Publications

@article{abbas_primary_2025,

title = {A primary human Gut/Liver microphysiological system to estimate human oral bioavailability},

author = {Yassen Abbas and Morné van Wyk and Hailey Sze and James Christophi and Ashley A. Spreen and Robert Jarrett Bliton and Elizabeth M. Boazak and Tomasz Kostrzewski},

url = {https://dmd.aspetjournals.org/article/S0090-9556(25)09139-1/fulltext},

doi = {10.1016/j.dmd.2025.100130},

issn = {0090-9556, 1521-009X},

year  = {2025},

date = {2025-09-01},

urldate = {2025-09-01},

journal = {Drug Metabolism and Disposition},

volume = {53},

number = {9},

note = {Publisher: Elsevier},

keywords = {absorption, Bioavailability, Biopharmaceutics Classification System, Fa, Fg, Gut liver microphysiological system, metabolism, microphysiological system, Organ-on-a-chip, Papp, pharmacokinetic},

pubstate = {published},

tppubtype = {article}

}

@article{peddibhotla_scalable_2025,

title = {A scalable human gut-immune co-culture model for evaluating inflammatory bowel disease anti-inflammatory therapies},

author = {Swetha Peddibhotla and Lauren A. Boone and Earnest L. Taylor and Bryan E. McQueen and Elizabeth M. Boazak},

url = {https://www.slas-discovery.org/article/S2472-5552(25)00041-3/fulltext},

doi = {10.1016/j.slasd.2025.100248},

issn = {2472-5552, 2472-5560},

year  = {2025},

date = {2025-09-01},

urldate = {2025-09-01},

journal = {SLAS Discovery},

volume = {35},

note = {Publisher: Elsevier},

keywords = {{THP}-1 macrophages, Co-culture model, Immune-epithelial interaction, Inflammatory bowel disease ({IBD}), Primary human intestinal cells},

pubstate = {published},

tppubtype = {article}

}

@article{hudson_comparing_2025,

title = {Comparing the Altis RepliGut Organoid System to MDCK Monolayers in Predicting the Oral Absorption of Lenalidomide},

author = {Cole S. Hudson and Jonathan Cheong and Jesse Yu and Eugene C. Chen and Laurent Salphati and Matthew R. Durk and Benjamin Lai and Karen Samy},

url = {https://www.mdpi.com/1999-4923/17/9/1140},

doi = {10.3390/pharmaceutics17091140},

issn = {1999-4923},

year  = {2025},

date = {2025-08-30},

journal = {Pharmaceutics},

volume = {17},

number = {9},

pages = {1140},

abstract = {Background: Predicting oral drug absorption in humans is critical during early drug development. Current in vitro systems to predict absorption (e.g., PAMPA and MDCK cells) are lacking for certain classes of drugs. Intestinal organoids are emerging as a promising alternative that offers several potential advantages. In this study, we utilized human intestinal organoid-derived monolayers to predict oral absorption of lenalidomide. Methods: Human jejunal organoids (RepliGut®) were cultured as monolayers on transwell plates and differentiated into intestinal epithelial cells. Lenalidomide permeability in the organoid system was compared with the permeability in the conventional Madin-Darby Canine Kidney cell (MDCK) monolayer system, as well as P-gp knockout, human P-gp overexpressing, and human BCRP overexpressing MDCK cells across a concentration range of 1 to 500 µM. Male Sprague Dawley rats were administered lenalidomide orally/intravenously, and concentrations in the serum, urine, and feces were measured and modeled in Phoenix WinNonlin. Results: Orally administered lenalidomide was well absorbed by rats at all doses (bioavailability = 68–120%). In the human jejunal organoid model, lenalidomide apparent permeability (Papp) was approximately 0.6 × 10−6 cm/s independent of the concentration used (1–500 µM). In contrast, lenalidomide Papp was significantly lower in gMDCK cell monolayers, approximately 0.2 × 10−6 cm/s. Additionally, lenalidomide was identified as a P-gp/BCRP substrate in intestinal organoids and gMDCK P-gp and BCRP overexpressing cells. Conclusions: Lenalidomide Papp was significantly lower in gMDCK monolayers than expected based on its high bioavailability. Our results suggest that organoid systems can better capture transporter and paracellularly mediated effects on drug permeability, which may allow for more accurate predictions of in vivo absorption.},

note = {Publisher: Multidisciplinary Digital Publishing Institute},

keywords = {{MDCK} cells, drug permeability, intestinal organoids, lenalidomide, oral bioavailability},

pubstate = {published},

tppubtype = {article}

}

@article{thomas_clostridioides_2025,

title = {Clostridioides difficile toxins alter host metabolic pathway and bile acid homeostasis gene expression in colonic epithelium},

author = {Stephanie A. Thomas and Colleen M. Pike and Cypress E. Perkins and Sean T. Brown and Xochilt M. Espinoza Jaen and Arthur S. McMillan and Casey M. Theriot},

url = {https://journals.asm.org/doi/full/10.1128/iai.00150-25},

doi = {10.1128/iai.00150-25},

year  = {2025},

date = {2025-06-30},

urldate = {2025-06-30},

journal = {Infection and Immunity},

volume = {93},

number = {8},

pages = {e00150–25},

note = {Publisher: American Society for Microbiology},

keywords = {intestinal microenvironment, microbiome},

pubstate = {published},

tppubtype = {article}

}

@article{pike_high-throughput_2025,

title = {High-throughput assay for predicting diarrhea risk using a 2D human intestinal stem cell-derived model},

