Biofabrication of primary patient tissue-derived inflammatory bowel disease (IBD) model by organoid three-dimensional culture

Biofabrication of primary patient tissue-derived inflammatory bowel disease (IBD) model by organoid three-dimensional culture | 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 Research Article Biofabrication of primary patient tissue-derived inflammatory bowel disease (IBD) model by organoid three-dimensional culture Roya Karimi, Mohsen Masoodi, Shahram Agah, Zeinab Hajmohammadi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7498761/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Inflammatory bowel disease is a chronic gastrointestinal disorder characterized by relapsing inflammation, disruption of the epithelial barrier, and dysregulated immune responses, leading to substantial morbidity. Conventional in vitro cell cultures and animal models often fail to reproduce the cellular heterogeneity, tissue architecture, and patient-specific features of the disease, limiting mechanistic understanding and therapeutic development. This study aimed to develop and characterize a patient-derived three-dimensional organoid model that closely recapitulates the structural, functional, and molecular hallmarks of the disease. Colonic biopsies from inflamed and non-inflamed regions of patients were embedded in alginate–gelatin scaffolds to generate organoids that maintain physiological tissue architecture and long-term viability. Results: Organoids from non-inflamed tissue maintained uniform spherical morphology with well-defined crypt-like domains, whereas organoids derived from inflamed tissue exhibited irregular architecture, disrupted epithelial junctions, and delayed recovery after passage. Scanning electron microscopy confirmed epithelial discontinuities and surface microfold irregularities in inflamed organoids. Functional viability assessments demonstrated an average survival rate of 71.0% at day 21. Gene expression analysis revealed significant downregulation of the intestinal stem cell marker LGR5 in inflamed organoids compared to controls (0.43 ± 0.05 vs. 1.03 ± 0.06, p = 0.0002), along with marked upregulation of inflammatory chemokines CXCL8 (3.65 ± 0.37, p = 0.0003), CCL2 (2.71 ± 0.17, p = 0.0001), and CXCL10 (4.28 ± 0.15, p < 0.0001). Conclusions: This patient-derived three-dimensional organoid system accurately models disease-associated structural deterioration, impaired regenerative capacity, and inflammatory signaling, providing a physiologically relevant and reproducible platform for mechanistic studies. The model enables high-throughput drug screening, evaluation of patient-specific therapeutic responses, and development of personalized interventions. By bridging the gap between conventional in vitro and in vivo systems, this organoid platform represents a significant advance in tissue engineering and translational gastrointestinal research, facilitating precise investigation of disease pathophysiology and accelerating the development of effective therapies for chronic inflammatory disorders. Inflammatory Bowel Disease Patient-Derived Organoids Organoids Three-Dimensional (3D) Culture Alginate-Gelatin Scaffold Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic, relapsing inflammatory disorder of the gastrointestinal (GI) tract. It commonly presents with abdominal pain, diarrhea, fatigue, and unintended weight loss, significantly impairing patients' quality of life and impose a substantial burden on healthcare systems globally. Current treatment strategies primarily rely on pharmacological agents such as amino-salicylates, corticosteroids, and immunosuppressants. While these therapies can reduce inflammation and induce remission, they are often associated with considerable adverse effects, including hepatotoxicity, gastrointestinal disturbances, and dermatologic immune reactions. To overcome these limitations, novel approaches such as targeted drug delivery systems—like nanoparticle-based carriers—are being developed to enhance therapeutic efficacy and minimize side effects. Additionally, non-pharmacologic interventions like leukocytapheresis are under investigation as potential alternatives for patients who exhibit poor tolerance to conventional medications. The etiology of IBD is multifactorial, involving genetic susceptibility, environmental influences, dysbiosis of gut microbiota, and dysregulated immune responses; however, the precise pathogenic mechanisms remain inadequately understood. As a result, ongoing research aims to establish more effective, less invasive, and better-tolerated treatment models, while also addressing the substantial global healthcare burden posed by these diseases ( 1 – 3 ). A variety of in vitro and ex vivo models have been developed to investigate the pathophysiology of inflammatory bowel disease (IBD), including both ulcerative colitis and Crohn’s disease. These models are increasingly favored over traditional in vivo animal models, which, despite offering valuable insights, fail to fully replicate the complexity of human IBD, prompting a shift towards cellular models that better simulate human gut physiology. Commonly used epithelial cell lines—such as Caco-2, HT29, and T84—represent different intestinal cell types and are widely employed to study barrier function, inflammation, and drug absorption. More advanced systems, such as intestinal organoids, enable the study of complex cellular differentiation and tissue architecture, closely reflecting the in vivo environment. Co-culture models, including the combination of absorptive Caco-2 cells with mucus-secreting HT29-MTX cells, provide a more physiologically relevant representation of the gut epithelium. These systems facilitate the study of cellular crosstalk, microbial interactions, and drug permeability—key aspects in understanding IBD pathogenesis ( 5 , 6 ). Additionally, platforms such as the Ussing chamber, everted gut sac, and microfluidic gut-on-chip devices offer dynamic and functional assessments of intestinal tissue. While no single model can fully recapitulate the multifaceted nature of IBD, an integrated approach utilizing multiple complementary systems holds promise for advancing our understanding of disease mechanisms and for accelerating the development of more effective therapies ( 4 ). Traditional two-dimensional (2D) cell culture models have significantly contributed to our understanding of the cellular and molecular mechanisms involved in inflammatory bowel disease (IBD). However, these models are limited by their inability to replicate the cellular heterogeneity, structural complexity, and dynamic microenvironment of the human intestinal tract, thereby reducing their physiological relevance ( 7 ). Similarly, while animal models have been instrumental in advancing our understanding of IBD pathogenesis and therapeutic responses, species-specific differences often limit their translational applicability to human disease ( 8 ). These limitations underscore the pressing need for more physiologically accurate experimental platforms to investigate IBD mechanisms and to facilitate the development of targeted therapeutic interventions. Recent advances in bioengineering and stem cell technologies have led to the emergence of three-dimensional (3D) culture systems, particularly organoids, which offer a more faithful representation of native human tissues. Derived from pluripotent or adult stem cells, organoids are capable of self-organizing into miniature, organ-like structures that recapitulate key features of the intestinal epithelium, including multiple differentiated cell types and spatial organization. This advancement has positioned organoids as a powerful and reproducible model system for