflammatory bowel disease (IBD) human intestinal enteroids (HIEs) from these regions are largely underrepresented. To our knowledge, this study is the first to characterize pediatric patient-derived HIEs from South Asia, offering a unique perspective on IBD biology in a region where the incidence and disease phenotype are increasingly recognized but insufficiently explored. The HIE growth curve analysis revealed significant variability in proliferative capacity among patient-derived lines, with the Crohn's-derived line (ED-81) exhibiting the steepest growth trajectory, while the healthy control (ED-19) demonstrated the slowest growth. These differences may reflect disease-specific epithelial adaptations or altered crypt biology associated with chronic inflammation, as previously observed in adult IBD studies[ 9 , 14 ] or could be individual-specific.[ 18 ] Similar variability has been observed in adult IBD organoids, where studies involving single-cell analysis and biobanks have identified growth differences linked to disease activity and variations between patients[ 19 – 21 ]. Additionally, epigenetic and transcriptional profiling have demonstrated that epithelial programs can categorize disease severity and phenotype[ 14 , 17 ], reinforcing the notion that the variability seen in pediatric HIEs represents authentic disease-associated adaptations. All three pediatric HIEs exhibited the anticipated epithelial lineages; however, HIEs derived from IBD demonstrated altered lineage and stemness markers, indicative of impaired maturation and increased proliferative capacity[ 9 ]. Similar variability in innate immune gene expression has been reported, largely attributable to donor heterogeneity and inflammatory state at biopsy[ 18 ]. Several studies have noted enrichment of Paneth cell markers (e.g., LYZ) and upregulation of antigen-presentation pathways in IBD colonoids, suggesting heightened epithelial immune activation[ 22 , 23 ]. Interestingly, Kelsen et al. observed reduced growth efficiency in pediatric IBD colonoids, measured as lower crypt-to-spheroid conversion, despite concurrent Paneth and immune marker upregulation[ 22 ]. This contrast may reflect differences between conversion efficiency and long-term expansion potential[ 24 , 25 ], as well as disease stage, inflammatory stress, or altered responsiveness to niche factors[ 18 ]. Collectively, these findings suggest that epithelial remodeling in inflammatory bowel disease (IBD) encompasses both the augmentation of immune lineage signatures and changes in growth dynamics, highlighting the complex nature of responses observed in patient-derived organoids. Histological analyses revealed that ED-71 monolayers show reduced epithelial height and flattened morphology compared to ED-19 (columnar) and ED-81 (intermediate), reflecting impaired differentiation. Similar morphological alterations in IBD-derived colonoids show reduced size, altered polarity, and loss of budding structures under inflammatory contexts[ 26 ]. Epithelial flattening in organoid and monolayer systems correlates with compromised barrier integrity and heightened susceptibility to injury, with such phenotypes reversed by treatment[ 26 ][ 27 ]. These changes mirror in vivo crypt atrophy and epithelial distortion—established markers of disease activity in IBD[ 28 – 30 ]. Our patient-derived monolayer phenotypes recapitulate the structural features seen in vivo, suggesting epithelial height metrics may indicate differentiation capacity and barrier functionality. The ultrastructural evidence of apoptosis in the enteroids of ED71 suggest increased cell death that could contribute to the compromised barrier functionality. Our intestinal permeability assays revealed donor-specific differences: ED-81 showed higher baseline permeability and greatest EGTA-induced FITC flux, suggesting tight-junction fragility. Similar barrier defects occur in IBD organoid models, where reduced TEER and increased flux correlate with impaired differentiation[ 31 , 32 ]. EGTA chelation probes tight-junction stability, with increased sensitivity reflecting weakened junctional integrity[ 33 ]. Elevated fecal calprotectin and MPO correlate with mucosal permeability[ 34 ], consistent with ED-81's barrier vulnerability. These findings support an epithelial contribution to IBD pathology beyond immune-mediated injury[ 31 , 35 ] Although ED81 HIE showed elevated expression of tight junction genes like Claudin-1 and ZO1, increased permeability in FITC-dextran assays suggests a disconnect between gene expression and barrier integrity. The increased expression of innate immune genes like TLR3, TLR4, and IFN-β indicates inflammatory pathway activation, which disrupts epithelial junctions through cytokine-mediated modulation and increased cell turnover. These inflammatory signals may override repair mechanisms, causing barrier dysfunction despite upregulated junctional transcripts. Animal studies using DSS-treated organoids confirmed that inflammatory insults degrade tight-junction integrity and increase paracellular permeability, shown by increased FITC-dextran flux and has shown increased expression of Claudin-1 [ 33 ]. In adult IBD colonoids, reduced TEER and disrupted tight junction integrity persist in remission samples[ 31 , 36 ]. The major limitation of this work is the small sample size, which restricts the generalizability of donor-specific findings. However, our results align with prior reports showing that patient-derived intestinal organoids retain donor- and disease-specific molecular and functional signatures. While our sample size is limited, the robust differences and reproducible results observed indicate feasibility of the approach and that additional studies with larger numbers of subjects are needed to make meaningful conclusions. Further these HIEs were derived from duodenal biopsies, HIEs derived from colonic biopsies could have provided more information. This exploratory study therefore supplements this growing body of literature by extending these observations to pediatric HIEs and highlighting the potential of this model to interrogate patient-level variation in epithelial physiology. Co-culture with immune cells or microbiota could further enhance the physiological relevance of this model. Future studies expanding patient numbers and incorporating transcriptomic and proteomic analyses will help identify molecular drivers of observed phenotypes. This platform could evaluate reactions to cytokines, microbial challenges, and therapeutic agents to reveal age and disease-specific vulnerabilities, aiding in precision therapy for paediatric IBD. Methods Patient Recruitment and Establishment of HIE Biobank A paediatric HIE biobank was established at Christian Medical College (CMC), Vellore, India between 2016 to 2018 as part of a study to establish HIE from children with environmental enteric dysfunction (EED). Briefly, children under the age of five years undergoing abdominal surgery (n = 26) or endoscopic biopsies (n = 25) for a range of clinical indications were recruited after written informed consent from an adult caregiver (parent or legal guardian) was obtained by a trained research nurse. Along with intestinal biopsy tissue, a stool sample and a blood sample were also collected, processed and banked. Derivation of enteroids from intestinal tissue were conducted in accordance with established protocols [ 37 , 38 ], as detailed in supplementary methods with 19 duodenal, 10 jejunal and 12 ileal HIE for a total of 41/51 successfully grown HIEs and banked in liquid nitrogen for further characterisation and future studies. IBD and control HIE for characterisation : Three duodenal HIEs were selected for in-depth characterisation: two from patients diagnosed with very early onset IBD (Crohn’s disease) and one without any abnormalities