author = {Colleen M. Pike and James A. Levi and Lauren A. Boone and Swetha Peddibhotla and Jacob Johnson and Bailey Zwarycz and Maureen K. Bunger and William Thelin and Elizabeth M. Boazak},

url = {https://www.sciencedirect.com/science/article/pii/S0887233325000347},

doi = {10.1016/j.tiv.2025.106040},

issn = {0887-2333},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

journal = {Toxicology In Vitro},

volume = {106},

pages = {106040},

abstract = {Gastrointestinal toxicities (GITs) in clinical trials often lead to dose-limitations that reduce drug efficacy and delay treatment optimization. Preclinical animal models do not accurately replicate human physiology, leaving few options for early detection of GITs, such as diarrhea, before human studies. Chemotherapeutic agents, known to cause clinical diarrhea, frequently target mitotic cells. Therefore, we hypothesized a model utilizing proliferative cell populations derived from human intestinal crypts would predict clinical diarrhea occurrence with high accuracy. Here, we describe the development of a diarrhea prediction assay utilizing RepliGut® Planar, a primary intestinal stem cell-derived platform. To evaluate the ability of this model to predict clinical diarrhea risk, we assessed toxicity of 30 marketed drugs by measuring cell proliferation (EdU incorporation), cell abundance (nuclei quantification), and barrier formation (TEER) in cells derived from three human donors. Dose response curves were generated for each drug, and the IC15 to Cmax ratio was used to identify a threshold for assay positivity. This model accurately predicted diarrhea potential, achieving an accuracy of 91 % for proliferation, 90 % for abundance, and 88 % for barrier formation. In vitro toxicity screening using primary proliferative cells may reduce clinical diarrhea and ultimately lead to safer and more effective treatments for patients.},

keywords = {Adverse events, Diarrhea, Epithelium, High throughput, In vitro model, Intestine},

pubstate = {published},

tppubtype = {article}

}

@article{corte_morphogenesis_2024,

title = {Morphogenesis of bacterial colonies in polymeric environments},

author = {Sebastian Gonzalez La Corte and Corey A. Stevens and Gerardo Cárcamo-Oyarce and Katharina Ribbeck and Ned S. Wingreen and Sujit S. Datta},

url = {https://www.science.org/doi/10.1126/sciadv.adq7797},

doi = {10.1126/sciadv.adq7797},

year  = {2025},

date = {2025-01-17},

urldate = {2024-04-22},

journal = {Science Advances},

volume = {11},

number = {3},

issue = {3},

pages = {eadq7797},

publisher = {bioRxiv},

abstract = {Many bacteria live in polymeric fluids, such as mucus, environmental polysaccharides, and extracellular polymers in biofilms. However, lab studies typically focus on cells in polymer-free fluids. Here, we show that interactions with polymers shape a fundamental feature of bacterial life—how they proliferate in space in multicellular colonies. Using experiments, we find that when polymer is sufficiently concentrated, cells generically and reversibly form large serpentine “cables” as they proliferate. By combining experiments with biophysical theory and simulations, we demonstrate that this distinctive form of colony morphogenesis arises from an interplay between polymer-induced entropic attraction between neighboring cells and their hindered ability to diffusely separate from each other in a viscous polymer solution. Our work thus reveals a pivotal role of polymers in sculpting proliferating bacterial colonies, with implications for how they interact with hosts and with the natural environment, and uncovers quantitative principles governing colony morphogenesis in such complex environments.},

note = {Pages: 2024.04.18.590088 

Section: New Results},

keywords = {microbiome, Mucus},

pubstate = {published},

tppubtype = {article}

}

@article{magnusson_exposure_2024,

title = {Exposure of Colon-Derived Epithelial Monolayers to Fecal Luminal Factors from Patients with Colon Cancer and Ulcerative Colitis Results in Distinct Gene Expression Patterns},

author = {Maria K. Magnusson and Anna Bas Forsberg and Alexandra Verveda and Maria Sapnara and Julie Lorent and Otto Savolainen and Yvonne Wettergren and Hans Strid and Magnus Simrén and Lena Öhman},

url = {https://www.mdpi.com/1422-0067/25/18/9886},

doi = {10.3390/ijms25189886},

issn = {1422-0067},

year  = {2024},

date = {2024-09-13},

urldate = {2024-09-13},

journal = {Int. J. Mol. Sci.},

volume = {25},

number = {18},

pages = {9886},

abstract = {Microbiota and luminal components may affect epithelial integrity and thus participate in the pathophysiology of colon cancer (CC) and inflammatory bowel disease (IBD). Therefore, we aimed to determine the effects of fecal luminal factors derived from patients with CC and ulcerative colitis (UC) on the colonic epithelium using a standardized colon-derived two-dimensional epithelial monolayer. The complex primary human stem cell-derived intestinal epithelium model, termed RepliGut® Planar, was expanded and passaged in a two-dimensional culture which underwent stimulation for 48 h with fecal supernatants (FS) from CC patients (n = 6), UC patients with active disease (n = 6), and healthy subjects (HS) (n = 6). mRNA sequencing of monolayers was performed and cytokine secretion in the basolateral cell culture compartment was measured. The addition of fecal supernatants did not impair the integrity of the colon-derived epithelial monolayer. However, monolayers stimulated with fecal supernatants from CC patients and UC patients presented distinct gene expression patterns. Comparing UC vs. CC, 29 genes were downregulated and 33 genes were upregulated, for CC vs. HS, 17 genes were downregulated and five genes were upregulated, and for UC vs. HS, three genes were downregulated and one gene was upregulated. The addition of FS increased secretion of IL8 with no difference between the study groups. Fecal luminal factors from CC patients and UC patients induce distinct colonic epithelial gene expression patterns, potentially reflecting the disease pathophysiology. The culture of colonic epithelial monolayers with fecal supernatants derived from patients may facilitate the exploration of IBD- and CC-related intestinal microenvironmental and barrier interactions.},