studying IBD pathophysiology and for testing therapeutic strategies in a controlled, human-relevant and reproducible manner ( 9 ). In the context of IBD, intestinal organoids represent a highly promising platform for studying disease mechanisms and evaluating therapeutic interventions. These 3D structures can be bioengineered to incorporate critical components of the intestinal microenvironment—including epithelial cells, immune populations, and microbial communities—thereby offering a more physiologically relevant and integrative model compared to traditional systems ( 10 ). Furthermore, organoids can be generated from patient-derived stem cells, allowing for the investigation of individual disease phenotypes and the development of personalized therapeutic approaches. This study aims to develop a biofabricated 3D culture model of IBD using organoid technology. By integrating advanced biofabrication techniques with organoid culture, we seek to create a robust and scalable model that faithfully mimics the pathological features of IBD. This model will not only enhance our understanding of IBD pathogenesis but also serve as a valuable platform for preclinical drug testing and the development of personalized therapeutic strategies ( 4 ). Materials and Methods Preparation of Solutions: Washing Solution: Phosphate-buffered saline (PBS) supplemented with 2% gentamycin and 5% penicillin-streptomycin. Complete Culture Medium: Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. 3% Alginate Solution: Prepared by dissolving 0.3 g of alginate in 10 mL of DMEM. 10% Gelatin Solution: Prepared by dissolving 0.1 g of gelatin in 10 mL of DMEM. Calcium Chloride (CaCl₂) Solution: 10 mM in sterile distilled water. Procedure: 2.1 Sample Collection Colon biopsies were obtained from patients undergoing colonoscopy for IBD diagnosis. Tissues were collected from both inflamed (IBD-affected) and non-inflamed (normal) regions of the colon. Samples were transported to the laboratory in sterile conditions for organoid culture. 2.2 Isolation and Organoid Culture a. Pre-coating of Culture Plates: The day prior to sample processing, 300 µL of 10% gelatin solution was added to each well of a 24-well culture plate. The plate was incubated overnight at 37°C in a 5% CO₂ atmosphere to allow gelatin coating. b. Tissue Handling: Immediately following collection, tissue samples were transferred into sterile 15 mL Falcon tubes containing ~3 mL of washing solution and transported to the cell culture facility. c. Plate Conditioning: The pre-coated culture plate was removed from the incubator and stored at 4°C until use. d. Tissue Washing: Samples were vortexed in the washing solution for 2 minutes, centrifuged at 2000 RPM for 10 minutes, and the supernatant discarded. This wash step was repeated once with fresh washing solution. e. Enzymatic Digestion: Tissue pellets were incubated in 1 mL of 1.5% collagenase solution at 37°C with 5% CO₂ for 45 minutes to facilitate digestion. f. Post-digestion Processing: Following digestion, an equal volume of complete media was added to neutralize collagenase activity. The mixture was centrifuged at 2000 RPM for 10 minutes, and the supernatant was discarded. g. Matrix Embedding: The pellet was resuspended in 500 µL of complete media, then mixed with 500 µL of 3% alginate solution. The suspension was pipetted gently to achieve a homogeneous cell suspension. h. 3D Culture Setup: Approximately 500 µL of the alginate-cell suspension was seeded into each well of the pre-coated 24-well plate. Subsequently, 1 mL of 10 mM CaCl₂ solution was added to crosslink the alginate matrix. The plate was incubated at 37°C with 5% CO₂ for 1 hour. i. Media Replacement: After incubation, the remaining CaCl₂ solution was discarded and replaced with 1 mL of fresh complete media per well. j. Maintenance: The first media change was performed after 48 hours. Subsequently, culture media was refreshed every 3 days using a semi-exchange method. 2.3 Morphological Analysis of Organoids Organoid morphology was assessed using both inverted light microscopy for routine monitoring and scanning electron microscopy (SEM) for detailed ultrastructural evaluation. a. Inverted light microscopy Daily morphological changes and growth patterns of the organoids were monitored using an inverted phase-contrast microscope (Olympus IX51 inverted microscope), allowing for non-invasive visualization of size, shape, and structural integrity throughout the culture period. b. Scanning electron microscopy (SEM) Organoids were fixed in Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) for 1–2 hours at room temperature. Samples were then dehydrated through a graded ethanol series: 30%, 50%, 70%, 90%, and 100% (twice), each for 5 minutes. Following dehydration, samples were dried using either critical point drying (45–60 minutes) or hexamethyldisilazane (HMDS) treatment. Dried specimens were mounted on aluminum SEM stubs using conductive carbon adhesive tape and further secured with conductive gold paint to ensure optimal electrical grounding. Samples were then sputter-coated with a thin layer of gold to enhance conductivity and minimize charging artifacts prior to imaging. 2.4 Viability assessment Organoid viability was assessed using a Live/Dead fluorescence assay. Fluorescein diacetate (FDA) was used to stain viable cells (green fluorescence), while propidium iodide (PI) stained non-viable cells (red fluorescence). Stained organoids were imaged using a fluorescence microscope, and viability was quantified by calculating the proportion of live versus dead cells. 2.5 Gene Expression Analysis Quantitative real-time polymerase chain reaction (qPCR) was performed to assess the expression of inflammation- and stem cell–related genes. Total RNA was extracted from organoids using an RNA isolation kit following the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using reverse transcription, and gene-specific primers were designed for LGR5, CXCL8, CXCL10, and CCL2. qPCR reactions were carried out using SYBR Green, and relative gene expression was calculated using the ΔΔCt method with appropriate housekeeping gene normalization. Results Organoid Morphology-light invert microscopy Within 24 hours of seeding, organoids from both normal and IBD colon samples formed compact three-dimensional spheroids. By day 5–7, crypt-like protrusions became evident. Normal tissue–derived organoids exhibited uniform spherical architecture and robust proliferation, whereas IBD-derived organoids often showed irregular contours, heterogeneous size distribution, and less compact morphology. By day 14–21, normal organoids maintained a well-organized structure, while IBD organoids displayed disorganized outer layers and occasional lumen collapse. After passaging, normal organoids readily re-established crypt-like domains, whereas IBD organoids showed delayed recovery (figure 1). Scanning Electron Microscopy (SEM) On day 14, organoids were fixed and analyzed using SEM. In both groups, SEM imaging confirmed the presence of differentiated epithelial surface structures. Normal organoids exhibited well-defined luminal borders, with tightly packed epithelial cells and intact epithelial morphology. In contrast, IBD-derived organoids demonstrated surface irregularities, disrupted cell–cell junctions, and discontinuities in the epithelial layer, indicative of compromised structural integrity. High-magnification views further revealed microfolds and areas of epithelial flattening in IBD samples, consistent with morphological deterioration (figure 2). Viability Assay On day 21, organoid viability was assessed using the Live/Dead assay, where green fluorescence indicated viable cells (figure 3). Quantitative image analysis revealed