in tissue histopathology, serving as a non-IBD age-matched control. The diagnosis of Crohn's disease was ascertained by gastroenterologists in accordance with the Asia Pacific criteria[ 39 ] with thorough assessment of clinical manifestations, endoscopic observations, histopathological evaluation, and radiological findings. Fecal calprotectin (Epitope Diagnostics, California) and myeloperoxidase (MPO, Immundiagnostik, IDK, Germany) were also tested. HIEs were revived from cryopreserved stocks at passage 3 or 4 and subsequently expanded as mentioned in the supplementary methods. For differentiation, expansion media was replaced with differentiation media at Day 4 and all assays were performed with differentiated HIE at Day 7. All experiments were performed using HIE lines at passage 6 with two technical replicates. To ensure reproducibility, gene expression and all functional assays were independently validated using three separate cryopreserved vials that were thawed and expanded separately. HIEs were grown as monolayers in transwells as described in the supplementary methods for cell height and permeability assays. Growth Curves Growth curve measurements were performed using Image J/Fiji software version 2.14.0/1.54f by calibrating images to a known scale bar (10 µm) and measuring intact 3D HIE diameter using the line tool. All measurements obtained from 10 randomly selected HIEs per replicate at each time point (days 1 to 7) were recorded in micrometres, and output data were saved as TIFF images and corresponding measurement files. LOESS smoothing was applied to visualise growth trends, and 95% confidence intervals were plotted to indicate variability. Transmission Electron Microscopy (TEM) of HIE Intact 3D HIEs were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 2 mM CaCl₂ for 3 hours at 4°C. After washing in sodium cacodylate-sucrose buffer, samples were post-fixed in 1% OsO₄ in 0.2 M sodium cacodylate buffer for 2 hours at 4°C. Samples were dehydrated through graded ethanol (50–100%), treated with propylene oxide, and infiltrated with epoxy resin over three changes. HIEs were embedded and polymerized at 60°C for 48 hours. Semi-thin sections (0.5–1 µm) were cut using Leica UCT ultramicrotome and stained with toluidine blue in borax. Ultrathin sections (~ 50–70 nm) were cut, mounted on copper grids, and stained with uranyl acetate and Reynold's lead citrate. Sections were examined using a FEI Tecnai T12 microscope at 120 keV. Immunofluorescence Staining and Confocal Imaging of HIE : Intact 3D HIEs grown in Matrigel domes were fixed with 4% formaldehyde for 1 hour at room temperature (RT), followed by three PBS (1X) washes. Samples were blocked and permeabilised with 5% fetal bovine serum (FBS, MP Biomedicals, Ohio) and 2% Triton X-100 for 2 hours at RT. Primary antibodies (1:50 in 5% FBS, 0.25% Triton) were added and incubated at RT in the dark with gentle shaking. After washing, secondary antibodies (1:1000) were applied for 3 hours at RT. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, California) (1:1000) for 30 minutes, followed by three PBS washes and clearing for 2 hours. Slides were mounted with Prolong gold antifade reagent (Invitrogen, California) and sealed. Imaging was performed using confocal microscope with appropriate fluorescent filters. Antibodies are listed in Supplementary Table 5. Samples were imaged using an Olympus Laser Scanning Confocal Microscope system (spectral version), model Olympus FV1000. Images were captured at 20X and 60X magnifications using Olympus Fluoview Ver.3.16 software. Gene Expression of HIE Intact 3D HIEs were incubated in cold water for 10 minutes, followed by adding 800 µL cold PBS and centrifugation at 800 × g for 3 minutes at 4°C. The pellet was lysed in 350 µL RLT (RNA Lysis Tissue) buffer with 1% β-mercaptoethanol, incubated for 10 minutes at room temperature, and mixed with 350 µL 70% ethanol. The lysate was processed using spin columns (Qiagen, Germany) for RNA isolation. RNA was quantified using a Nanodrop, and 500 ng–2 µg was reverse-transcribed into cDNA (cDNA REV TRANS KIT, ThermoScientific, USA) in a 20 µL reaction using manufacturer’s protocol. Gene expression was assessed by qPCR using 5 ng/µL diluted cDNA, TaqMan Fast Advanced Master Mix, and gene-specific primer-probe pairs. This included Sucrose Isomaltase (SI), Lysozyme (LYZ), Chromogranin A(CHGA), LGR5, CD44, KI67, Z0-1, Claudin-1,Toll-like receptor4 (TLR4), TLR3, CXCL-8, and Interferon β (IFN- β) (listed in Supplementary Table) Flex (Applied Biosystems ™ ) as per manufacturers protocols. Negative controls included no-template and no-reverse transcriptase reactions to confirm the absence of contamination. GAPDH was used for normalisation. Cell Height Measurement Cell height was assessed in Hematoxylin and Eosin (H&E)-stained sections of differentiated HIE monolayers grown in transwells and then mounted on slides. Only typical enterocytes were included; cells with inclusions, vacuoles, or multilayered stacking were excluded. Heights were measured from the base of the cell to the apical surface, bisecting the nucleus. Three cells were measured per image for each sample across three separate images. Measurements were conducted using ImageJ/Fiji version 2.14.0/1.54f software after setting the image scale using the embedded scale bar. A blinded analyst performed all analyses to eliminate bias. Intestinal Permeability Assays : Transepithelial electrical resistance (TEER) of differentiated HIEs grown on transwells (Corning, New York) was recorded using Millicell ERS-2 before and after a 2-hour treatment with 10mM EGTA (Millipore, Merck, USA). FITC-Dextran (Sigma-Aldrich, USA) serial dilutions (5 mg/ml) for the standard curve and a 1:100 working dilution were prepared during incubation. Following treatment, the media was replaced with 100 µL of diluted FITC-Dextran and incubated for two additional hours. Final TEER was measured, and 50 µL each of apical, basolateral, and standard curve samples were transferred to a 96-well plate. To assess permeability, fluorescence was read at 490 ± 9 nm excitation and 520 ± 9 nm emission using TECAN Infinite M200 PRO reader. Statistical Analysis All analyses were conducted using R version 4.4., and all images were analysed using ImageJ/Fiji version 2.14.0/1.54f. Data from 3 biological and technical replicates were included, and for imaging-based analyses, at least 10 measurements per replicate were obtained to ensure statistical robustness. For experiments involving continuous variables such as HIE diameter, cell height, gene expression, and permeability assays, data were analysed using descriptive statistics (mean ± standard deviation). For comparison between control and IBD HIE lines, unpaired two-tailed Student’s t-test was used. P-values < 0.05 were considered statistically significant. This HIE biobank establishment and this study was approved by the institutional ethics committee at Christian Medical College, Vellore. All methods were carried out in accordance with relevant ethical guidelines and regulations. Declarations Acknowledgements We thank the CSCR Confocal Imaging Facility at CMC ,Vellore for technical support, and the Mathais Zilbauer Lab for valuable guidance in further optimising cell culture media. We also thank study nurse Charlet Suresh, and our colleagues Roshni Parameswaran, Shanmugam, Xi-Lei Zeng, Lakshmi Nirmal Somasundaram, and Ketki Patil for their assistance during the project. Most importantly, we are grateful to the infants and children and their parents for enrolling in this study and providing samples. Author contributions B.V. contributed to study design, performed laboratory work and data analysis, and drafted the manuscript. B.S., S.B., and A.P. conducted laboratory experiments and A.J.J, A.K.D., and E.S. were involved in clinical data acquisition. M.E. H.W and S.R. contributed to study design and provided training and resources. S.S.R.A. supervised the study, contributed to study design, provided resources and finalised the manuscript. All authors meet the criteria for authorship, reviewed the manuscript, and approved the final version for submission. Data availability statement Data available with the corresponding author on request. Additional Information Conflict of Interest The authors have declared no conflicts of interest. Funding sources The EED study that led to the development of the HIE biobank was funded by the Gates Foundation OPP1108228 to Tufts Medical Center, Boston, MA, USA; Baylor College of Medicine, TX, USA and Christian Medical College, Vellore, India. Characterisation of HIE from IBD cases was supported by institutional funds at the Wellcome Trust Research Laboratory, CMC, Vellore. References Ng, S. C. et al. Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn’s and colitis epidemiology study. Gastroenterology 145 , 158-165.e2 (2013). Ng, S. C. et al. Population Density and Risk of Inflammatory Bowel Disease: A Prospective Population-Based Study in 13 Countries or Regions in Asia-Pacific. Am. J. Gastroenterol. 114 , 107–115 (2019). Gordon, H. & Langholz, E. The EpiCom Survey-Registries Across Europe, Epidemiological Research and Beyond. J. Crohns Colitis 11 , 1019–1021 (2017). Yewale, R. V. et al. 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intervals.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/a7b7dc1a107ccd65f337ac85.png\"},{\"id\":96243052,\"identity\":\"2e1648a0-4e15-4d27-8572-3dad676c8d5b\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:15:21\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":471472,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eConfocal imaging of immunofluorescence staining of differentiated pediatric HIEs. Representative confocal microscopy images showing expression of intestinal epithelial lineage markers in differentiated HIEs. (A) Sucrase-isomaltase (SI, green) indicates brush border enterocytes(ED81). (B) MUC2 (red) indicates goblet cells(ED81) (C) LYZ (lysozyme, red) indicates Paneth cells, with punctate expression near crypt regions(ED19) (D) CHGA(ED81) (chromogranin A, red) identifies sparse enteroendocrine cells. Nuclei are counterstained with DAPI (blue). Scale bars = 20 µm for 60X magnification and 60 µm for 20X magnification as indicated\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/a5fbea815f398d138da5c8fa.png\"},{\"id\":95895754,\"identity\":\"a9e093b9-6ce3-44e2-8b11-25e691f1c6b5\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:14:48\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":151493,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAltered gene expression in pediatric IBD HIEs compared to healthy control.\\u003cbr\\u003e\\nqPCR analysis of epithelial and immune markers in human HIEs derived from healthy control (ED19, red) and IBD patients (ED71, green; ED81, blue). Shown are relative expression levels normalised to GAPDH (2^-ΔCt) for enterocyte (SI), Paneth cell (LYZ), enteroendocrine (CHGA), stemness (LGR5), proliferation (KI67), and innate immune response genes (TLR4, TLR3, CXCL-8, β-IFNB). Data represent mean ± SD of triplicate qPCR measurements. Significance *p \\u0026lt; 0.05; **p \\u0026lt; 0.01; ***p \\u0026lt; 0.001; ****p \\u0026lt; 0.0001; ns = not significant .\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/12daf7f0e18c20379ae55a52.png\"},{\"id\":96243181,\"identity\":\"16c20d83-8787-43ca-95c3-7b897d36f21d\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:15:49\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":102159,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA, Cell Height Measurements of HIE Monolayers. Box plot showing epithelial cell height (in µm). Statistical significance indicated by asterisks (p \\u0026lt; 0.05). Outliers are shown as dots. B, C TEER Measurement and FITC-Dextran Permeability Assay in HIE Monolayers: Mean trans-epithelial electrical resistance (TEER) values of HIE monolayers derived from ED19, ED71, and ED81 measured at 2 hrs post-treatment. Error bars represent standard deviation. Mean FITC-dextran flux (µg/mL) across ED19, ED71, and ED81 HIE monolayers in untreated and treated conditions at 2 hours post-treatment\\u003cstrong\\u003e.\\u003c/strong\\u003e qPCR analysis of tight junction markers in human HIEs derived from healthy control (ED19, red) and IBD patients (ED71, green; ED81, blue). Shown are relative expression levels normalised with GAPDH (2^-ΔCt) for tight junction (ZO, Claudin-1). Data represent mean ± SD of triplicate qPCR measurements.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/699da1571170077fde1cebc9.png\"},{\"id\":95895753,\"identity\":\"8b39bcb3-8284-42c5-83ee-c6bec749a198\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:14:48\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":323150,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTransmission electron microscopy (TEM) images of ultra-thin sections: Representative images from enteroids demonstrate rings and nests. (A) Part of a ring lined by flattened to cuboidal epithelium, with rudimentary apical microvilli (ED81). (B) Part of an epithelial cell nest with small lumina lined by short to tall microvilli (green arrow)(ED19). (C,D) Tall columnar cells, lining rings, with prominent microvilli(green arrows) suggestive of enterocytes. Other cells show apical mucin vacuoles (orange arrows), suggestive of goblet cell differentiation(ED19).. (E) Epithelial cells with nuclear condensation or pyknosis indicative of early apoptosis (ED71). (F) Shrunken apoptotic cells being extruded into the lumen of a ring (ED 71). Scale bar: 1-5 μm (red).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/bd61e41c4cd914eded9676f5.png\"},{\"id\":96362984,\"identity\":\"ea64c7a3-862b-4735-b268-21c39978b58f\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 10:03:29\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1834260,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/8f873c32-2167-4289-b018-412d912a3c4e.pdf\"},{\"id\":96242650,\"identity\":\"fcb8f46b-fb6e-4037-a96d-55080e8c47ad\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:13:54\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":32356,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementary.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034357/v1/efd37705802d9f283dcce232.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Paediatric Intestinal Enteroids Derived from Very Early Onset Inflammatory Bowel Disease Reveal Differences in Growth, Morphology, and Barrier Function\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eInflammatory bowel disease (IBD), encompassing Crohn\\u0026rsquo;s disease (CD) and Ulcerative colitis (UC), presents significant challenges, affecting patients and burdening the healthcare system globally. Despite historically being reported more often in Europe and North America, the geographic distribution of IBD has since expanded, affecting populations in countries previously considered low-risk areas[\\u003cspan additionalcitationids=\\\"CR2 CR3\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. This rising trend in the incidence of IBD incidence has been attributed to multiple a range of factors such as increased awareness, improved diagnostics, and better access to specialized healthcare, and societal changes such as urbanization, adoption of westernized diet, and improved hygiene[\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]., which This underscores the need for a deeper understanding of the pathogenesis and progression of IBD in these populations. This is even more important in pediatric IBD, where disease characteristics and treatment responses vary from those in adults[\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Pediatric IBD is a distinct and often more aggressive subtype that is frequently associated with extensive intestinal involvement, growth failure, and complex therapeutic challenges[\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. The increasing prevalence of pediatric IBD is particularly significant among younger children, under the age of ten[\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. However, the study of pediatric IBD pathophysiology remains limited by the difficulty in obtaining sufficient patient-specific tissue, the absence of suitable animal models, and the inadequacy of conventional cell culture techniques to replicate the complexity of the intestinal epithelium.