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Item Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection(Springer Science and Business Media LLC, 2023-10) Hashimi, Marziah; Sebrell, T. Andrew; Hedges, Jodi F.; Snyder, Deann; Lyon, Katrina N.; Byrum, Stephanie D.; Mackintosh, Samuel G.; Crowley, Dan; Cherne, Michelle D.; Skwarchuk, David; Robison, Amanda; Sidar, Barkan; Kunze, Anja; Loveday, Emma K.; Taylor, Matthew P.; Chang, Connie B.; Wilking, James N.; Walk, Seth T.; Schountz, Tony; Jutila, Mark A.; Bimczok, DianeBats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.Item Gastrointestinal organoid structure and transport(Montana State University - Bozeman, College of Engineering, 2019) Sidar, Barkan; Chairperson, Graduate Committee: James Wilking; Thomas A. Sebrell was an author and Rachel Bruns, Royce A. Wilkinson, Blake Wiedenheft, Paul J. Taylor, Brian A. Perrino, Linda C. Samuelson, James N. Wilking and Diane Bimczok were co-authors of the article, 'Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation, and fusion events' in the journal 'Cell and tissue research' which is contained within this dissertation.; Thomas A. Sebrell, Bengisu Kilic, David Brown, Mert Aytac, Brian A. Perrino, Linda C. Samuelson, Henry Fu, Diane Bimzcok, James N. Wilking were co-authors of the article, 'Rupturing of human gastric organoids' which is contained within this dissertation.; Brittany R. Jenkins, Sha Huang, Jason R. Spence, Seth T. Walk and James N. Wilking were co-authors of the article, 'Flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip)' submitted to the journal 'Lab on a Chip' which is contained within this dissertation.; Dissertation contains two articles of which Barkan Sidar is not the main author.Organoids are three-dimensional (3D) self-assembled, mammalian tissue cultures derived from stem cells that differentiate to contain multiple cell types. These cells spatially organize within the 3D structure and are capable of recapitulating the structure and function of a particular organ. Organoids offer a variety of existing and potential applications in medicine and biotechnology, including drug formulation testing, regenerative medicine, and microbiome research. Despite their value, knowledge of how organoid structure impacts dynamics, mechanics, and transport is lacking. This is particularly true for gastrointestinal organoids, which are composed of a monolayer-thick epithelial sheet wrapped into a closed sphere. The primary goals of this dissertation are to understand the impact of gastrointestinal organoid structure on organoid function, develop a millifluidic chip platform to improve their viability and reliability as a model system and to explore their uses as model co-culture systems. To achieve this, we use a combination of time-lapse microscopy, image analysis, modeling, and fluidics fabrication techniques to develop an understanding of organoid growth and development in addition to expanding current uses as model systems. Our observations revealed that human gastric organoid growth was associated with cyclic rupture of the epithelial shell, rotational movement around their axes within the Matrigel matrix and luminal fusion by adjacent organoids. Furthermore, the rupture events are an indirect result of osmotic swelling carried out by the diffusion of water due to osmolyte concentration regulation by the epithelial shell. To overcome the advection limitation due to the topologically closed spherical structure of the organoids, we developed a millifluidic device called the Gut Organoid Flow Chip (GOFlowChip). This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid. Given that organoids show great potential as model systems, established co-culture system consisting of dendritic cells (DC) with infected human gastric organoids shows the gastric epithelium actively recruits DCs for immunosurveillance with increased recruitment upon active Helicobacter pylori infection. Finally, investigation on CD103 attachment protein in gastric DCs revealed that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin but is not a significant driver of DC adhesion to gastrointestinal epithelia.Item Long-Term Flow through Human Intestinal Organoids with the Gut Organoid Flow Chip (GOFlowChip)(2019-09) Sidar, Barkan; Jenkins, Brittany R.; Huang, Sha; Spence, Jason R.; Walk, Seth T.Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to “port” HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.Item A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium(2019-03) Sebrell, Thomas A.; Hashimi, Marziah; Sidar, Barkan; Wilkinson, Royce A.; Kirpotina, Liliya; Quinn, Mark T.; Malkoc, Zeynep; Taylor, Paul J.; Wilking, James N.; Bimczok, DianeBackground & Aims Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection.Item CD103 (aE integrin) undergoes endosomal trafficking in human dendritic cells, but does not mediate epithelial adhesion(2018-12) Swain, Steve; Roe, Mandi M.; Sebrell, T. Andrew; Sidar, Barkan; Dankoff, Jennifer; VanAusdol, Rachel; Smythies, Lesley E.; Smith, Phillip D.; Bimczok, DianeDendritic cell (DC) expression of CD103, the α subunit of αEβ7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to β1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia.Item Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events(2018-02) Sebrell, T. Andrew; Sidar, Barkan; Bruns, Rachel; Wilkinson, Royce A.; Wiedenheft, Blake A.; Taylor, Brian A.; Samuelson, Linda C.; Wilking, James N.; Bimczok, DianeThree-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 μm after 12 days, with the largest spheroids reaching diameters of >1000 μm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC–Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.