that viable cells covered approximately 71% of the total organoid area. The mean viability across samples was 71.03%. Detailed image-based metrics are presented in Table 1. Table 1. Live/Dead assay quantification at day 21. Count Total Area (µm²) Average Size (µm²) %Area Mean viability % 841 1017613 1210.004 20.195 71.029 Gene Expression analysis qPCR analysis of LGR5, CXCL8, CCL2, and CXCL10 revealed distinct transcriptional profiles. LGR5, a stem cell marker, was significantly downregulated in IBD organoids (p = 0.0002), suggesting reduced stemness potential and impaired regeneration capacity in the inflamed tissue. Conversely, inflammatory mediators CXCL8 (p = 0.0003), CCL2 (p = 0.0001), and CXCL10 (p < 0.0001) were markedly upregulated in IBD organoids relative to normal controls, consistent with an inflammatory activation signature (table 2 and figure 4). Table 2. Relative gene expression (fold change, mean ± SD) in normal vs. IBD-derived colon organoids. gene Fold change mean ± SD (normal) Fold change mean ± SD (IBD) p-value LGR5 1.03 ± 0.06 0.43 ± 0.05 0.0002 CXCL8 1.00 ± 0.08 3.65 ± 0.37 0.0003 CCL2 1.00 ± 0.02 2.71 ± 0.17 0.0001 CXCL10 1.00 ± 0.05 4.28 ± 0.15 0.0000 Discussion Inflammatory bowel disease (IBD), encompassing Crohn’s disease and ulcerative colitis, is a multifactorial, relapsing inflammatory disorder of the gastrointestinal tract with complex interactions between genetic predisposition, immune dysregulation, microbiota alterations, and environmental triggers ( 1 – 3 ). Existing in vitro and in vivo models have advanced our understanding of IBD pathogenesis and drug responses, but their translational fidelity remains limited ( 4 , 7 , 8 , 13 ). Animal models, while valuable for dissecting immune and epithelial mechanisms, are constrained by species-specific differences in immune architecture and gut physiology ( 13 , 14 ), and conventional 2D cultures fail to capture the spatial complexity, multicellular diversity, and dynamic microenvironment of the human intestine ( 4 , 7 ). This translational gap has driven the development of patient-derived organoid systems, which offer a physiologically relevant, manipulable, and reproducible platform for disease modeling ( 9 , 10 , 16 – 18 ). In this study, we successfully biofabricated patient-derived 3D colon organoids from inflamed (IBD) and non-inflamed (control) tissues, demonstrating that the IBD organoids retain disease-relevant structural and molecular phenotypes. Morphologically, IBD organoids displayed irregular contours, lumen collapse, and delayed regeneration after passaging, in contrast to the uniform crypt-like morphology of normal organoids. These features are consistent with previous reports showing that IBD-derived organoids often exhibit reduced budding efficiency, abnormal epithelial polarity, and compromised barrier properties due to altered stem cell activity and persistent inflammatory signaling ( 11 , 12 , 16 ). SEM analysis in our study revealed disrupted cell–cell junctions and surface discontinuities in IBD organoids. Such ultrastructural deterioration mirrors the epithelial damage and tight junction disruption observed in patient biopsy specimens, which are known to impair barrier function and promote translocation of luminal antigens, perpetuating inflammation ( 11 , 14 ). The persistence of these features ex vivo supports the hypothesis that epithelial defects in IBD are at least partly cell-intrinsic and not solely dependent on the in vivo inflammatory milieu. Viability assays indicated an average of 71% live cell area in IBD organoids at day 21, which—although supportive of long-term culture—suggests sustained cellular stress or altered proliferative capacity in the disease state. This aligns with previous findings that inflammatory cytokines and oxidative stress can impair organoid growth and viability over extended culture periods ( 12 , 16 ). At the transcriptional level, our organoids demonstrated significant downregulation of LGR5 (0.43 ± 0.05 vs. 1.03 ± 0.06, p = 0.0002) in IBD samples, indicative of a depleted or dysfunctional intestinal stem cell compartment. This observation is consistent with Buttó et al. ( 11 ), who reported defective stem cell niche function and impaired crypt regeneration in Crohn’s disease-like ileitis models. The robust upregulation of CXCL8 (3.65 ± 0.37, p = 0.0003) and CXCL10 (4.28 ± 0.15, p < 0.0001) supports prior evidence that these chemokines are key amplifiers of the mucosal inflammatory cascade, driving recruitment of neutrophils and Th1-polarized T cells to the epithelium ( 12 , 14 ). Notably, CCL2 was also significantly increased (2.71 ± 0.17, p = 0.0001), suggesting active monocyte/macrophage chemoattraction, a feature variably reported in prior organoid studies ( 12 ) but well-documented in IBD tissue transcriptomics. This elevation may reflect the retention of innate immune signaling programs within the epithelial compartment, independent of direct immune cell presence. The novelty of our approach lies in the integration of patient-derived primary tissue, a biofabrication strategy using alginate-gelatin scaffolding, and multi-modal characterization, combining morphology, ultrastructure, viability, and targeted inflammatory gene profiling—in a single IBD organoid platform. While prior studies have described IBD organoids, few have systematically correlated morphological deterioration with molecular signatures in a reproducible, scaffold-supported culture system that can sustain long-term growth and repeated passaging. Our use of alginate-gelatin hydrogel as a supportive extracellular matrix substitute offers a cost-effective and tunable alternative to animal-derived matrices, facilitating scalability for future drug screening and mechanistic studies. Furthermore, by preserving patient-specific epithelial phenotypes and inflammatory transcriptional profiles ex vivo , this model offers opportunities for precision medicine applications. For instance, organoids could be exposed to candidate therapeutics to assess patient-specific drug responsiveness or resistance before clinical initiation—an approach already gaining traction in oncology and now poised for translation in gastroenterology ( 16 – 18 ). Implications for Future Research Our findings support the growing consensus that organoid-based systems can bridge the translational gap between reductionist cell culture models and complex animal systems. Future iterations of this platform could incorporate immune components (e.g., macrophages, T cells) and commensal or pathogenic microbes to further recapitulate the intestinal microenvironment. Additionally, integration with microfluidic "gut-on-a-chip" devices may enable simulation of mechanical forces and nutrient/microbe gradients absent in static cultures ( 4 , 16 ). Longitudinal studies assessing stability of disease phenotypes across passages, as well as comparative transcriptomic profiling with matched patient biopsies, will be critical for validating the fidelity of the model. Conclusion This study demonstrates that patient-derived, scaffold-supported colon organoids can preserve key morphological, ultrastructural, and inflammatory features of IBD, including stem cell niche impairment and chemokine-driven inflammatory signaling. By combining physiological relevance, experimental accessibility, and potential for patient-specific modeling, this platform represents a significant step toward more predictive preclinical systems for understanding IBD pathogenesis and advancing personalized therapeutic strategies. Declarations Acknowledgements Not applicable. Authors’ contributions RK conceived and designed the study. MM and SA provided the biopsies. RK, ZH and AA performed experiments and data analysis. ZR and SK contributed to interpretation of results. ZR drafted the manuscript. All authors reviewed, revised, and approved the final version of the manuscript. Ethical considerations This study was approved by the Research Ethics committees of Iran University of Medical Sciences under approval number IR.IUMS.REC.1402.132. Consent to participate Written informed consent was obtained from all participants prior to sample collection. Consent for publication Not applicable. Declaration of conflicting interest The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding statement This research received no external funding Data availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut. 2015;64(6):911-20. Kaplan GG, Windsor JW. 