\\u003c/p\\u003e\\u003cp\\u003eRecent advances in three-dimensional intestinal organoid culture systems from patient-derived intestinal crypts or pluripotent stem cells have provided unprecedented opportunities to model human intestinal physiology and pathology \\u003cem\\u003eex vivo\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Tissue-derived human intestinal enteroids (HIEs) closely mimic the cellular diversity, polarity, and functional characteristics of the native intestinal epithelium and can be expanded over time while maintaining donor-specific traits[\\u003cspan additionalcitationids=\\\"CR12\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. These have proven especially useful in the study of epithelial barrier function, host\\u0026ndash;microbe interactions, and responses to pharmacologic agents in IBD[\\u003cspan additionalcitationids=\\\"CR15 CR16\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Yet, paediatric-derived HIEs are underutilised despite their potential to reveal age-specific disease mechanisms and inform targeted therapy. Additionally, there is poor representation of available HIE from low-and-middle income countries (LMIC).\\u003c/p\\u003e\\u003cp\\u003eIn this study, we utilised an established HIE biobank to characterise duodenal enteroids from paediatric IBD cases and a non-IBD age-matched control in southern India. We employed morphological and intestinal permeability assays to compare epithelial cell properties across IBD and control HIE lines. By integrating these methods, we aim to advance the use of paediatric enteroids as a translational platform for intestinal disease biology in IBD, evaluate therapeutic responses and guide precision therapy.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eClinical History, Tissue Histopathology and Other Laboratory Parameters of IBD cases and control\\u003c/strong\\u003e\\u003cp\\u003eThree paediatric subjects who were enrolled for duodenal tissue-derived HIE generation were included and comprised two IBD cases (derived HIEs ED-71 and ED-81) and one non-IBD control (ED-19).\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003e​ED-71 was a 1-year-old female who underwent endoscopy for complaints of chronic blood mixed diarrhoea and failure to thrive. Colonoscopy showed multiple ulcers in the entire colon except the descending colon, and biopsy changes indicated Crohn\\u0026rsquo;s disease. Duodenal biopsy showed minimal chronic duodenitis and increased intraepithelial lymphocytes. There was no stricture or fistula, and he improved with immunomodulator therapy(azathioprine and mesalazine). ED-81 was a 2-year-old male who presented with a history of fever, diarrhoea with occasional blood in the stool and failure to thrive. Colonoscopy showed multiple colonic ulcers from caecum to sigmoid colon and histopathology showed features of Crohn\\u0026rsquo;s disease. Duodenal biopsy showed moderate chronic duodenitis. He also had no strictures or fistula and was treated with immunomodulators(methotrexate and mesalazine)​. Both ED-71 and ED-81 cases are currently 9 and 10 years old, and at the last follow-up (2025 and 2024, respectively) were continuing treatment for Crohn\\u0026rsquo;s disease. The control, ED-19, was an 11-month-old male who underwent intestinal surgery for neonatal intestinal obstruction and was diagnosed postoperatively with duodenal atresia requiring duodenoplasty. Duodenal mucosal biopsy did not show chronic inflammation. On the last follow-up at the age of 9 in 2025, he was healthy, and laboratory parameters were normal. Laboratory parameters for the two cases and the control are shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eClinical and Biomarker Profile of Study Participants\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"4\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eED-71\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eED-81\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eED-19\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAge\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1yr\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2 yr\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e11months\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eSex\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eFemale\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eMale\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eMale\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eDiagnosis\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCrohn\\u0026rsquo;s disease\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eCrohn\\u0026rsquo;s disease\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eIntestinal Obstruction due to duodenal web\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eBiopsy reports\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDuodenum: Minimal chronic duodenitis with focal increase in intraepithelial lymphocytes with minimal activity (~\\u0026thinsp;30/100 enterocytes).\\u003c/p\\u003e\\u003cp\\u003eIleum: Mild chronic active ileitis with mild villous atrophy and increased intraepithelial lymphocytes\\u003c/p\\u003e\\u003cp\\u003eCecum to rectum: Mild to patchy moderate colitis with mild activity\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eDuodenum: Mild to moderate chronic active duodenitis with patchy regenerative change.\\u003c/p\\u003e\\u003cp\\u003eIleum: No significant lesion\\u003c/p\\u003e\\u003cp\\u003eCecum to rectum: Patchy chronic active colitis with chronic ulceration and fibrosis.\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eConsistent with duodenal web with normal mucosa and submucosa (excision biopsy)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFecal Calprotectin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e\\u0026gt;\\u0026thinsp;2000\\u0026micro;g/g\\u003c/p\\u003e \\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e\\u0026gt;\\u0026thinsp;2000\\u0026micro;g/g\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e60.62\\u0026micro;g/g\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMyeloperoxidase (MPO)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e8962.06ng/mL\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e101381.33ng/mL\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eBelow detection limit\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eGrowth Curve Analysis\\u003c/strong\\u003e\\u003cp\\u003eTo evaluate the proliferative capacity of the paediatric duodenal tissue-derived intact HIEs, we monitored growth over a 7-day culture period. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, all HIE lines exhibited time-dependent increases in diameter of the enteroids, indicating sustained growth. ED-81 HIEs showed the steepest growth trajectory, particularly between Days 4 and 6. In contrast, ED-71 HIEs showed moderate growth, while ED-19 HIEs displayed the slowest growth trajectory. These differences suggest inherent variability in growth potential among donor-derived lines.