note = {Publisher: Multidisciplinary Digital Publishing Institute},

keywords = {colon cancer, epithelial barrier, epithelial monolayers, inflammatory bowel disease, intestinal microenvironment},

pubstate = {published},

tppubtype = {article}

}

@article{pike_characterization_2024,

title = {Characterization and optimization of variability in a human colonic epithelium culture model},

author = {Colleen M. Pike and Bailey Zwarycz and Bryan E. McQueen and Mariana Castillo and Catherine Barron and Jeremy M. Morowitz and James A. Levi and Dhiral Phadke and Michele Balik-Meisner and Deepak Mav and Ruchir Shah and Danielle L. Cunningham Glasspoole and Ron Laetham and William Thelin and Maureen K. Bunger and Elizabeth M. Boazak},

url = {https://www.altex.org/index.php/altex/article/view/2686},

doi = {10.14573/altex.2309221},

issn = {1868-8551},

year  = {2024},

date = {2024-07-16},

urldate = {2024-07-16},

journal = {ALTEX},

volume = {41},

number = {3},

pages = {425–438},

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 inad­equately replicate the complexities of the human gut, making them poor predictors of how humans respond to new drugs. Models using human stem cells are closer to human GI physiology, but their responses are not uniform owing to variability in the stem cells. We looked for the sources of this variability in the primary stem-cell derived RepliGut® Planar model. We found that limiting how long the cells were kept in culture reduced their variability and improved the physiological relevance of the model. These findings highlight key assay design considerations that also can be applied to other primary cell-derived systems. Reliable and physiologically relevant cell-based models can reduce animal testing, improve research accuracy, and ensure new treatments are more relevant and effective for patients.},

note = {Number: 3},

keywords = {epithelial cell culture, in vitro models, intestinal barrier, microphysiological systems, permeability},

pubstate = {published},

tppubtype = {article}

}

@article{debad_trust_2024,

title = {Trust your gut: Establishing confidence in gastrointestinal models – An overview of the state of the science and contexts of use},

author = {Susan Debad and David Allen and Omari Bandele and Colin Bishop and Michaela Blaylock and Paul Brown and Maureen K. Bunger and Julia Y. Co and Lynn Crosby and Amber B. Daniel and Steve S. Ferguson and Kevin Ford and Gonçalo Gamboa da Costa and Kristin H. Gilchrist and Matthew W. Grogg and Maureen Gwinn and Thomas Hartung and Simon P. Hogan and Ye Eun Jeong and George E. N. Kass and Elaina Kenyon and Nicole C. Kleinstreuer and Ville Kujala and Patrik Lundquist and Joanna Matheson and Shaun D. McCullough and Angela Melton-Celsa and Steven Musser and Ilung Oh and Oluwakemi B. Oyetade and Sarita U. Patil and Elijah J. Petersen and Nakissa Sadrieh and Christie M. Sayes and Benjamin S. Scruggs and Yu-Mei Tan and Bill Thelin and M. Tyler Nelson and José V. Tarazona and John F. Wambaugh and Jun-Young Yang and Changwoo Yu and Suzanne Fitzpatrick},

url = {https://altex.org/index.php/altex/article/view/2787},

doi = {10.14573/altex.2403261},

issn = {1868-8551},

year  = {2024},

date = {2024-07-16},

urldate = {2024-07-16},

journal = {ALTEX},

volume = {41},

number = {3},

pages = {402–424},

abstract = {The webinar series and workshop titled “Trust Your Gut: Establishing Confidence in Gastrointestinal Models – An Overview of the State of the Science and Contexts of Use” was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting. 

Plain language summaryNon-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.},

keywords = {Adverse events, Co-culture model, Enteroendocrine Cells, epithelial barrier, Gut barrier function, Gut liver microphysiological system, in vitro models, intestinal barrier, microphysiological system, microphysiological systems, organ-on-chips},

pubstate = {published},

tppubtype = {article}

}

@article{sharma_developing_2024,

title = {Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans},

author = {Abhinav Sharma and Liang Jin and Xue Wang and Yue-Ting Wang and David M. Stresser},

url = {https://pubs.rsc.org/en/content/articlelanding/2024/lc/d3lc00843f},

doi = {10.1039/D3LC00843F},

issn = {1473-0189},

year  = {2024},

date = {2024-01-17},

urldate = {2024-01-17},

journal = {Lab Chip},

volume = {24},

number = {2},

pages = {339–355},

abstract = {Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration–time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.},

note = {Publisher: The Royal Society of Chemistry},

keywords = {absorption, Bioavailability, Biological Transport, Caco-2 Cells, drug absorption, drug metabolising enzymes ({DME}), drug permeability, intestinal barrier, oral bioavailability, permeability},

pubstate = {published},

tppubtype = {article}

}

@article{sarma_design_2023,

title = {Design of 8-mer Peptides that Block Clostridioides difficile Toxin A in Intestinal Cells},

author = {Sudeep Sarma and Carly M. Catella and Ellyce T. San Pedro and Xingqing Xiao and Deniz Durmusoglu and Stefano Menegatti and Nathan Crook and Scott T. Magness and Carol K. Hall},

doi = {10.1101/2023.01.10.523493},

year  = {2023},

date = {2023-01-12},

urldate = {2023-01-12},

pages = {2023.01.10.523493},

abstract = {Clostridioides difficile ( C. diff .) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff . infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a pep tide b inding d esign algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR). 