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Huang Y, Huang Z, Tang Z, Chen Y, Huang M, Liu H, et al. Research progress, challenges, and breakthroughs of organoids as disease models. Frontiers in Cell and Developmental Biology. 2021;9:740574. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7498761","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514359032,"identity":"d76b30db-fedc-4891-a4d6-46d41f260cf3","order_by":0,"name":"Roya Karimi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYBAC+wYQeYCBgY2BseHAh4oaOTD/AR4tBgcQWhoPzjhzzBjMTyBGCxAwH+ZtY04EW4tXy/Eeww8MZ+zy+cQONxzmOcOWPj/s8EOgLXZyug04/NJzxliC4UayZZt0YsPBORUyuRtvpxkAtSQbmx3ArsVOIsdAguEDswEbUMuBN2fYcjfOTgBpOZC4DYcWY4kc4x8MH+ohWoB+STecnf4BrxbDGTlmQIcdBms5CNSSIC+dg98WgzPHyiwSzhyHaAEGsuEG6ZyCAwkGuP1icLx5840Px6oN5GenP/4AjEp5IGMzkGEnh0sLAwOHAWosQGLKAJdyEGB/gMqXb8CnehSMglEwCkYiAACFi2wm29wIlQAAAABJRU5ErkJggg==","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Roya","middleName":"","lastName":"Karimi","suffix":""},{"id":514359033,"identity":"2c807b55-de33-4ecf-ab50-dae729320d5d","order_by":1,"name":"Mohsen Masoodi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohsen","middleName":"","lastName":"Masoodi","suffix":""},{"id":514359034,"identity":"ba310d37-8a6a-40a6-ae0e-8538cf5ffe12","order_by":2,"name":"Shahram Agah","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shahram","middleName":"","lastName":"Agah","suffix":""},{"id":514359035,"identity":"e49478d7-ed86-42b6-a98c-b22138840b08","order_by":3,"name":"Zeinab Hajmohammadi","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Hajmohammadi","suffix":""},{"id":514359036,"identity":"9c141491-46df-4ff7-b9fd-d4f75a6bea9e","order_by":4,"name":"Zahra Rahimi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Rahimi","suffix":""},{"id":514359037,"identity":"a498c6a1-2bdf-40de-8034-259c7ccc72cd","order_by":5,"name":"Abolfazl Akbari","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abolfazl","middleName":"","lastName":"Akbari","suffix":""},{"id":514359038,"identity":"1905fadf-b106-45a2-a5c5-27839e264d72","order_by":6,"name":"Shaghayegh Karimi","email":"","orcid":"","institution":"Behbahan Faculty of medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shaghayegh","middleName":"","lastName":"Karimi","suffix":""}],"badges":[],"createdAt":"2025-08-31 06:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7498761/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7498761/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91330097,"identity":"68d468f4-f72d-447c-8dc6-7b098fa4d473","added_by":"auto","created_at":"2025-09-15 10:49:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126272,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative phase-contrast images of IBD organoid development: A) Day 0, B) Day 1, C) Day 5, D) Day 7, E) Day 14, F) Day 21, G) Day 21 post-passage, and H) Day 28 at harvesting. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7498761/v1/e40239b17c96cff790aaf413.jpg"},{"id":91330098,"identity":"73db687c-ce77-43e3-812e-c276b714d984","added_by":"auto","created_at":"2025-09-15 10:49:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52776,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) images showing the surface ultrastructure of the IBD organoid at varying magnifications. (A) 1,000× (scale bar: 20 µm), (B) 2,000× (scale bar: 10 µm), and (C) 5,000× (scale bar: 5 µm).\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7498761/v1/f81c156b43813eefdd5a4a02.jpg"},{"id":91330100,"identity":"8fcc1170-e199-4a80-9577-0c28a89d1a43","added_by":"auto","created_at":"2025-09-15 10:49:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140070,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology and viability assessment of patient-derived IBD colon organoids.\u003cem\u003e (Left)\u003c/em\u003e Phase-contrast micrograph showing the three-dimensional morphology of organoids cultured from patient-derived colonic tissue. Organoids display heterogeneous size and shape distribution with discernible epithelial boundaries. \u003cem\u003e(Right)\u003c/em\u003e Live/Dead fluorescence assay performed at day 21 of culture, with fluorescein diacetate (green) indicating viable cells. The high prevalence of green fluorescence reflects a predominance of viable cells within the culture.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7498761/v1/5d4f3677aaf62561775a8179.jpg"},{"id":91330102,"identity":"a3319ae6-e87b-4bcd-9bda-cfe2057894db","added_by":"auto","created_at":"2025-09-15 10:49:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32804,"visible":true,"origin":"","legend":"\u003cp\u003eBar graphs of normalized gene expression in normal vs. IBD organoids. Data represent mean ± SD. Statistical significance determined by Student’s t-test.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7498761/v1/7cebc8fc66c5e19cacc2156a.jpg"},{"id":91798216,"identity":"e89bea06-a653-4165-bdd6-b4a83b647d82","added_by":"auto","created_at":"2025-09-21 17:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":849560,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7498761/v1/ce98bf44-748a-4f48-ba6f-973015c677f7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biofabrication of primary patient tissue-derived inflammatory bowel disease (IBD) model by organoid three-dimensional culture","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eInflammatory bowel disease (IBD), encompassing Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), is a chronic, relapsing inflammatory disorder of the gastrointestinal (GI) tract. It commonly presents with abdominal pain, diarrhea, fatigue, and unintended weight loss, significantly impairing patients' quality of life and impose a substantial burden on healthcare systems globally. Current treatment strategies primarily rely on pharmacological agents such as amino-salicylates, corticosteroids, and immunosuppressants. While these therapies can reduce inflammation and induce remission, they are often associated with considerable adverse effects, including hepatotoxicity, gastrointestinal disturbances, and dermatologic immune reactions. To overcome these limitations, novel approaches such as targeted drug delivery systems\u0026mdash;like nanoparticle-based carriers\u0026mdash;are being developed to enhance therapeutic efficacy and minimize side effects. Additionally, non-pharmacologic interventions like leukocytapheresis are under investigation as potential alternatives for patients who exhibit poor tolerance to conventional medications. The etiology of IBD is multifactorial, involving genetic susceptibility, environmental influences, dysbiosis of gut microbiota, and dysregulated immune responses; however, the precise pathogenic mechanisms remain inadequately understood. As a result, ongoing research aims to establish more effective, less invasive, and better-tolerated treatment models, while also addressing the substantial global healthcare burden posed by these diseases (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA variety