Characterization and optimization of variability in a human colonic epithelium culture model
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Abstract
Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem-cell-based cultures makes development of useful models a challenge; the stochastic nature of stem-cell differentiation interferes with the ability to build and validate reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling cell passage number reduces variability and maximizes physiological relevance of the model. In a case study where passage number was optimized, distinct cytokine responses were observed among four human donors, indicating that biological variability can be detected in cell cultures originating from diverse human sources. These findings highlight key considerations for designing assays that can be applied to additional primary-cell derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.
Plain language summary
Animal models are frequently used as tools for studying gastrointestinal (GI) disease, but they poorly replicate the complexities of the human gut limiting the clinical translation of new therapeutics in development. Human stem cell derived models can better recapitulate human GI physiology, but the inherent dynamic nature of stem cells introduces variability in culture performance. We identified sources of variability in the primary stem-cell derived RepliGut® Planar model to develop robust and reliable assays that can improve preclinical therapeutic development for GI disease. Analysis of barrier formation, gene expression, and cytokine responses demonstrated that controlling cell passage number reduces variability and maximizes physiological relevance of the model. These findings highlight key assay design considerations that can be applied to additional primary-cell derived systems. Availability of reliable and physiologically relevant cell-based models can reduce animal testing, improve research accuracy, and make new treatments more relevant and effective for patients.
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Ahmad, A. A. et al. (2014). Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications, Journal of Biological Engineering 8, 9. doi:10.1186/1754-1611-8-9
Al-Bawardy, B., Shivashankar, R. and Proctor, D. D. (2021). Novel and Emerging Therapies for Inflammatory Bowel Disease, Frontiers in Pharmacology, 12. doi:10.3389/fphar.2021.651415
Andreou, N.-P., Legaki, E. and Gazouli, M. (2020). Inflammatory bowel disease pathobiology: the role of the interferon signature, Annals of Gastroenterology, 33(2), pp. 125–133. doi:10.20524/aog.2020.0457
Antunes, J. C. et al. (2021). Drug Targeting of Inflammatory Bowel Diseases by Biomolecules, Nanomaterials, 11(8), p. 2035. doi:10.3390/nano11082035
Apostolou, A. et al. (2021). A Novel Microphysiological Colon Platform to Decipher Mechanisms Driving Human Intestinal Permeability, Cellular and Molecular Gastroenterology and Hepatology, 12(5), pp. 1719–1741. doi:10.1016/j.jcmgh.2021.07.004
Bank, S. et al. (2014). Polymorphisms in the Inflammatory Pathway Genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG Are Associated with Susceptibility of Inflammatory Bowel Disease in a Danish Cohort, PLoS ONE. Edited by M.M. Heimesaat, 9(6), p. e98815. doi:10.1371/journal.pone.0098815
Beaurivage, C. et al. (2019). Development of a Gut-on-a-Chip Model for High Throughput Disease Modeling and Drug Discovery, International Journal of Molecular Sciences, 20(22), p. 5661. doi:10.3390/ijms20225661
Benjamini, Y. and Hochberg, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society: Series B 57, 289-300. doi:10.1111/j.2517-6161.1995.tb02031.x