SIGNIFICANCE STATEMENT: Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat C. diff . infection. We describe an approach that combines a Peptide B inding D esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.},

keywords = {Adverse events, epithelial barrier, In vitro model, inflammatory bowel disease, intestinal organoids, intestinal stem cells, microbiome},

pubstate = {published},

tppubtype = {article}

}

@article{bolster_impact_2022,

title = {Impact of selected novel plant bioactives on improvement of impaired gut barrier function using human primary cell intestinal epithelium:},

author = {Doug Bolster and Lee Chae and Jan-Willem van Klinken and Swati Kalgaonkar},

url = {http://www.isnff-jfb.com/index.php/JFB/article/view/301},

doi = {10.31665/JFB.2022.18324},

issn = {2637-8779},

year  = {2022},

date = {2022-12-30},

urldate = {2022-12-30},

journal = {Journal of Food Bioactives},

volume = {20},

abstract = {Gut barrier function is compromised in the obese state. The N-trans caffeoyltyramine (NCT) and N-trans feruloyltyramine (NFT), two naturally occurring bioactive compounds in hemp hulls, identified using in silico approaches, have the potential to improve gut barrier function and their effects were studied here in vitro. Proliferative human transverse colon epithelial cells were plated and co-cultured with tumor necrosis factor (TNF) along with NCT, NFT or NCT/NFT (2.2 ratio) post-differentiation, over a 48-hour period to induce inflammation and to observe the effects of NCT and NFT. A decrease in transepithelial electrical resistance (TEER) and increase in the intestinal permeability were observed with increased addition of TNF. Co-administration of NCT and NFT demonstrated a dose-dependent and statistically significant reversal of impaired TEER and intestinal permeability. NCT and NFT demonstrated a physiologically relevant reversal of impaired gut barrier function in the setting of inflammation via significant improvement in TEER and percent permeability.},

keywords = {Bioactives, Gut barrier function, Gut permeability, Hepatic nuclear factor 4α, In vitro model, inflammatory bowel disease, intestinal barrier, N-Trans-caffeoyltyramine, N-Trans-feruloyltyramine},

pubstate = {published},

tppubtype = {article}

}

@article{villegas-novoa_development_2022,

title = {Development of a Primary Human Intestinal Epithelium Enriched in L-Cells for Assay of GLP-1 Secretion},

author = {Cecilia Villegas-Novoa and Yuli Wang and Christopher E. Sims and Nancy L. Allbritton},

doi = {10.1021/acs.analchem.2c00912},

issn = {1520-6882},

year  = {2022},

date = {2022-07-12},

urldate = {2022-07-12},

journal = {Development},

volume = {94},

number = {27},

pages = {9648–9655},

abstract = {Type 2 diabetes mellitus is a chronic disease associated with obesity and dysregulated human feeding behavior. The hormone glucagon-like peptide 1 (GLP-1), a critical regulator of body weight, food intake, and blood glucose levels, is secreted by enteroendocrine L-cells. The paucity of L-cells in primary intestinal cell cultures including organoids and monolayers has made assays of GLP-1 secretion from primary human cells challenging. In the current paper, an analytical assay pipeline consisting of an optimized human intestinal tissue construct enriched in L-cells paired with standard antibody-based GLP-1 assays was developed to screen compounds for the development of pharmaceuticals to modulate L-cell signaling. The addition of the serotonin receptor agonist Bimu 8, optimization of R-spondin and Noggin concentrations, and utilization of vasoactive intestinal peptide (VIP) increased the density of L-cells in a primary human colonic epithelial monolayer. Additionally, the incorporation of an air-liquid interface culture format increased the L-cell number so that the signal-to-noise ratio of conventional enzyme-linked immunoassays could be used to monitor GLP-1 secretion in compound screens. To demonstrate the utility of the optimized analytical method, 21 types of beverage sweeteners were screened for their ability to stimulate GLP-1 secretion. Stevioside and cyclamate were found to be the most potent inducers of GLP-1 secretion. This platform enables the quantification of GLP-1 secretion from human primary L-cells and will have broad application in understanding L-cell formation and physiology and will improve the identification of modulators of human feeding behavior.},

keywords = {Diabetes Mellitus, Enteroendocrine Cells, Glucagon-Like Peptide 1, Intestinal Mucosa, L Cells},

pubstate = {published},

tppubtype = {article}

}

@article{breau_efficient_2022,

title = {Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells},

author = {Keith A. Breau and Meryem T. Ok and Ismael Gomez-Martinez and Joseph Burclaff and Nathan P. Kohn and Scott T. Magness},