of in vitro and ex vivo models have been developed to investigate the pathophysiology of inflammatory bowel disease (IBD), including both ulcerative colitis and Crohn\u0026rsquo;s disease. These models are increasingly favored over traditional in vivo animal models, which, despite offering valuable insights, fail to fully replicate the complexity of human IBD, prompting a shift towards cellular models that better simulate human gut physiology. Commonly used epithelial cell lines\u0026mdash;such as Caco-2, HT29, and T84\u0026mdash;represent different intestinal cell types and are widely employed to study barrier function, inflammation, and drug absorption. More advanced systems, such as intestinal organoids, enable the study of complex cellular differentiation and tissue architecture, closely reflecting the in vivo environment. Co-culture models, including the combination of absorptive Caco-2 cells with mucus-secreting HT29-MTX cells, provide a more physiologically relevant representation of the gut epithelium. These systems facilitate the study of cellular crosstalk, microbial interactions, and drug permeability\u0026mdash;key aspects in understanding IBD pathogenesis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Additionally, platforms such as the Ussing chamber, everted gut sac, and microfluidic gut-on-chip devices offer dynamic and functional assessments of intestinal tissue. While no single model can fully recapitulate the multifaceted nature of IBD, an integrated approach utilizing multiple complementary systems holds promise for advancing our understanding of disease mechanisms and for accelerating the development of more effective therapies (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTraditional two-dimensional (2D) cell culture models have significantly contributed to our understanding of the cellular and molecular mechanisms involved in inflammatory bowel disease (IBD). However, these models are limited by their inability to replicate the cellular heterogeneity, structural complexity, and dynamic microenvironment of the human intestinal tract, thereby reducing their physiological relevance (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Similarly, while animal models have been instrumental in advancing our understanding of IBD pathogenesis and therapeutic responses, species-specific differences often limit their translational applicability to human disease (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These limitations underscore the pressing need for more physiologically accurate experimental platforms to investigate IBD mechanisms and to facilitate the development of targeted therapeutic interventions.\u003c/p\u003e\u003cp\u003eRecent advances in bioengineering and stem cell technologies have led to the emergence of three-dimensional (3D) culture systems, particularly organoids, which offer a more faithful representation of native human tissues. Derived from pluripotent or adult stem cells, organoids are capable of self-organizing into miniature, organ-like structures that recapitulate key features of the intestinal epithelium, including multiple differentiated cell types and spatial organization. This advancement has positioned organoids as a powerful and reproducible model system for studying IBD pathophysiology and for testing therapeutic strategies in a controlled, human-relevant and reproducible manner (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the context of IBD, intestinal organoids represent a highly promising platform for studying disease mechanisms and evaluating therapeutic interventions. These 3D structures can be bioengineered to incorporate critical components of the intestinal microenvironment\u0026mdash;including epithelial cells, immune populations, and microbial communities\u0026mdash;thereby offering a more physiologically relevant and integrative model compared to traditional systems (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Furthermore, organoids can be generated from patient-derived stem cells, allowing for the investigation of individual disease phenotypes and the development of personalized therapeutic approaches.\u003c/p\u003e\u003cp\u003eThis study aims to develop a biofabricated 3D culture model of IBD using organoid technology. By integrating advanced biofabrication techniques with organoid culture, we seek to create a robust and scalable model that faithfully mimics the pathological features of IBD. This model will not only enhance our understanding of IBD pathogenesis but also serve as a valuable platform for preclinical drug testing and the development of personalized therapeutic strategies (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003col\u003e\n \u003cli\u003ePreparation of Solutions:\u003col class=\"decimal_type\" style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003eWashing Solution: Phosphate-buffered saline (PBS) supplemented with 2% gentamycin and 5% penicillin-streptomycin.\u003c/li\u003e\n \u003cli\u003eComplete Culture Medium: Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.\u003c/li\u003e\n \u003cli\u003e3% Alginate Solution: Prepared by dissolving 0.3 g of alginate in 10 mL of DMEM.\u003c/li\u003e\n \u003cli\u003e10% Gelatin Solution: Prepared by dissolving 0.1 g of gelatin in 10 mL of DMEM.\u003c/li\u003e\n \u003cli\u003eCalcium Chloride (CaCl₂) Solution: 10 mM in sterile distilled water.\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003col class=\"decimal_type\" start=\"2\"\u003e\n \u003cli\u003eProcedure:\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e2.1 Sample Collection\u003c/p\u003e\n\u003cp\u003eColon biopsies were obtained from patients undergoing colonoscopy for IBD diagnosis. Tissues were collected from both inflamed (IBD-affected) and non-inflamed (normal) regions of the colon. Samples were transported to the laboratory in sterile conditions for organoid culture.\u003c/p\u003e\n\u003cp\u003e2.2 Isolation and Organoid Culture\u003c/p\u003e\n\u003cp\u003ea. Pre-coating of Culture Plates:\u003c/p\u003e\n\u003cp\u003eThe day prior to sample processing, 300 \u0026micro;L of 10% gelatin solution was added to each well of a 24-well culture plate. The plate was incubated overnight at 37\u0026deg;C in a 5% CO₂ atmosphere to allow gelatin coating.\u003c/p\u003e\n\u003cp\u003eb. Tissue Handling:\u003c/p\u003e\n\u003cp\u003eImmediately following collection, tissue samples were transferred into sterile 15 mL Falcon tubes containing ~3 mL of washing solution and transported to the cell culture facility.\u003c/p\u003e\n\u003cp\u003ec. Plate Conditioning:\u003c/p\u003e\n\u003cp\u003eThe pre-coated culture plate was removed from the incubator and stored at 4\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003ed. Tissue Washing:\u003c/p\u003e\n\u003cp\u003eSamples were vortexed in the washing solution for 2 minutes, centrifuged at 2000 RPM for 10 minutes, and the supernatant discarded. This wash step was repeated once with fresh washing solution.\u003c/p\u003e\n\u003cp\u003ee. Enzymatic Digestion:\u003c/p\u003e\n\u003cp\u003eTissue pellets were incubated in 1 mL of 1.5% collagenase solution at 37\u0026deg;C with 5% CO₂ for 45 minutes to facilitate digestion.\u003c/p\u003e\n\u003cp\u003ef. Post-digestion Processing:\u003c/p\u003e\n\u003cp\u003eFollowing digestion, an equal volume of complete media was added to neutralize collagenase activity. The mixture was centrifuged at 2000 RPM for 10 minutes, and the supernatant was discarded.\u003c/p\u003e\n\u003cp\u003eg. Matrix Embedding:\u003c/p\u003e\n\u003cp\u003eThe pellet was resuspended in 500 \u0026micro;L of complete media, then mixed with 500 \u0026micro;L of 3% alginate solution. The suspension was pipetted gently to achieve a homogeneous cell suspension.