A NEW INTESTINE-CHIP MODEL TO STUDY HUMAN INTESTINAL PHYSIOLOGY AND PATHOPHYSIOLOGY
Magdalena Julia Kasendra1, Nasia Apostolou1, Raymond Luc1, Mark Donowitz2, Katia Karalis1
1Emulate Inc., Boston, Massachusetts, United States; 2Department of Medicine, Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States;
Background: Human intestinal diseases, such as irritable bowel syndrome, inflammatory bowel disease, and bacterial and viral infection, affect millions of people worldwide. A lack of reliable preclinical models that adequately reflect critical aspects of human intestinal physiology and pathophysiology limits our understanding of disease pathogenesis and the associated development of new drug therapies. To address this compelling need, we have used our Organs-on-Chips technology to recreate the appropriate microenvironment of the human intestine in our microengineered Intestine-Chip.
Methods-Approach: Our approach involves the use of Organ-Chips that are approximately the size of a AA battery and are composed of a clear flexible polymer. The chips contain microchannels lined with living, human cells. Inside the chips we can recapitulate normal tissue-tissue interfaces and recreate the complex physical and biochemical microenvironment of living, human organs. We have previously used this technology to develop a human Intestine-Chip that utilized Caco-2 cells (a human colon tumor-derived intestinal cell line), that emulates many features of human intestinal structure and function. Here we present a more advanced Intestine-Chip that contains human primary intestinal epithelium cells derived from duodenal biopsies, along with intestinal tissue-specific microvascular endothelial cells. This system provides a more physiological model as it recreates the complex dynamic state of the native human intestine required for normal cell architecture and function, including appropriate extracellular matrix, tissue-tissue interface, and the mechanical forces that mimic both intestinal peristalsis and blood flow.
Results: The Intestine-Chip was shown to recapitulate intestinal barrier function, multi-lineage differentiation, and response to external stimuli (e.g., drugs, cytokines). In addition we demonstrated drug metabolism capacity (expression of drug metabolism enzyme CYP3A4), as well as, expression of the major drug transporters that are present in the small intestine in vivo. Importantly, expression of CYP3A4 in the Intestine-Chip was much higher than in the previous Caco-2 cell-based chips and closer to levels found in vivo. In addition we showed that CYP3A4 levels were induced upon exposure to different drug treatments, such as rifampicin and 1,25-dihydroxyvitamin D3, known CYP340 inducers in humans.
Conclusion: In conclusion, we believe that the described primary Intestine-Chip provides a human-relevant system for studying intestinal physiology and pathophysiology. Our findings provide evidence that the Intestine-Chip may provide a much-needed platform for pre-clinical testing of drug safety and efficacy.
DEVELOPMENT OF A COLON-CHIP TO STUDY HOST-MICROBIOME INTERACTIONS IN HUMANS
Athanasia P. Apostolou1,2, S. Jordan Kerns1, Jenifer Lea Obrigewitch1, Geraldine Alexandra Hamilton1, Katia Karalis1
1 Emulate Inc., Boston, Massachusetts, United States;
2 Department of Medicine, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece;
Background: Recent studies reveal the complexity of the gut microbiome and its symbiotic interactions with the host in both healthy and disease states. While no direct causality has been defined, alterations in the diversity and abundance of intestinal bacterial strains have been linked to metabolic diseases, cancer, inflammation, and even specific brain functions. We applied our Organs-on-Chips technology to develop a human, colonoid-based microengineered system that enables the culture of human epithelial and endothelial cells while exposing them to an in vivo relevant microenvironment that includes relevant mechanical forces. Development of colonoid-derived epithelial monolayers within the chip allows for direct exposure of the intestinal lumen surface to co-culture with symbiotic bacteria.
Methods: We developed a Colon-Chip model that includes: epithelial monolayers from expanded organoid-derived colonic epithelial cells obtained from healthy donors, human intestinal microvascular endothelial cells (HIMECs), the obligate anaerobic commensal bacterial strain Clostridia symbiosum (C. symbiosum) and the facultative anaerobe Lactobacillus rhamnosus GG (LGG). The chip was maintained for four days in the expansion phase under the presence of Wnt3a, Noggin, and Rspo1 signaling, followed by four days of differentiation where Wnt3a was omitted analogously to published protocols. Results: Formation of continuous intestinal monolayer within the chip was evident on day four as shown by brightfield microscopy. The integrity of the epithelial barrier permeability was demonstrated and we confirmed the presence of proliferative cells during the expansion phase, as indicated by EdU pulse. The lineage-specific differentiation capacity of the epithelial cells in the Colon-Chip was also demonstrated as shown by identification of Villin positive absorptive enterocytes and Mucin 2 positive Goblet cells via immunofluorescence. To assess the sensitivity of Colon-Chip to bacterial antigens, we apically challenged it with Pam2CSK4, a TLR2 agonist. Quantitative polymerase chain reaction revealed increased expression of Reg3 beta gene, a C-type antibacterial lectin, following Pam2CSK4, but not vehicle, treatment. Finally, we maintained the C. symbiosum bacterial strain in symbiosis with the differentiated epithelial cells in an in vivo relevant hypoxic microenvironment (5% O2) for 48 hours.