doi = {10.1016/j.stemcr.2022.04.005},

issn = {2213-6711},

year  = {2022},

date = {2022-06-14},

urldate = {2022-06-14},

volume = {17},

number = {6},

pages = {1493–1506},

abstract = {Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.},

keywords = {2D monolayer cultures, electroporation, Gene Editing, Gene Targeting, human {ISC} marker, Humans, Intestine, microphysiological device, Organoids, planar crypt-microarray, Small, stem cells, transfection, transgenic},

pubstate = {published},

tppubtype = {article}

}

@article{burclaff_proximal–distal_2022,

title = {A Proximal-to-Distal Survey of Healthy Adult Human Small Intestine and Colon Epithelium by Single-Cell Transcriptomics},

author = {Joseph Burclaff and R. Jarrett Bliton and Keith A. Breau and Meryem T. Ok and Ismael Gomez-Martinez and Jolene S. Ranek and Aadra P. Bhatt and Jeremy E. Purvis and John T. Woosley and Scott T. Magness},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9043569/},

doi = {10.1016/j.jcmgh.2022.02.007},

issn = {2352-345X},

year  = {2022},

date = {2022-02-15},

urldate = {2022-02-15},

journal = {Cell Mol Gastroenterol Hepatol},

volume = {13},

number = {5},

pages = {1554–1589},

abstract = {Background & Aims 

Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 human beings. 

Methods 

A total of 12,590 single epithelial cells from 3 independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and their capacity for response to extrinsic signals along the gut axis across different human beings. 

Results 

Cells were assigned to 25 epithelial lineage clusters. Multiple accepted intestinal stem cell markers do not specifically mark all human intestinal stem cells. Lysozyme expression is not unique to human Paneth cells, and Paneth cells lack expression of expected niche factors. Bestrophin 4 (BEST4)+ cells express Neuropeptide Y (NPY) and show maturational differences between the small intestine and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell junctions, and nutrient absorption genes show unappreciated regional expression differences across lineages. The differential expression of receptors and drug targets across lineages show biological variation and the potential for variegated responses. 

Conclusions 

Our study identifies novel lineage marker genes, covers regional differences, shows important differences between mouse and human gut epithelium, and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomic regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.},

keywords = {Differentiate Cell Lineage, Enterochromaffin Cells, Enterocytes, Enteroendocrine Cells, Gut barrier function, Intestinal Epithelial Cells},

pubstate = {published},

tppubtype = {article}

}

@article{gomez-martinez_planar_2022,

title = {A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export},

author = {Ismael Gomez-Martinez and R. Jarrett Bliton and Keith A. Breau and Michael J. Czerwinski and Ian A. Williamson and Jia Wen and John F. Rawls and Scott T. Magness},

doi = {10.1016/j.jcmgh.2022.04.009},

issn = {2352-345X},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Cellular and Molecular Gastroenterology and Hepatology},

volume = {14},

number = {2},

pages = {409–434},

abstract = {BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin. 

METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography. 

RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes. 

CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.},

keywords = {Absorptive Enterocyte Monolayers, Caco-2 Cells, Diabetes Mellitus, Drug Screening, Enterocytes, Fatty Acid Oxidation, Fatty Acids, Humans, Hypoglycemic Agents, intestinal stem cells, Metformin, Type 2},

pubstate = {published},

tppubtype = {article}

}

@article{howard_biochemical_2021,

title = {Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus},

author = {R. Logan Howard and Matthew Markovetz and Yuli Wang and Camille Ehre and Shehzad Z. Sheikh and Nancy L. Allbritton and David B. Hill},

url = {https://www.cell.com/biophysj/abstract/S0006-3495(21)00883-3},

doi = {10.1016/j.bpj.2021.10.024},

issn = {0006-3495},

year  = {2021},

date = {2021-12-07},

urldate = {2021-12-07},

journal = {Biophysical Journal},

volume = {120},

number = {23},

pages = {5384–5394},

note = {Publisher: Elsevier},

keywords = {Mucus},

pubstate = {published},

tppubtype = {article}

}

@article{dutton_hyperglycemia_2021,

title = {Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction},

author = {Johanna S Dutton and Samuel S Hinman and Raehyun Kim and Peter J Attayek and Mallory Maurer and Christopher S Sims and Nancy L Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204630/},

doi = {10.1093/intbio/zyab008},

issn = {1757-9694},

year  = {2021},

date = {2021-05-15},

urldate = {2023-07-03},

volume = {13},

number = {6},

pages = {139–152},

abstract = {Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{lemmens_drug_2021,

title = {Drug Disposition in the Lower Gastrointestinal Tract: Targeting and Monitoring},

author = {Glenn Lemmens and Arno Van Camp and Stephanie Kourula and Tim Vanuytsel and Patrick Augustijns},

url = {https://www.mdpi.com/1999-4923/13/2/161},

doi = {10.3390/pharmaceutics13020161},

issn = {1999-4923},

year  = {2021},

date = {2021-01-26},

urldate = {2021-01-26},

journal = {Pharmaceutics},

volume = {13},

number = {2},

pages = {161},

abstract = {The increasing prevalence of colonic diseases calls for a better understanding of the various colonic drug absorption barriers of colon-targeted formulations, and for reliable in vitro tools that accurately predict local drug disposition. In vivo relevant incubation conditions have been shown to better capture the composition of the limited colonic fluid and have resulted in relevant degradation and dissolution kinetics of drugs and formulations. Furthermore, drug hurdles such as efflux transporters and metabolising enzymes, and the presence of mucus and microbiome are slowly integrated into drug stability- and permeation assays. Traditionally, the well characterized Caco-2 cell line and the Ussing chamber technique are used to assess the absorption characteristics of small drug molecules. Recently, various stem cell-derived intestinal systems have emerged, closely mimicking epithelial physiology. Models that can assess microbiome-mediated drug metabolism or enable coculturing of gut microbiome with epithelial cells are also increasingly explored. Here we provide a comprehensive overview of the colonic physiology in relation to drug absorption, and review colon-targeting formulation strategies and in vitro tools to characterize colonic drug disposition.},