Bhatt, A. P., Gunasekara, D. B., Speer, J., Reed, M. I., Peña, A. N., Midkiff, B. R., Magness, S. T., Bultman, S. J., Allbritton, N. L., & Redinbo, M. R. (2018). Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells. ACS infectious diseases, 4(1), 46–52. doi:10.1021/acsinfecdis.7b00139
Biagini, F. et al. (2023). Designs and methodologies to recreate in vitro human gut microbiota models, Bio-Design and Manufacturing, 6(3), pp. 298–318. doi:10.1007/s42242-022-00210-6
Cai, Z., Wang, S. and Li, J. (2021). Treatment of Inflammatory Bowel Disease: A Comprehensive Review, Frontiers in Medicine, 8. doi:10.3389/fmed.2021.765474
Creff, J., Malaquin, L. and Besson, A. (2021). In vitro models of intestinal epithelium: Toward bioengineered systems, Journal of Tissue Engineering, 12, p. 204173142098520. doi:10.1177/2041731420985202
Dutton, J. S. et al. (2019). Primary Cell-Derived Intestinal Models: Recapitulating Physiology, Trends in biotechnology, 37(7), p. 744. doi:10.1016/j.tibtech.2018.12.001
Dwinell, M. B. et al. (2001). Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells, Gastroenterology, 120(1), pp. 49–59. doi:10.1053/gast.2001.20914
Franco, Y. L., Da Silva, L. and Cristofoletti, R. (2021). Navigating Through Cell-Based In vitro Models Available for Prediction of Intestinal Permeability and Metabolism: Are We Ready for 3D?, The AAPS Journal, 24(1), p. 2. doi:10.1208/s12248-021-00665-y
Friedrich, M., Pohin, M. and Powrie, F. (2019). Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease, Immunity, 50(4), pp. 992–1006. doi:10.1016/j.immuni.2019.03.017
Gareb, B. et al. (2020). Review: Local Tumor Necrosis Factor-α Inhibition in Inflammatory Bowel Disease, Pharmaceutics, 12(6), p. 539. doi:10.3390/pharmaceutics12060539
Gracz, A.D. and Magness, S. T. (2014). Defining hierarchies of stemness in the intestine: evidence from biomarkers and regulatory pathways, American Journal of Physiology-Gastrointestinal and Liver Physiology, 307(3), pp. G260–G273. doi:10.1152/ajpgi.00066.2014
Grossmann, J. et al. (1998). New isolation technique to study apoptosis in human intestinal epithelial cells, The American journal of pathology, 153(1), pp. 53–62. doi:10.1016/s0002-9440(10)65545-9
Grossmann, J. et al. (2003) .Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC), European Journal of Cell Biology, 82(5), pp. 262–270. doi:10.1078/0171-9335-00312
Gunasekara, D. B. et al. (2018). A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies, Analytical Chemistry, 90(22), pp. 13331–13340. doi:10.1021/acs.analchem.8b02845
Khan, I. et al. (2019). Alteration of Gut Microbiota in Inflammatory Bowel Disease (IBD): Cause or Consequence? IBD Treatment Targeting the Gut Microbiome, Pathogens, 8(3), p. 126. doi:10.3390/pathogens8030126
Kucharzik, T. et al. (2005). Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury, Gut, 54(11), pp. 1565–1572. doi:10.1136/gut.2004.061168
Larregieu, C. A. and Benet, L. Z. (2013). Drug Discovery and Regulatory Considerations for Improving In Silico and In Vitro Predictions that Use Caco-2 as a Surrogate for Human Intestinal Permeability Measurements, The AAPS Journal, 15(2), pp. 483–497. doi:10.1208/s12248-013-9456-8
Lennernäs, H. (2007). Intestinal permeability and its relevance for absorption and elimination, Xenobiotica, 37(10–11), pp. 1015–1051. doi:10.1080/00498250701704819
Liu, L. and Rando, T. A. (2011). Manifestations and mechanisms of stem cell aging, The Journal of Cell Biology, 193(2), pp. 257–266. doi:10.1083/jcb.201010131
Marrero, D. et al. (2021). Gut-on-a-chip: Mimicking and monitoring the human intestine, Biosensors and Bioelectronics, 181, p. 113156. doi:10.1016/j.bios.2021.113156
Mohammadi, S. et al. (2021). Assessing donor-to-donor variability in human intestinal organoid cultures, Stem Cell Reports, 16(9), pp. 2364–2378. doi:10.1016/j.stemcr.2021.07.016