\u003c/p\u003e\n\u003cp\u003eh. 3D Culture Setup:\u003c/p\u003e\n\u003cp\u003eApproximately 500 \u0026micro;L of the alginate-cell suspension was seeded into each well of the pre-coated 24-well plate. Subsequently, 1 mL of 10 mM CaCl₂ solution was added to crosslink the alginate matrix. The plate was incubated at 37\u0026deg;C with 5% CO₂ for 1 hour.\u003c/p\u003e\n\u003cp\u003ei. Media Replacement:\u003c/p\u003e\n\u003cp\u003eAfter incubation, the remaining CaCl₂ solution was discarded and replaced with 1 mL of fresh complete media per well.\u003c/p\u003e\n\u003cp\u003ej. Maintenance:\u003c/p\u003e\n\u003cp\u003eThe first media change was performed after 48 hours. Subsequently, culture media was refreshed every 3 days using a semi-exchange method.\u003c/p\u003e\n\u003cp\u003e2.3 Morphological Analysis of Organoids\u003c/p\u003e\n\u003cp\u003eOrganoid morphology was assessed using both inverted light microscopy for routine monitoring and scanning electron microscopy (SEM) for detailed ultrastructural evaluation.\u003c/p\u003e\n\u003cp\u003ea. Inverted light microscopy\u003c/p\u003e\n\u003cp\u003eDaily morphological changes and growth patterns of the organoids were monitored using an inverted phase-contrast microscope (Olympus IX51 inverted microscope), allowing for non-invasive visualization of size, shape, and structural integrity throughout the culture period.\u003c/p\u003e\n\u003cp\u003eb. Scanning electron microscopy (SEM)\u003c/p\u003e\n\u003cp\u003eOrganoids were fixed in Karnovsky\u0026rsquo;s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) for 1\u0026ndash;2 hours at room temperature. Samples were then dehydrated through a graded ethanol series: 30%, 50%, 70%, 90%, and 100% (twice), each for 5 minutes. Following dehydration, samples were dried using either critical point drying (45\u0026ndash;60 minutes) or hexamethyldisilazane (HMDS) treatment. Dried specimens were mounted on aluminum SEM stubs using conductive carbon adhesive tape and further secured with conductive gold paint to ensure optimal electrical grounding. Samples were then sputter-coated with a thin layer of gold to enhance conductivity and minimize charging artifacts prior to imaging.\u003c/p\u003e\n\u003cp\u003e2.4 Viability assessment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOrganoid viability was assessed using a Live/Dead fluorescence assay. Fluorescein diacetate (FDA) was used to stain viable cells (green fluorescence), while propidium iodide (PI) stained non-viable cells (red fluorescence). Stained organoids were imaged using a fluorescence microscope, and viability was quantified by calculating the proportion of live versus dead cells.\u003c/p\u003e\n\u003cp\u003e2.5 Gene Expression Analysis\u003c/p\u003e\n\u003cp\u003eQuantitative real-time polymerase chain reaction (qPCR) was performed to assess the expression of inflammation- and stem cell\u0026ndash;related genes. Total RNA was extracted from organoids using an RNA isolation kit following the manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized using reverse transcription, and gene-specific primers were designed for LGR5, CXCL8, CXCL10, and CCL2. qPCR reactions were carried out using SYBR Green, and relative gene expression was calculated using the \u0026Delta;\u0026Delta;Ct method with appropriate housekeeping gene normalization.\u003c/p\u003e"},{"header":"Results","content":"\u003col\u003e\n \u003cli\u003eOrganoid Morphology-light invert microscopy\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eWithin 24 hours of seeding, organoids from both normal and IBD colon samples formed compact three-dimensional spheroids. By day 5\u0026ndash;7, crypt-like protrusions became evident. Normal tissue\u0026ndash;derived organoids exhibited uniform spherical architecture and robust proliferation, whereas IBD-derived organoids often showed irregular contours, heterogeneous size distribution, and less compact morphology. By day 14\u0026ndash;21, normal organoids maintained a well-organized structure, while IBD organoids displayed disorganized outer layers and occasional lumen collapse. After passaging, normal organoids readily re-established crypt-like domains, whereas IBD organoids showed delayed recovery (figure 1).\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eScanning Electron Microscopy (SEM)\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eOn day 14, organoids were fixed and analyzed using SEM. In both groups, SEM imaging confirmed the presence of differentiated epithelial surface structures. Normal organoids exhibited well-defined luminal borders, with tightly packed epithelial cells and intact epithelial morphology. In contrast, IBD-derived organoids demonstrated surface irregularities, disrupted cell\u0026ndash;cell junctions, and discontinuities in the epithelial layer, indicative of compromised structural integrity. High-magnification views further revealed microfolds and areas of epithelial flattening in IBD samples, consistent with morphological deterioration (figure 2).\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003eViability Assay\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eOn day 21, organoid viability was assessed using the Live/Dead assay, where green fluorescence indicated viable cells (figure 3). Quantitative image analysis revealed that viable cells covered approximately 71% of the total organoid area. The mean viability across samples was 71.03%. Detailed image-based metrics are presented in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Live/Dead assay quantification at day 21.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"320\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eCount\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eTotal Area (\u0026micro;m\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eAverage Size (\u0026micro;m\u0026sup2;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e%Area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003eMean viability %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1017613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1210.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20.195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e71.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003e\u0026nbsp;Gene Expression analysis\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eqPCR analysis of LGR5, CXCL8, CCL2, and CXCL10 revealed distinct transcriptional profiles. LGR5, a stem cell marker, was significantly downregulated in IBD organoids (p = 0.0002), suggesting reduced stemness potential and impaired regeneration capacity in the inflamed tissue. Conversely, inflammatory mediators CXCL8 (p = 0.0003), CCL2 (p = 0.0001), and CXCL10 (p \u0026lt; 0.0001) were markedly upregulated in IBD organoids relative to normal controls, consistent with an inflammatory activation signature (table 2 and figure 4).\u003c/p\u003e\n\u003cp\u003eTable 2. Relative gene expression (fold change, mean \u0026plusmn; SD) in normal vs. IBD-derived colon organoids.