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eDifferentiation of HIE\\u003c/strong\\u003e\\u003cp\\u003eTo evaluate epithelial lineage differentiation in the pediatric HIEs, we performed immunofluorescence staining and confocal imaging of lineage-specific markers on HIEs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Enterocytes of all three HIE lines identified by sucrase-isomaltase (SI) staining localized prominently to the apical brush border of the epithelium, confirming mature absorptive cell differentiation. Goblet cells detected using mucin-2 (MUC2) staining showed cytoplasmic staining and localization towards the luminal aspect of the HIEs, indicative of mucin production. Paneth cells visualized by lysozyme (LYZ) staining, showed a discrete punctate red signal. Enteroendocrine cells, marked by chromogranin A (CHGA) staining, were sparsely distributed but positive within the epithelial layer.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003eTo investigate epithelial alterations in pediatric IBD, we assessed gene expression markers representing epithelial lineages and stemness compared to controls. SI expression, an enterocyte differentiation marker, was significantly reduced in both ED71 and ED81 IBD cases (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) compared to the ED-19 control, indicating impaired epithelial maturation in IBD HIEs. The Paneth cell marker LYZ and the goblet cell marker MUC2 were significantly elevated in ED81 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) while CHGA, an enteroendocrine marker, was downregulated in both ED71 and ED81 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001). LGR5 which is a stem cell marker showed increased expression in ED81 and decreased expression in ED71 compared to the ED-19 control. KI67, a marker of proliferation, was significantly elevated in ED71 and 81 compared to ED19 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), indicating hyperproliferative responses in IBD HIE (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). TLR4, TLR3, CXCL-8, and IFN-β gene expression was significantly upregulated in ED81 (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 to p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), whereas they were downregulated in ED71. These variations could be attributed to donor-specific differences in innate immune gene expression or polymorphisms or potentially to the disease stage of IBD.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eBarrier function and structural integrity\\u003c/strong\\u003e\\u003cp\\u003eCell height measurements revealed significant variability among the pediatric tissue-derived HIE monolayers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Quantitative analysis demonstrated that ED19 monolayers had the highest mean cell height, followed by ED81, while ED71 exhibited the lowest mean height. Statistical analysis indicated that the cell height of ED71 was significantly reduced compared to both ED19 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and ED81 (\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). These findings are supported by hematoxylin and eosin (H\\u0026amp;E) staining in intact HIEs (not shown), which showed taller and more columnar epithelial morphology in ED19. The variation in cell height may reflect differences in epithelial differentiation and structural integrity among the ED lines, potentially correlating with disease severity or barrier function.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003eTrans-epithelial electrical resistance (TEER) and FITC-dextran flux assays were performed on differentiated HIE monolayers (grown in transwells) derived from ED19, ED71, and ED81 to evaluate epithelial barrier integrity. At baseline (0hrs) all three HIE lines exhibited high TEER values, indicating intact monolayers. After 2hrs, TEER values decreased significantly on EGTA treatment in all three HIE lines, suggesting a potential compromise in barrier function (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). FITC-dextran flux revealed significant differences in permeability upon treatment with EGTA. Under untreated conditions, all three lines maintained low permeability; however, baseline FITC-dextran flux was slightly higher in ED81 than ED19 and ED71, suggesting inherently weaker barrier properties in the former. Upon exposure to treatment with EGTA, all HIEs exhibited a marked increase in FITC-dextran flux (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). Notably, ED81 showed the highest permeability, followed by ED71 and ED19. These results indicate that the ED81-derived monolayer is the most susceptible to barrier disruption, with elevated baseline permeability and heightened sensitivity to EGTA treatment. Paradoxically, an upregulation in the expression of genes associated with tight junctions, specifically Claudin-1 and ZO-1, was observed in ED81 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD).\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eUltrastructure of HIE\\u003c/strong\\u003e\\u003cp\\u003eElectron microscopy of all three HIEs showed cells arranged in rings and nests. Some of the rings were lined by flattened to cuboidal epithelium with rudimentary microvilli on the inner luminal surface. Others were lined by tall columnar epithelium with more prominent microvilli. Some of the columnar cells had mucin vacuoles in the apical cytoplasm, suggesting differentiation towards a goblet cell phenotype. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, C, D). Apart from rings, \\u0026lsquo;nests\\u0026rsquo; of epithelial cells were also seen. Some of the nests had small lumina between the tall columnar cells, the luminal surface of which showed prominent microvilli (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). All epithelial cells in both rings and nests showed apical intercellular tight junctions and desmosomes were seen between lateral cell margins. ED71 HIEs showed rings and nests with focal prominent clumping of nuclear chromatin, consistent with pyknosis, an early change of apoptosis. These cells also showed increased size and number of lysosomes. Many shrunken, dark cells with nuclear fragments containing condensed chromatin and degenerate organelles suggestive of apoptosis were also seen in ED71, some being extruded into the lumen of rings or smaller lumina of nests (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE, F).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we characterised paediatric patient-derived HIEs from two cases with IBD and one non-IBD control, generating a comparative platform to assess characteristics of the intestinal epithelium pertinent to paediatric IBD. By combining morphological, molecular, and permeability assays, we offer a thorough examination of HIE characteristics in both IBD and non-IBD conditions, highlighting the value of paediatric HIEs for translational research in IBD. Reports from LMICs remain limited, and pediatric inflammatory bowel disease (IBD) human intestinal enteroids (HIEs) from these regions are largely underrepresented. To our knowledge, this study is the first to characterize pediatric patient-derived HIEs from South Asia, offering a unique perspective on IBD biology in a region where the incidence and disease phenotype are increasingly recognized but insufficiently explored.\\u003c/p\\u003e\\u003cp\\u003eThe HIE growth curve analysis revealed significant variability in proliferative capacity among patient-derived lines, with the Crohn's-derived line (ED-81) exhibiting the steepest growth trajectory, while the healthy control (ED-19) demonstrated the slowest growth. These differences may reflect disease-specific epithelial adaptations or altered crypt biology associated with chronic inflammation, as previously observed in adult IBD studies[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e] or could be individual-specific.