Monticello, T. M. et al. (2017). Current nonclinical testing paradigm enables safe entry to First-In-Human clinical trials: The IQ consortium nonclinical to clinical translational database, Toxicology and Applied Pharmacology, 334, pp. 100–109. doi:10.1016/j.taap.2017.09.006
Olson, H. et al. (2000). Concordance of the toxicity of pharmaceuticals in humans and in animals, Regulatory toxicology and pharmacology: RTP, 32(1), pp. 56–67. doi:10.1006/rtph.2000.1399
Parlesak, A. (2004). Modulation of cytokine release by differentiated CACO‐2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria., Scandinavian journal of immunology, 60(5), pp. 477–485. doi:10.1111/j.0300-9475.2004.01495.x
Peters, M. F. et al. (2019). Human 3D Gastrointestinal Microtissue Barrier Function As a Predictor of Drug-Induced Diarrhea, Toxicological Sciences, 168(1), pp. 3–17. doi:10.1093/toxsci/kfy268
Press, B. and Grandi, D. D. (2008). Permeability for Intestinal Absorption: Caco-2 Assay and Related Issues, Current Drug Metabolism, 9(9), pp. 893–900. doi:10.2174/138920008786485119
Rees, W. D. et al. (2020). Regenerative Intestinal Stem Cells Induced by Acute and Chronic Injury: The Saving Grace of the Epithelium?, Frontiers in Cell and Developmental Biology, 8. doi:10.3389/fcell.2020.583919
Reynolds, A. et al. (2014). Canonical Wnt signals combined with suppressed TGFβ/BMP pathways promote renewal of the native human colonic epithelium, Gut, 63(4), pp. 610–621. doi:10.1136/gutjnl-2012-304067.
Sambuy, Y. et al. (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics, Cell Biology and Toxicology, 21(1), pp. 1–26. doi:10.1007/s10565-005-0085-6.
Sashio, H. et al. (2002). Polymorphisms of the TNF gene and the TNF receptor superfamily member 1B gene are associated with susceptibility to ulcerative colitis and Crohn’s disease, respectively, (53), pp. 1020–1027. doi:10.1007/s00251-001-0423-7
Sato, T. et al. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche, Nature, 459(7244), pp. 262–265. doi:10.1038/nature07935
Snippert, H. J. et al. (2010). Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells, Cell, 143(1), pp. 134–144. doi:10.1016/j.cell.2010.09.016
Sonnier, D. I. et al. (2010). TNF-α Induces Vectorial Secretion of IL-8 in Caco-2 Cells, Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract, 14(10), pp. 1592–1599. doi:10.1007/s11605-010-1321-9
Sun, H. et al. (2008). The Caco-2 cell monolayer: usefulness and limitations, Expert Opinion on Drug Metabolism & Toxicology, 4(4), pp. 395–411. doi:10.1517/17425255.4.4.395
Treede, I. et al. (2009). TNF-α-induced up-regulation of pro-inflammatory cytokines is reduced by phosphatidylcholine in intestinal epithelial cells, BMC Gastroenterology, 9(1), p. 53. doi:10.1186/1471-230X-9-53
Trujillo-de Santiago, G. et al. (2018). Gut-microbiota-on-a-chip: an enabling field for physiological research, Microphysiological Systems, 1, pp. 1–1. doi:10.21037/mps.2018.09.01
VanDussen, K. L. et al. (2015). Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays, Gut, 64(6), pp. 911–920. doi:10.1136/gutjnl-2013-306651
Wang, Q. et al. (2022). Applications of human organoids in the personalized treatment for digestive diseases, Signal Transduction and Targeted Therapy, 7(1), p. 336. doi:10.1038/s41392-022-01194-6
Wang, Y. et al. (2017). Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells, Cellular and Molecular Gastroenterology and Hepatology, 4(1), pp. 165-182.e7. doi:10.1016/j.jcmgh.2017.02.011
Yoo, J.-H. and Donowitz, M. (2019). Intestinal enteroids/organoids: A novel platform for drug discovery in inflammatory bowel diseases, World Journal of Gastroenterology, 25(30), pp. 4125–4147. doi:10.3748/wjg.v25.i30.4125