Conclusions: We provide evidence that we have developed a microengineered, human colonoid-based Colon-Chip that is able to accommodate, maintain, and recapitulate phenotypic characteristics and physiological responses of primary colonic epithelial cells. Ongoing studies aim to further develop and optimize this chip as a platform for studying host-pathogen interactions and facilitate a better understanding of the associated immune functions across different donors.
A NOVEL MODEL OF HUMAN ENTEROIDS IN THE INTESTINE-CHIP (I-C) PLATFORM THAT INCLUDES DYNAMIC FLOW AND CYCLIC STRAIN FOR PHYSIOLOGICAL AND PHAMACOLOGICAL STUDIES OF INTESTINAL ION TRANSPORT
Jianyi Yin1, Laxmi Sunuwar1, Magdalena Julia Kasendra2, Huimin Yu1, Michael Sun4, Ming Tse1, Peter Haggie3, Alan S. Verkman3, Katia Karalis2, Mark Donowitz1
1Johns Hopkins University School of Medicine, Baltimore, Maryland, United States; 2Emulate Inc, Boston, Massachusetts, United States; 3UCSF, San Francisco, California, United States; 4Johns Hopkins University, Baltimore, Maryland, United States;
Background: The development of I-C technology combined with use of human enteroid monolayers provides a new platform to study human intestinal health and disease, including diarrheal diseases. The aim of the present study was to establish/characterize a novel model of normal human proximal Small Intestine-Chip that includes dynamic flow and cyclic stretch and to define the state of differentiation and ion transport function emphasizing intestinal NaCl absorption, which is affected in most diarrheal diseases.
Methods: Normal human jejunal enteroids and human umbilical vein endothelial cells were seeded into the two microchannels of a novel fluidic microphysiological system (Emulate Inc, Boston, MA) with the two microchannels separated by a semi-permeable membrane. Chips were maintained in a Human Emulation System that allows gassing plus continuous flow mimicking gut lumen and blood flow and provides mechanical stretch mimicking peristalsis. Enteroid monolayers grown in I-Cs were compared to jejunal enteroid monolayers grown on Transwell inserts under static conditions without endothelial cells. Basal NHE3 and DRA activity were studied by two-photon microscopy/pH-sensitive SNARF dyes.
Results: From day 2 to day 8 post-plating in the I-C, there was a rapid but time-dependent elevation in the mRNA levels of SI and DRA, and conversely, a reduction of LGR5, Ki67, NKCC1. This indicates a transition into a differentiated phenotype. The mRNA levels of SI and DRA in enteroid monolayers in I-Cs (day 6-8) were higher than undifferentiated/differentiated enteroid monolayers on Transwell inserts. Enteroids in the I-Cs that were rhythmically stretched had even higher mRNA levels of DRA and SI at post-plating day 2 than those not stretched, suggesting an enhanced effect of mechanical stretch on cell differentiation. Immunofluorescence studies confirmed the localization of NHE3 and DRA on the apical surface. At post-plating day 6, enteroid monolayers in the I-C had basal NHE3 activity that was stimulated by LPA and inhibited by the specific NHE3 inhibitor, tenapanor. Similarly, basal DRA activity was detected and inhibited by a novel pharmacologic DRA inhibitor.
Conclusions: We have developed a novel normal human enteroid-based Intestine-Chip model that includes apical and basolateral fluid flow and peristalsis-like strain that produces differentiated enteroids that are similar to small intestinal villus epithelial cells. We demonstrate that both fluid flow and mechanical strain increase the rate of differentiation of normal human jejunal enteroids in this platform. Enteroids in the Intestine-Chip platform express NHE3 and DRA and have transport activity and physiological regulation. This model provides a potentially powerful tool for evaluating human intestinal transport physiology and the pathophysiology of diarrheal diseases that affect villus enterocytes.