[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e] Similar variability has been observed in adult IBD organoids, where studies involving single-cell analysis and biobanks have identified growth differences linked to disease activity and variations between patients[\\u003cspan additionalcitationids=\\\"CR20\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Additionally, epigenetic and transcriptional profiling have demonstrated that epithelial programs can categorize disease severity and phenotype[\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], reinforcing the notion that the variability seen in pediatric HIEs represents authentic disease-associated adaptations.\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eAll three pediatric HIEs exhibited the anticipated epithelial lineages; however, HIEs derived from IBD demonstrated altered lineage and stemness markers, indicative of impaired maturation and increased proliferative capacity[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Similar variability in innate immune gene expression has been reported, largely attributable to donor heterogeneity and inflammatory state at biopsy[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Several studies have noted enrichment of Paneth cell markers (e.g., LYZ) and upregulation of antigen-presentation pathways in IBD colonoids, suggesting heightened epithelial immune activation[\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. Interestingly, Kelsen et al. observed reduced growth efficiency in pediatric IBD colonoids, measured as lower crypt-to-spheroid conversion, despite concurrent Paneth and immune marker upregulation[\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. This contrast may reflect differences between conversion efficiency and long-term expansion potential[\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], as well as disease stage, inflammatory stress, or altered responsiveness to niche factors[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Collectively, these findings suggest that epithelial remodeling in inflammatory bowel disease (IBD) encompasses both the augmentation of immune lineage signatures and changes in growth dynamics, highlighting the complex nature of responses observed in patient-derived organoids.\\u003c/p\\u003e\\u003cp\\u003eHistological analyses revealed that ED-71 monolayers show reduced epithelial height and flattened morphology compared to ED-19 (columnar) and ED-81 (intermediate), reflecting impaired differentiation. Similar morphological alterations in IBD-derived colonoids show reduced size, altered polarity, and loss of budding structures under inflammatory contexts[\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Epithelial flattening in organoid and monolayer systems correlates with compromised barrier integrity and heightened susceptibility to injury, with such phenotypes reversed by treatment[\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. These changes mirror in vivo crypt atrophy and epithelial distortion\\u0026mdash;established markers of disease activity in IBD[\\u003cspan additionalcitationids=\\\"CR29\\\" citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Our patient-derived monolayer phenotypes recapitulate the structural features seen in vivo, suggesting epithelial height metrics may indicate differentiation capacity and barrier functionality. The ultrastructural evidence of apoptosis in the enteroids of ED71 suggest increased cell death that could contribute to the compromised barrier functionality.\\u003c/p\\u003e\\u003cp\\u003eOur intestinal permeability assays revealed donor-specific differences: ED-81 showed higher baseline permeability and greatest EGTA-induced FITC flux, suggesting tight-junction fragility. Similar barrier defects occur in IBD organoid models, where reduced TEER and increased flux correlate with impaired differentiation[\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. EGTA chelation probes tight-junction stability, with increased sensitivity reflecting weakened junctional integrity[\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Elevated fecal calprotectin and MPO correlate with mucosal permeability[\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], consistent with ED-81's barrier vulnerability. These findings support an epithelial contribution to IBD pathology beyond immune-mediated injury[\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eAlthough ED81 HIE showed elevated expression of tight junction genes like Claudin-1 and ZO1, increased permeability in FITC-dextran assays suggests a disconnect between gene expression and barrier integrity. The increased expression of innate immune genes like TLR3, TLR4, and IFN-β indicates inflammatory pathway activation, which disrupts epithelial junctions through cytokine-mediated modulation and increased cell turnover. These inflammatory signals may override repair mechanisms, causing barrier dysfunction despite upregulated junctional transcripts. Animal studies using DSS-treated organoids confirmed that inflammatory insults degrade tight-junction integrity and increase paracellular permeability, shown by increased FITC-dextran flux and has shown increased expression of Claudin-1 [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. In adult IBD colonoids, reduced TEER and disrupted tight junction integrity persist in remission samples[\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe major limitation of this work is the small sample size, which restricts the generalizability of donor-specific findings. However, our results align with prior reports showing that patient-derived intestinal organoids retain donor- and disease-specific molecular and functional signatures. While our sample size is limited, the robust differences and reproducible results observed indicate feasibility of the approach and that additional studies with larger numbers of subjects are needed to make meaningful conclusions. Further these HIEs were derived from duodenal biopsies, HIEs derived from colonic biopsies could have provided more information.\\u003c/p\\u003e\\u003cp\\u003eThis exploratory study therefore supplements this growing body of literature by extending these observations to pediatric HIEs and highlighting the potential of this model to interrogate patient-level variation in epithelial physiology. Co-culture with immune cells or microbiota could further enhance the physiological relevance of this model. Future studies expanding patient numbers and incorporating transcriptomic and proteomic analyses will help identify molecular drivers of observed phenotypes. This platform could evaluate reactions to cytokines, microbial challenges, and therapeutic agents to reveal age and disease-specific vulnerabilities, aiding in precision therapy for paediatric IBD.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003ePatient Recruitment and Establishment of HIE Biobank\\u003c/strong\\u003e\\u003cp\\u003eA paediatric HIE biobank was established at Christian Medical College (CMC), Vellore, India between 2016 to 2018 as part of a study to establish HIE from children with environmental enteric dysfunction (EED). Briefly, children under the age of five years undergoing abdominal surgery (n\\u0026thinsp;=\\u0026thinsp;26) or endoscopic biopsies (n\\u0026thinsp;=\\u0026thinsp;25) for a range of clinical indications were recruited after written informed consent from an adult caregiver (parent or legal guardian) was obtained by a trained research nurse. Along with intestinal biopsy tissue, a stool sample and a blood sample were also collected, processed and banked. Derivation of enteroids from intestinal tissue were conducted in accordance with established protocols [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e], as detailed in supplementary methods with 19 duodenal, 10 jejunal and 12 ileal HIE for a total of 41/51 successfully grown HIEs and banked in liquid nitrogen for further characterisation and future studies.