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003egene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eFold change mean \u0026plusmn; SD (normal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eFold change mean \u0026plusmn; SD (IBD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eLGR5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e1.03 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.43 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eCXCL8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e3.65 \u0026plusmn; 0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eCCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e2.71 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eCXCL10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e4.28 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eInflammatory bowel disease (IBD), encompassing Crohn\u0026rsquo;s disease and ulcerative colitis, is a multifactorial, relapsing inflammatory disorder of the gastrointestinal tract with complex interactions between genetic predisposition, immune dysregulation, microbiota alterations, and environmental triggers (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Existing \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models have advanced our understanding of IBD pathogenesis and drug responses, but their translational fidelity remains limited (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Animal models, while valuable for dissecting immune and epithelial mechanisms, are constrained by species-specific differences in immune architecture and gut physiology (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), and conventional 2D cultures fail to capture the spatial complexity, multicellular diversity, and dynamic microenvironment of the human intestine (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This translational gap has driven the development of patient-derived organoid systems, which offer a physiologically relevant, manipulable, and reproducible platform for disease modeling (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we successfully biofabricated patient-derived 3D colon organoids from inflamed (IBD) and non-inflamed (control) tissues, demonstrating that the IBD organoids retain disease-relevant structural and molecular phenotypes. Morphologically, IBD organoids displayed irregular contours, lumen collapse, and delayed regeneration after passaging, in contrast to the uniform crypt-like morphology of normal organoids. These features are consistent with previous reports showing that IBD-derived organoids often exhibit reduced budding efficiency, abnormal epithelial polarity, and compromised barrier properties due to altered stem cell activity and persistent inflammatory signaling (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSEM analysis in our study revealed disrupted cell\u0026ndash;cell junctions and surface discontinuities in IBD organoids. Such ultrastructural deterioration mirrors the epithelial damage and tight junction disruption observed in patient biopsy specimens, which are known to impair barrier function and promote translocation of luminal antigens, perpetuating inflammation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The persistence of these features \u003cem\u003eex vivo\u003c/em\u003e supports the hypothesis that epithelial defects in IBD are at least partly cell-intrinsic and not solely dependent on the in vivo inflammatory milieu.\u003c/p\u003e\u003cp\u003eViability assays indicated an average of 71% live cell area in IBD organoids at day 21, which\u0026mdash;although supportive of long-term culture\u0026mdash;suggests sustained cellular stress or altered proliferative capacity in the disease state. This aligns with previous findings that inflammatory cytokines and oxidative stress can impair organoid growth and viability over extended culture periods (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAt the transcriptional level, our organoids demonstrated significant downregulation of LGR5 (0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs. 1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) in IBD samples, indicative of a depleted or dysfunctional intestinal stem cell compartment. This observation is consistent with Butt\u0026oacute; et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), who reported defective stem cell niche function and impaired crypt regeneration in Crohn\u0026rsquo;s disease-like ileitis models. The robust upregulation of CXCL8 (3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003) and CXCL10 (4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) supports prior evidence that these chemokines are key amplifiers of the mucosal inflammatory cascade, driving recruitment of neutrophils and Th1-polarized T cells to the epithelium (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Notably, CCL2 was also significantly increased (2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001), suggesting active monocyte/macrophage chemoattraction, a feature variably reported in prior organoid studies (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) but well-documented in IBD tissue transcriptomics. This elevation may reflect the retention of innate immune signaling programs within the epithelial compartment, independent of direct immune cell presence.\u003c/p\u003e\u003cp\u003eThe novelty of our approach lies in the integration of patient-derived primary tissue, a biofabrication strategy using alginate-gelatin scaffolding, and multi-modal characterization, combining morphology, ultrastructure, viability, and targeted inflammatory gene profiling\u0026mdash;in a single IBD organoid platform. While prior studies have described IBD organoids, few have systematically correlated morphological deterioration with molecular signatures in a reproducible, scaffold-supported culture system that can sustain long-term growth and repeated passaging. Our use of alginate-gelatin hydrogel as a supportive extracellular matrix substitute offers a cost-effective and tunable alternative to animal-derived matrices, facilitating scalability for future drug screening and mechanistic studies.\u003c/p\u003e\u003cp\u003eFurthermore, by preserving patient-specific epithelial phenotypes and inflammatory transcriptional profiles \u003cem\u003eex vivo\u003c/em\u003e, this model offers opportunities for precision medicine applications. For instance, organoids could be exposed to candidate therapeutics to assess patient-specific drug responsiveness or resistance before clinical initiation\u0026mdash;an approach already gaining traction in oncology and now poised for translation in gastroenterology (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eImplications for Future Research\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur findings support the growing consensus that organoid-based systems can bridge the translational gap between reductionist cell culture models and complex animal systems. Future iterations of this platform could incorporate immune components (e.g., macrophages, T cells) and commensal or pathogenic microbes to further recapitulate the intestinal microenvironment. Additionally, integration with microfluidic \"gut-on-a-chip\" devices may enable simulation of mechanical forces and nutrient/microbe gradients absent in static cultures (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Longitudinal studies assessing stability of disease phenotypes across passages, as well as comparative transcriptomic profiling with matched patient biopsies, will be critical for validating the fidelity of the model.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study demonstrates that patient-derived, scaffold-supported colon organoids can preserve key morphological, ultrastructural, and inflammatory features of IBD, including stem cell niche impairment and chemokine-driven inflammatory signaling. By combining physiological relevance, experimental accessibility, and potential for patient-specific modeling, this platform represents a significant step toward more predictive preclinical systems for understanding IBD pathogenesis and advancing personalized therapeutic strategies.