note = {Number: 2 

Publisher: Multidisciplinary Digital Publishing Institute},

keywords = {colon drug delivery, colonic drug disposition, colonic physiology, drug absorption, drug metabolising enzymes ({DME}), intestinal in vitro models, microbiome, microphysiological systems ({MPS})},

pubstate = {published},

tppubtype = {article}

}

@article{hinman_vitro_2021,

title = {In Vitro Generation of Self-Renewing Human Intestinal Epithelia over Planar and Shaped Collagen Hydrogels},

author = {Samuel S. Hinman and Yuli Wang and Raehyun Kim and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420814/},

doi = {10.1038/s41596-020-00419-8},

issn = {1754-2189},

year  = {2021},

date = {2021-01-01},

journal = {Nature Protocols},

volume = {16},

number = {1},

pages = {352–382},

abstract = {The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a three-dimensional shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication and soft lithography protocols necessary to shape these hydrogels to match the dimensions and density of in vivo crypts. Additionally, stem-cell scale-up protocols are provided so that even ultrasmall primary samples can be used as starting material. Initially, these cells are seeded as a proliferative monolayer over the shaped scaffold and cultured as stem/proliferative cells to expand the cell number and cover the scaffold surface with the crypt-shaped structures. To convert these immature crypts into fully polarized, functional units with a basal stem cell niche and luminal differentiated cell zone, stable, linear gradients of growth factors are formed across the crypts. These gradients imposed along the crypt long axis support a basal stem cell niche with transit amplifying cells migrating upward along this axis to form a luminal differentiated cell zone as in vivo. This platform supports the formation of a multitude of chemical gradients across the crypts including those of growth and differentiation factors, inflammatory compounds, bile and food metabolites, and bacterial products. All microfabrication and device assembly steps are expected to take 8 days if performing for the first time, with the primary cells cultured for 12 days to form mature in vitro crypts. Compatibility of the in vitro crypt arrays with standard microscopy methods, as well as the accessible luminal and basal reservoirs, facilitate mechanistic studies and screenings with sufficient replicates and statistical power to test hypotheses or survey large numbers of compounds (and bacteria) for their impact on epithelial homeostasis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{wang_enterochromaffin_2020,

title = {Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells},

author = {Yuli Wang and Christopher E. Sims and Nancy L. Allbritton},

doi = {10.1021/acs.analchem.0c02016},

issn = {1520-6882},

year  = {2020},

date = {2020-09-15},

volume = {92},

number = {18},

pages = {12330–12337},

abstract = {Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.},

keywords = {Enterochromaffin Cells, Humans, Intestinal Mucosa, Serotonin},

pubstate = {published},

tppubtype = {article}

}

@article{speer_evaluation_2019,

title = {Evaluation of human primary intestinal monolayers for drug metabolizing capabilities},

author = {Jennifer E. Speer and Yuli Wang and John K. Fallon and Philip C. Smith and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829970/},

doi = {10.1186/s13036-019-0212-1},

issn = {1754-1611},

year  = {2019},

date = {2019-11-04},

urldate = {2019-11-04},

journal = {J. Biol. Eng},

volume = {13},

pages = {82},

abstract = {Background 

The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo. 

Results 

The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes. 

Conclusions 

The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells. 

Graphical abstract},

keywords = {Absorptive Enterocyte Monolayers, Bioavailability, drug metabolising enzymes ({DME}), epithelial barrier, Fg, Intestinal Epithelial Cells, permeability},

pubstate = {published},

tppubtype = {article}

}

@article{dutton_primary_2019,

title = {Primary Cell-Derived Intestinal Models: Recapitulating Physiology},

author = {Johanna S. Dutton and Samuel S. Hinman and Raehyun Kim and Yuli Wang and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571163/},

doi = {10.1016/j.tibtech.2018.12.001},

year  = {2019},

date = {2019-07-01},

journal = {Trends in Biotechnology},

volume = {37},

number = {7},

pages = {744},

abstract = {The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods, has enabled successful recapitulation of key features of intestinal physiology. These advances when paired with engineering methods, ...},

note = {Publisher: NIH Public Access},

keywords = {Biological, Cell Culture Techniques, Cells, Cultured, Humans, in vitro models, Intestine, Intestines, Models, monolayers, organ-on-chips, Organoids, stem cells, Tissue Engineering},

pubstate = {published},

tppubtype = {article}

}

@article{speer_molecular_2019,

title = {Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking},

author = {Jennifer E. Speer and Dulan B. Gunasekara and Yuli Wang and John K. Fallon and Peter J. Attayek and Philip C. Smith and Christopher E. Sims and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487070/},

doi = {10.1186/s13036-019-0165-4},

issn = {1754-1611},

year  = {2019},

date = {2019-04-27},

urldate = {2023-07-03},

volume = {13},

pages = {36},

abstract = {Background 

The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. 