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eIBD and control HIE for characterisation\\u003c/b\\u003e: Three duodenal HIEs were selected for in-depth characterisation: two from patients diagnosed with very early onset IBD (Crohn\\u0026rsquo;s disease) and one without any abnormalities in tissue histopathology, serving as a non-IBD age-matched control. The diagnosis of Crohn's disease was ascertained by gastroenterologists in accordance with the Asia Pacific criteria[\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e] with thorough assessment of clinical manifestations, endoscopic observations, histopathological evaluation, and radiological findings. Fecal calprotectin (Epitope Diagnostics, California) and myeloperoxidase (MPO, Immundiagnostik, IDK, Germany) were also tested.\\u003c/p\\u003e\\u003cp\\u003eHIEs were revived from cryopreserved stocks at passage 3 or 4 and subsequently expanded as mentioned in the supplementary methods. For differentiation, expansion media was replaced with differentiation media at Day 4 and all assays were performed with differentiated HIE at Day 7. All experiments were performed using HIE lines at passage 6 with two technical replicates. To ensure reproducibility, gene expression and all functional assays were independently validated using three separate cryopreserved vials that were thawed and expanded separately. HIEs were grown as monolayers in transwells as described in the supplementary methods for cell height and permeability assays.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eGrowth Curves\\u003c/strong\\u003e\\u003cp\\u003eGrowth curve measurements were performed using Image J/Fiji software version 2.14.0/1.54f by calibrating images to a known scale bar (10 \\u0026micro;m) and measuring intact 3D HIE diameter using the line tool. All measurements obtained from 10 randomly selected HIEs per replicate at each time point (days 1 to 7) were recorded in micrometres, and output data were saved as TIFF images and corresponding measurement files. LOESS smoothing was applied to visualise growth trends, and 95% confidence intervals were plotted to indicate variability.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eTransmission Electron Microscopy (TEM) of HIE\\u003c/strong\\u003e\\u003cp\\u003eIntact 3D HIEs were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 2 mM CaCl₂ for 3 hours at 4\\u0026deg;C. After washing in sodium cacodylate-sucrose buffer, samples were post-fixed in 1% OsO₄ in 0.2 M sodium cacodylate buffer for 2 hours at 4\\u0026deg;C. Samples were dehydrated through graded ethanol (50\\u0026ndash;100%), treated with propylene oxide, and infiltrated with epoxy resin over three changes. HIEs were embedded and polymerized at 60\\u0026deg;C for 48 hours. Semi-thin sections (0.5\\u0026ndash;1 \\u0026micro;m) were cut using Leica UCT ultramicrotome and stained with toluidine blue in borax. Ultrathin sections (~\\u0026thinsp;50\\u0026ndash;70 nm) were cut, mounted on copper grids, and stained with uranyl acetate and Reynold's lead citrate. Sections were examined using a FEI Tecnai T12 microscope at 120 keV.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eImmunofluorescence Staining and Confocal Imaging of HIE\\u003c/b\\u003e: Intact 3D HIEs grown in Matrigel domes were fixed with 4% formaldehyde for 1 hour at room temperature (RT), followed by three PBS (1X) washes. Samples were blocked and permeabilised with 5% fetal bovine serum (FBS, MP Biomedicals, Ohio) and 2% Triton X-100 for 2 hours at RT. Primary antibodies (1:50 in 5% FBS, 0.25% Triton) were added and incubated at RT in the dark with gentle shaking. After washing, secondary antibodies (1:1000) were applied for 3 hours at RT. Nuclei were counterstained with 4\\u0026rsquo;,6-diamidino-2-phenylindole (DAPI, Invitrogen, California) (1:1000) for 30 minutes, followed by three PBS washes and clearing for 2 hours. Slides were mounted with Prolong gold antifade reagent (Invitrogen, California) and sealed. Imaging was performed using confocal microscope with appropriate fluorescent filters. Antibodies are listed in Supplementary Table\\u0026nbsp;5. Samples were imaged using an Olympus Laser Scanning Confocal Microscope system (spectral version), model Olympus FV1000. Images were captured at 20X and 60X magnifications using Olympus Fluoview Ver.3.16 software.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eGene Expression of HIE\\u003c/strong\\u003e\\u003cp\\u003eIntact 3D HIEs were incubated in cold water for 10 minutes, followed by adding 800 \\u0026micro;L cold PBS and centrifugation at 800 \\u0026times; g for 3 minutes at 4\\u0026deg;C. The pellet was lysed in 350 \\u0026micro;L RLT (RNA Lysis Tissue) buffer with 1% β-mercaptoethanol, incubated for 10 minutes at room temperature, and mixed with 350 \\u0026micro;L 70% ethanol. The lysate was processed using spin columns (Qiagen, Germany) for RNA isolation. RNA was quantified using a Nanodrop, and 500 ng\\u0026ndash;2 \\u0026micro;g was reverse-transcribed into cDNA (cDNA REV TRANS KIT, ThermoScientific, USA) in a 20 \\u0026micro;L reaction using manufacturer\\u0026rsquo;s protocol. Gene expression was assessed by qPCR using 5 ng/\\u0026micro;L diluted cDNA, TaqMan Fast Advanced Master Mix, and gene-specific primer-probe pairs. This included Sucrose Isomaltase (SI), Lysozyme (LYZ), Chromogranin A(CHGA), LGR5, CD44, KI67, Z0-1, Claudin-1,Toll-like receptor4 (TLR4), TLR3, CXCL-8, and Interferon β (IFN- β) (listed in Supplementary Table) Flex (Applied Biosystems\\u003csup\\u003e\\u0026trade;\\u003c/sup\\u003e) as per manufacturers protocols. Negative controls included no-template and no-reverse transcriptase reactions to confirm the absence of contamination. GAPDH was used for normalisation.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eCell Height Measurement\\u003c/strong\\u003e\\u003cp\\u003eCell height was assessed in Hematoxylin and Eosin (H\\u0026amp;E)-stained sections of differentiated HIE monolayers grown in transwells and then mounted on slides. Only typical enterocytes were included; cells with inclusions, vacuoles, or multilayered stacking were excluded. Heights were measured from the base of the cell to the apical surface, bisecting the nucleus. Three cells were measured per image for each sample across three separate images. Measurements were conducted using ImageJ/Fiji version 2.14.0/1.54f software after setting the image scale using the embedded scale bar. A blinded analyst performed all analyses to eliminate bias.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eIntestinal Permeability Assays\\u003c/b\\u003e: Transepithelial electrical resistance (TEER) of differentiated HIEs grown on transwells (Corning, New York) was recorded using Millicell ERS-2 before and after a 2-hour treatment with 10mM EGTA (Millipore, Merck, USA). FITC-Dextran (Sigma-Aldrich, USA) serial dilutions (5 mg/ml) for the standard curve and a 1:100 working dilution were prepared during incubation. Following treatment, the media was replaced with 100 \\u0026micro;L of diluted FITC-Dextran and incubated for two additional hours. Final TEER was measured, and 50 \\u0026micro;L each of apical, basolateral, and standard curve samples were transferred to a 96-well plate. To assess permeability, fluorescence was read at 490\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9 nm excitation and 520\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9 nm emission using TECAN Infinite M200 PRO reader.