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRK conceived and designed the study. MM and SA provided the biopsies. RK, ZH and AA performed experiments and data analysis. ZR and SK contributed to interpretation of results. ZR drafted the manuscript. All authors reviewed, revised, and approved the final version of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Research Ethics committees of Iran University of Medical Sciences under approval number IR.IUMS.REC.1402.132.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all participants prior to sample collection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, et al. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut. 2015;64(6):911-20.\u003c/li\u003e\n\u003cli\u003eKaplan GG, Windsor JW. The four epidemiological stages in the global evolution of inflammatory bowel disease. Nature reviews Gastroenterology \u0026amp; hepatology. 2021;18(1):56-66.\u003c/li\u003e\n\u003cli\u003eDel Sordo R, Lougaris V, Bassotti G, Armuzzi A, Villanacci V. Therapeutic agents affecting the immune system and drug-induced inflammatory bowel disease (IBD): A review on etiological and pathogenetic aspects. Clinical Immunology. 2022;234:108916.\u003c/li\u003e\n\u003cli\u003eJoshi A, Soni A, Acharya S. In vitro models and ex vivo systems used in inflammatory bowel disease. In vitro models. 2022;1(3):213-27.\u003c/li\u003e\n\u003cli\u003eAdamczak MI, Hagesaether E, Smistad G, Hiorth M. An in vitro study of mucoadhesion and biocompatibility of polymer coated liposomes on HT29-MTX mucus-producing cells. International journal of pharmaceutics. 2016;498(1-2):225-33.\u003c/li\u003e\n\u003cli\u003eLi C, Lun W, Zhao X, Lei S, Guo Y, Ma J, et al. Allicin alleviates inflammation of trinitrobenzenesulfonic acid‐induced rats and suppresses P38 and JNK pathways in Caco‐2 cells. Mediators of inflammation. 2015;2015(1):434692.\u003c/li\u003e\n\u003cli\u003eKiesler P, Fuss IJ, Strober W. Experimental models of inflammatory bowel diseases. Cellular and molecular gastroenterology and hepatology. 2015;1(2):154-70.\u003c/li\u003e\n\u003cli\u003eWen C, Chen D, Zhong R, Peng X. Animal models of inflammatory bowel disease: category and evaluation indexes. Gastroenterology Report. 2024;12:goae021.\u003c/li\u003e\n\u003cli\u003eHolloway EM, Capeling MM, Spence JR. Biologically inspired approaches to enhance human organoid complexity. Development. 2019;146(8):dev166173.\u003c/li\u003e\n\u003cli\u003eYin X, Farin HF, Van Es JH, Clevers H, Langer R, Karp JM. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nature methods. 2014;11(1):106-12.\u003c/li\u003e\n\u003cli\u003eButt\u0026oacute; LF, Pelletier A, More SK, Zhao N, Osme A, Hager CL, et al. Intestinal stem cell niche defects result in impaired 3D organoid formation in mouse models of Crohn\u0026apos;s disease-like ileitis. Stem cell reports. 2020;15(2):389-407.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Connell L, Winter DC, Aherne CM. The role of organoids as a novel platform for modeling of inflammatory bowel disease. Frontiers in Pediatrics. 2021;9:624045.\u003c/li\u003e\n\u003cli\u003eXu Y, Shrestha N, Pr\u0026eacute;at V, Beloqui A. An overview of in vitro, ex vivo and in vivo models for studying the transport of drugs across intestinal barriers. Advanced Drug Delivery Reviews. 2021;175:113795.\u003c/li\u003e\n\u003cli\u003eHartwig O, Boushehri MAS, Shalaby KS, Loretz B, Lamprecht A, Lehr C-M. Drug delivery to the inflamed intestinal mucosa\u0026ndash;targeting technologies and human cell culture models for better therapies of IBD. Advanced Drug Delivery Reviews. 2021;175:113828.\u003c/li\u003e\n\u003cli\u003evan der Sloot KW, Weersma RK, Alizadeh BZ, Dijkstra G. Identification of environmental risk factors associated with the development of inflammatory bowel disease. Journal of Crohn\u0026apos;s and Colitis. 2020;14(12):1662-71.\u003c/li\u003e\n\u003cli\u003eWakisaka Y, Sugimoto S, Sato T. Organoid medicine for inflammatory bowel disease. Stem Cells. 2022;40(2):123-32.\u003c/li\u003e\n\u003cli\u003eOkamoto R, Shimizu H, Suzuki K, Kawamoto A, Takahashi J, Kawai M, et al. Organoid-based regenerative medicine for inflammatory bowel disease. Regenerative therapy. 2020;13:1-6.\u003c/li\u003e\n\u003cli\u003eHuang Y, Huang Z, Tang Z, Chen Y, Huang M, Liu H, et al. Research progress, challenges, and breakthroughs of organoids as disease models. Frontiers in Cell and Developmental Biology. 2021;9:740574.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inflammatory Bowel Disease, Patient-Derived Organoids, Organoids, Three-Dimensional (3D) Culture, Alginate-Gelatin Scaffold","lastPublishedDoi":"10.21203/rs.3.rs-7498761/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7498761/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Inflammatory bowel disease is a chronic gastrointestinal disorder characterized by relapsing inflammation, disruption of the epithelial barrier, and dysregulated immune responses, leading to substantial morbidity. Conventional in vitro cell cultures and animal models often fail to reproduce the cellular heterogeneity, tissue architecture, and patient-specific features of the disease, limiting mechanistic understanding and therapeutic development. This study aimed to develop and characterize a patient-derived three-dimensional organoid model that closely recapitulates the structural, functional, and molecular hallmarks of the disease. Colonic biopsies from inflamed and non-inflamed regions of patients were embedded in alginate–gelatin scaffolds to generate organoids that maintain physiological tissue architecture and long-term viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Organoids from non-inflamed tissue maintained uniform spherical morphology with well-defined crypt-like domains, whereas organoids derived from inflamed tissue exhibited irregular architecture, disrupted epithelial junctions, and delayed recovery after passage. Scanning electron microscopy confirmed epithelial discontinuities and surface microfold irregularities in inflamed organoids. Functional viability assessments demonstrated an average survival rate of 71.0% at day 21. Gene expression analysis revealed significant downregulation of the intestinal stem cell marker LGR5 in inflamed organoids compared to controls (0.43 ± 0.05 vs. 1.03 ± 0.06, p = 0.0002), along with marked upregulation of inflammatory chemokines CXCL8 (3.65 ± 0.37, p = 0.0003), CCL2 (2.71 ± 0.17, p = 0.0001), and CXCL10 (4.28 ± 0.15, p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This patient-derived three-dimensional organoid system accurately models disease-associated structural deterioration, impaired regenerative capacity, and inflammatory signaling, providing a physiologically relevant and reproducible platform for mechanistic studies. The model enables high-throughput drug screening, evaluation of patient-specific therapeutic responses, and development of personalized interventions. By bridging the gap between conventional in vitro and in vivo systems, this organoid platform represents a significant advance in tissue engineering and translational gastrointestinal research, facilitating precise investigation of disease pathophysiology and accelerating the development of effective therapies for chronic inflammatory disorders.\u003c/p\u003e","manuscriptTitle":"Biofabrication of primary patient tissue-derived inflammatory bowel disease (IBD) model by organoid three-dimensional culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 10:49:32","doi":"10.21203/rs.3.rs-7498761/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec51596a-838b-47fe-9cf4-9dbdf0a5d6c4","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-21T17:08:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-15 10:49:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7498761","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7498761","identity":"rs-7498761","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}