Results 

The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. 

Conclusions 

The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function. 

Graphical abstract 

Electronic supplementary material 

The online version of this article (10.1186/s13036-019-0165-4) contains supplementary material, which is available to authorized users.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{wang_building_2019,

title = {Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models},

author = {Y. Wang and R. Kim and C. E. Sims and N. L. Allbritton},

doi = {10.1016/j.jcmgh.2019.07.009},

issn = {2352-345X},

year  = {2019},

date = {2019-01-01},

journal = {Cellular and Molecular Gastroenterology and Hepatology},

volume = {8},

number = {4},

pages = {653–655.e5},

keywords = {Cell Culture Techniques, Colon, Humans, Hydrogels, Intestinal Mucosa, Mucus},

pubstate = {published},

tppubtype = {article}

}

@article{gunasekara_monolayer_2018,

title = {A monolayer of primary colonic epithelium generated on a scaffold with a gradient of stiffness for drug transport studies},

author = {Dulan B. Gunasekara and Jennifer Speer and Yuli Wang and Daniel L. Nguyen and Mark I. Reed and Nicole M. Smiddy and Joel S. Parker and John K. Fallon and Philip C. Smith and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339567/},

doi = {10.1021/acs.analchem.8b02845},

year  = {2018},

date = {2018-11-11},

urldate = {2018-11-11},

volume = {90},

number = {22},

pages = {13331},

abstract = {Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as achieve tighter control of experimental variables than can be achieved with ...},

note = {Publisher: NIH Public Access},

keywords = {Atenolol, Biological Transport, Caco-2 Cells, Colon, Epithelium, Humans, Propranolol, Riboflavin, Tissue Engineering},

pubstate = {published},

tppubtype = {article}

}

@article{wang_analysis_2018,

title = {Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human Intestinal Epithelial Monolayer Platform},

author = {Yuli Wang and Raehyun Kim and Shee-Hwan J. Hwang and Johanna Dutton and Christopher E. Sims and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309958/},

doi = {10.1021/acs.analchem.8b02835},

issn = {0003-2700},

year  = {2018},

date = {2018-10-02},

urldate = {2023-07-03},

volume = {90},

number = {19},

pages = {11523–11530},

abstract = {In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer with a twenty-six-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor necrosis factor alpha (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer, but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate, gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effect while the dietary factor forskolin significantly increased production. These results demonstrate that the described human intestinal monolayer platform has the potential for assay and screening of metabolites and compounds that alter the inflammatory response of the intestine.,},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{gunasekara_development_2018,

title = {Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins},

author = {Dulan B. Gunasekara and Matthew DiSalvo and Yuli Wang and Daniel L. Nguyen and Mark I. Reed and Jennifer Speer and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://doi.org/10.1021/acs.analchem.7b04032},

doi = {10.1021/acs.analchem.7b04032},

issn = {0003-2700},

year  = {2018},

date = {2018-02-06},

urldate = {2024-08-20},

volume = {90},

number = {3},

pages = {1941–1950},

abstract = {Enterotoxins increase intestinal fluid secretion through modulation of ion channels as well as activation of the enteric nervous and immune systems. Colonic organoids, also known as colonoids, are functionally and phenotypically similar to in vivo colonic epithelium and have been used to study intestinal ion transport and subsequent water flux in physiology and disease models. In conventional cultures, organoids exist as spheroids embedded within a hydrogel patty of extracellular matrix, and they form at multiple depths, impairing efficient imaging necessary to capture data from statistically relevant sample sizes. To overcome these limitations, an analytical platform with colonic organoids localized to the planar surface of a hydrogel layer was developed. The arrays of densely packed colonoids (140 μm average diameter, 4 colonoids/mm2) were generated in a 96-well plate, enabling assay of the response of hundreds of organoids so that organoid subpopulations with distinct behaviors were identifiable. Organoid cell types, monolayer polarity, and growth were similar to those embedded in hydrogel. An automated imaging and analysis platform efficiently tracked over time swelling due to forskolin and fluid movement across the cell monolayer stimulated by cholera toxin. The platform was used to screen compounds associated with the enteric nervous and immune systems for their effect on fluid movement across epithelial cells. Prostaglandin E2 promoted increased water flux in a subset of organoids that resulted in organoid swelling, confirming a role for this inflammatory mediator in diarrheal conditions but also illustrating organoid differences in response to an identical stimulus. By allowing sampling of a large number of organoids, the arrayed organoid platform permits identification of organoid subpopulations intermixed within a larger group of nonresponding organoids. This technique will enable automated, large-scale screening of the impact of drugs, toxins, and other compounds on colonic physiology.},

note = {Publisher: American Chemical Society},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{bhatt_nsaid-induced_2018,

title = {NSAID-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells},

author = {Aadra P. Bhatt and Dulan B. Gunasekara and Jennifer Speer and Mark I. Reed and Alexis N. Peña and Bentley R. Midkiff and Scott T. Magness and Scott J. Bultman and Nancy L. Allbritton and Matthew R. Redinbo},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013262/},

doi = {10.1021/acsinfecdis.7b00139},

issn = {2373-8227},

year  = {2018},

date = {2018-01-12},

urldate = {2023-02-20},

volume = {4},

number = {1},

pages = {46–52},

abstract = {The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Non-steroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics, and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations and longitudinal ulcers in the gut. Here we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions., Primary human small intestinal monolayers for modelling NSAID-induced damage,},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{wang_formation_2018,