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eStatistical Analysis\\u003c/strong\\u003e\\u003cp\\u003eAll analyses were conducted using R version 4.4., and all images were analysed using ImageJ/Fiji version 2.14.0/1.54f. Data from 3 biological and technical replicates were included, and for imaging-based analyses, at least 10 measurements per replicate were obtained to ensure statistical robustness. For experiments involving continuous variables such as HIE diameter, cell height, gene expression, and permeability assays, data were analysed using descriptive statistics (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation). For comparison between control and IBD HIE lines, unpaired two-tailed Student\\u0026rsquo;s t-test was used. P-values\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 were considered statistically significant. This HIE biobank establishment and this study was approved by the institutional ethics committee at Christian Medical College, Vellore. All methods were carried out in accordance with relevant ethical guidelines and regulations.\\u003c/p\\u003e\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank the CSCR Confocal Imaging Facility at CMC ,Vellore for technical support, and the Mathais Zilbauer Lab for valuable guidance in further optimising cell culture media. We also thank study nurse Charlet Suresh, and our colleagues Roshni Parameswaran, Shanmugam, Xi-Lei Zeng, Lakshmi Nirmal Somasundaram, and Ketki Patil for their assistance during the project. Most importantly, we are grateful to the infants and children and their parents for enrolling in this study and providing samples.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eB.V. contributed to study design, performed laboratory work and data analysis, and drafted the manuscript. B.S., S.B., and A.P. conducted laboratory experiments and A.J.J, A.K.D., and E.S. were involved in clinical data acquisition. M.E. H.W and S.R. contributed to study design and provided training and resources. S.S.R.A. supervised the study, contributed to study design, provided resources and finalised the manuscript. All authors meet the criteria for authorship, reviewed the manuscript, and approved the final version for submission.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eData available with the corresponding author on request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional Information\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\u003c/ul\\u003e\\n\\u003cp\\u003eThe authors have declared no conflicts of interest.\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eFunding sources\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\u003c/ul\\u003e\\n\\u003cp\\u003eThe EED study that led to the development of the HIE biobank was funded by the Gates Foundation OPP1108228 to Tufts Medical Center, Boston, MA, USA; Baylor College of Medicine, TX, USA and Christian Medical College, Vellore, India. Characterisation of HIE from IBD cases was supported by institutional funds at the Wellcome Trust Research Laboratory, CMC, Vellore.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eNg, S. C. \\u003cem\\u003eet al.\\u003c/em\\u003e Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn\\u0026rsquo;s and colitis epidemiology study. \\u003cem\\u003eGastroenterology\\u003c/em\\u003e \\u003cstrong\\u003e145\\u003c/strong\\u003e, 158-165.e2 (2013).\\u003c/li\\u003e\\n\\u003cli\\u003eNg, S. C. \\u003cem\\u003eet al.\\u003c/em\\u003e Population Density and Risk of Inflammatory Bowel Disease: A Prospective Population-Based Study in 13 Countries or Regions in Asia-Pacific. \\u003cem\\u003eAm. J. Gastroenterol.\\u003c/em\\u003e \\u003cstrong\\u003e114\\u003c/strong\\u003e, 107\\u0026ndash;115 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eGordon, H. \\u0026amp; Langholz, E. 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Hepatol.\\u003c/em\\u003e \\u003cstrong\\u003e31\\u003c/strong\\u003e, 45\\u0026ndash;55 (2016).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Inflammatory Bowel Disease, Patient-Derived Enteroids, Intestinal Permeability, Inflammation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8034357/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8034357/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e​Paediatric inflammatory bowel disease (IBD) presents more aggressively than adult-onset disease; however,​ epithelial pathophysiology remains poorly understood due to limited access to patient tissue. Human intestinal enteroids (HIEs) are a powerful model for investigating disease phenotypes and heterogeneity. We characterized duodenal HIEs from two children with Crohn's disease(ED-71 and ED-81) and one control with intestinal obstruction(ED-19), all aged\\u0026thinsp;\\u0026lt;\\u0026thinsp;2years. Analyses included growth curves, epithelial cell heights, immunofluorescence, histology, electron microscopy, gene expression and intestinal permeability. All HIEs maintained consistent growth \\u003cem\\u003ein vitro\\u003c/em\\u003e, with ED-81 displaying the steepest trajectory. Morphological examination revealed variations in epithelial cell height, with ED-71 and ED-81[median(95%CI) 10.2(7.76\\u0026ndash;11.4) and 12.5(11.5\\u0026ndash;15) \\u0026micro;m] displaying a more flattened appearance than ED-19[18.7 (16.8\\u0026ndash;21.0) \\u0026micro;m, p-value:\\u0026lt;0.001]. HIE differentiation into intestinal cell types was confirmed by gene expression and microscopy. Intestinal permeability assays indicated compromised barrier integrity in IBD-derived monolayers, with ED-81 exhibiting the highest baseline permeability(5.27%) and EGTA-induced disruption (50.03%) compared to ED-19(3.88% and 36.82%). Pediatric intestinal enteroids in IBD demonstrate differences in epithelial growth, morphology, and barrier function. HIEs serve as a potential translational model for pediatric IBD, facilitating the study of epithelial pathophysiology and guiding precision therapy. Further studies with more pediatric HIEs are needed to confirm these findings.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Paediatric Intestinal Enteroids Derived from Very Early Onset Inflammatory Bowel Disease Reveal Differences in Growth, Morphology, and Barrier Function\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-14 07:14:43\",\"doi\":\"10.21203/rs.3.rs-8034357/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-04-20T05:01:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-04-14T05:04:14+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-04-09T15:55:12+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"201178088734985431198215478248438522467\",\"date\":\"2026-04-05T20:12:33+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"167905283779215953444266606088582604812\",\"date\":\"2026-04-04T21:27:02+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"272593376141154202442754751299785927437\",\"date\":\"2026-04-03T17:42:54+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-25T18:51:26+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-02-24T10:24:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-11-07T16:56:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-11-07T03:53:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2025-11-07T03:50:44+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"b6201425-eae3-4ed5-9aba-44defc4427fb\",\"owner\":[],\"postedDate\":\"November 14th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":57940142,\"name\":\"Health sciences/Diseases\"},{\"id\":57940143,\"name\":\"Health sciences/Gastroenterology\"},{\"id\":57940144,\"name\":\"Health sciences/Medical research\"}],\"tags\":[],\"updatedAt\":\"2026-05-05T12:55:22+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-14 07:14:43\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8034357\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8034357\",\"identity\":\"rs-8034357\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}