Publications
Browse peer-reviewed literature, posters, webinars, blog articles, and more showing how we and others are using RepliGut Systems to support discovery.
2024
Pike, Colleen M.; Zwarycz, Bailey; McQueen, Bryan E.; Castillo, Mariana; Barron, Catherine; Morowitz, Jeremy M.; Levi, James A.; Phadke, Dhiral; Balik-Meisner, Michele; Mav, Deepak; Shah, Ruchir; Glasspoole, Danielle L. Cunningham; Laetham, Ron; Thelin, William; Bunger, Maureen K.; Boazak, Elizabeth M.
Characterization and optimization of variability in a human colonic epithelium culture model Journal Article
In: ALTEX, vol. 41, no. 3, pp. 425–438, 2024, ISSN: 1868-8551, (Number: 3).
Abstract | Links | BibTeX | Tags: epithelial cell culture, in vitro models, intestinal barrier, microphysiological systems, permeability
@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}
}
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.
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.
Debad, Susan; Allen, David; Bandele, Omari; Bishop, Colin; Blaylock, Michaela; Brown, Paul; Bunger, Maureen K.; Co, Julia Y.; Crosby, Lynn; Daniel, Amber B.; Ferguson, Steve S.; Ford, Kevin; da Costa, Gonçalo Gamboa; Gilchrist, Kristin H.; Grogg, Matthew W.; Gwinn, Maureen; Hartung, Thomas; Hogan, Simon P.; Jeong, Ye Eun; Kass, George E. N.; Kenyon, Elaina; Kleinstreuer, Nicole C.; Kujala, Ville; Lundquist, Patrik; Matheson, Joanna; McCullough, Shaun D.; Melton-Celsa, Angela; Musser, Steven; Oh, Ilung; Oyetade, Oluwakemi B.; Patil, Sarita U.; Petersen, Elijah J.; Sadrieh, Nakissa; Sayes, Christie M.; Scruggs, Benjamin S.; Tan, Yu-Mei; Thelin, Bill; Nelson, M. Tyler; Tarazona, José V.; Wambaugh, John F.; Yang, Jun-Young; Yu, Changwoo; Fitzpatrick, Suzanne
Trust your gut: Establishing confidence in gastrointestinal models – An overview of the state of the science and contexts of use Journal Article
In: ALTEX, vol. 41, no. 3, pp. 402–424, 2024, ISSN: 1868-8551.
Abstract | Links | BibTeX | Tags: 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
@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}
}
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.
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.
Sharma, Abhinav; Jin, Liang; Wang, Xue; Wang, Yue-Ting; Stresser, David M.
Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans Journal Article
In: Lab Chip, vol. 24, no. 2, pp. 339–355, 2024, ISSN: 1473-0189, (Publisher: The Royal Society of Chemistry).
Abstract | Links | BibTeX | Tags: absorption, Bioavailability, Biological Transport, Caco-2 Cells, drug absorption, drug metabolising enzymes ({DME}), drug permeability, intestinal barrier, oral bioavailability, permeability
@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}
}
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.
2022
Bolster, Doug; Chae, Lee; van Klinken, Jan-Willem; Kalgaonkar, Swati
Impact of selected novel plant bioactives on improvement of impaired gut barrier function using human primary cell intestinal epithelium: Journal Article
In: Journal of Food Bioactives, vol. 20, 2022, ISSN: 2637-8779.
Abstract | Links | BibTeX | Tags: Bioactives, Gut barrier function, Gut permeability, Hepatic nuclear factor 4α, In vitro model, inflammatory bowel disease, intestinal barrier, N-Trans-caffeoyltyramine, N-Trans-feruloyltyramine
@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}
}
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.