title = {Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer},

author = {Yuli Wang and Raehyun Kim and Dulan B. Gunasekara and Mark I. Reed and Matthew DiSalvo and Daniel L. Nguyen and Scott J. Bultman and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.cmghjournal.org/article/S2352-345X(17)30154-6/fulltext},

doi = {10.1016/j.jcmgh.2017.10.007},

issn = {2352-345X},

year  = {2018},

date = {2018-01-01},

urldate = {2018-01-01},

volume = {5},

number = {2},

pages = {113–130},

note = {Publisher: Elsevier},

keywords = {Intestinal Epithelial Cells, Intestine-On-A-Chip, Muc2, N-hydroxysuccinimide, olfactomedin-4, Polarized Crypt, short-chain fatty acid, Stem Cell Niche},

pubstate = {published},

tppubtype = {article}

}

@article{wang_self-renewing_2017,

title = {Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells},

author = {Yuli Wang and Matthew DiSalvo and Dulan B. Gunasekara and Johanna Dutton and Angela Proctor and Michael S. Lebhar and Ian A. Williamson and Jennifer Speer and Riley L. Howard and Nicole M. Smiddy and Scott J. Bultman and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.sciencedirect.com/science/article/pii/S2352345X17300462},

doi = {10.1016/j.jcmgh.2017.02.011},

issn = {2352-345X},

year  = {2017},

date = {2017-07-01},

urldate = {2023-02-20},

volume = {4},

number = {1},

pages = {165–182.e7},

abstract = {Background & Aims 

Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. 

Methods 

The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. 

Results 

The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. 

Conclusions 

This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.},

keywords = {Colonic Epithelial Cells, Compound Screening, Monolayer, Organoids},

pubstate = {published},

tppubtype = {article}

}

@article{wang_microengineered_2017,

title = {A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium},

author = {Yuli Wang and Dulan B. Gunasekara and Mark I. Reed and Matthew DiSalvo and Scott J. Bultman and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.sciencedirect.com/science/article/pii/S0142961217301412},

doi = {10.1016/j.biomaterials.2017.03.005},

issn = {0142-9612},

year  = {2017},

date = {2017-06-01},

urldate = {2023-02-20},

volume = {128},

pages = {44–55},

abstract = {The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.},

keywords = {Crypt, Intestine, Microfabrication, Scaffold, Stem cell, Villus},

pubstate = {published},

tppubtype = {article}

}

@article{gracz_defining_2014,

title = {Defining hierarchies of stemness in the intestine: evidence from biomarkers and regulatory pathways},

author = {A. D. Gracz and S. T. Magness},

url = {https://journals.physiology.org/doi/full/10.1152/ajpgi.00066.2014},

doi = {10.1152/ajpgi.00066.2014},

issn = {0193-1857},

year  = {2014},

date = {2014-06-12},

journal = {Am J Physiol Gastrointest Liver Physio},

volume = {307},

number = {3},

pages = {G260–G273},

abstract = {For decades, the rapid proliferation and well-defined cellular lineages of the small intestinal epithelium have driven an interest in the biology of the intestinal stem cells (ISCs) and progenitors that produce the functional cells of the epithelium. Recent and significant advances in ISC biomarker discovery have established the small intestinal epithelium as a powerful model system for studying general paradigms in somatic stem cell biology and facilitated elegant genetic and functional studies of stemness in the intestine. However, this newfound wealth of ISC biomarkers raises important questions of marker specificity. Furthermore, the ISC field must now begin to reconcile biomarker status with functional stemness, a challenge that is made more complex by emerging evidence that cellular hierarchies in the intestinal epithelium are more plastic than previously imagined, with some progenitor populations capable of dedifferentiating and functioning as ISCs following damage. In this review, we discuss the state of the ISC field in terms of biomarkers, tissue dynamics, and cellular hierarchies, and how these processes might be informed by earlier studies into signaling networks in the small intestine.},

note = {Publisher: American Physiological Society},

keywords = {Bmp signaling, cell fate, differentiation, intestinal stem cells, Wnt signaling},

pubstate = {published},

tppubtype = {article}

}

@article{wang_vitro_2014,

title = {In vitro generation of colonic epithelium from primary cells guided by microstructures},

author = {Yuli Wang and Asad A. Ahmad and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037563/},

doi = {10.1039/c3lc51353j},

issn = {1473-0197},

year  = {2014},

date = {2014-05-07},

urldate = {2023-02-20},

volume = {14},

number = {9},

pages = {1622–1631},

abstract = {The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ahmad_optimization_2014,

title = {Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications},

author = {Asad A. Ahmad and Yuli Wang and Adam D. Gracz and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://doi.org/10.1186/1754-1611-8-9},

doi = {10.1186/1754-1611-8-9},

issn = {1754-1611},

year  = {2014},

date = {2014-04-01},

urldate = {2023-02-20},

volume = {8},

number = {1},

pages = {9},

abstract = {New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Here murine crypt isolation yield and quality are optimized, and colonoid culture efficiency measured in microfabricated culture devices.},

keywords = {{PDMS}, {PDMS} Substrate, Colonic Crypt, Crypt Cell, Differentiate Cell Lineage},

pubstate = {published},

tppubtype = {article}

}