Microscale Symposium Abstracts Session 3: Advances in Organ-on-chip Platforms

Chair: Ioannis Zervantonakis, Ph.D.(University of Pittsburgh)

Use of an In-Vitro Gut-on-Chip System to Investigate the Effect of X-Ray Irradiation on the Human GI Tract
and Gut Microbiome – Nicole Sherwood, B.S. (University of Arizona College of Medicine Phoenix)

Abstract: The investigation of radiation’s effects on the GI tract, the identification of dosimetry biomarkers, and the testing of new radiation countermeasure drugs are all limited in humans due to the restricted access to human samples or the use of animal models that are not representative of human physiology. Thus, there is a need for improved in vitro models to elucidate the effects of x-ray radiation on the human GI system that mimic the in vivo physiological environment and interaction between human GI epithelium and gut microbiome. Here, we developed a Gut-on-Chip system (HuMiX) to reproduce multiple in vivo parameters classically associated with human GI following acute irradiation. The HuMiX device is a co-culture system consisting of three chambers separated by two porous polycarbonate membranes, delimiting the microbial, intestinal, and vascular compartments. Previous design iterations of HuMiX have demonstrated that the system can imitate the in vivo immunologic, metabolic, and transcriptional responses to commensal gut bacteria [Shah et al, 2016]. In the newly designed system, ~16,000 Caco-2 cells/mm2 were seeded on a collagen-treated membrane in the middle chamber and incubated under normal cell culture incubator conditions. The middle and bottom chambers were then flowed with Caco-2 cell medium at 67 ul/min for 6 days, when 8 x 107/mL of mixed bacterial flora, isolated from human stool samples from 6 different donors, were injected into the top chamber. Post 12 hours co-culture, the devices were sham- or 8 Gy-irradiated at 1 Gy/min using 320 keV x-ray beam with 2 mm Al filter. Twenty-four hours after irradiation, the supernatant was collected for inflammatory cytokine detection, the bacterial cells were collected from the top chamber for microbiome profiling and Caco2 cells were stained. Preliminary microbiome profiling identified the presence of three phyla: Bacteroides, FAFV (Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia), and Proteobacteria. DNA concentration, species richness, and species diversity obtained from the devices decreased as compared to those profiles isolated from direct fecal bacteria isolation and/or cultured in bacterial culture broths, but still maintained highly diverse profiles. Current analyses are on-going to identify if radiation modified the Firmicutes/Bacteriodetes ratio as shown by in vivo studies. The Caco2 phenotype revealed the formation of villi, the presence of cell-cell tight junctions and the production of mucus, demonstrating the establishment of physiological intestinal hallmarks. Irradiation showed ability to disrupt cell junctions and to permeabilize the intestinal barrier as suggested by dextran assay. Citrulline, an amino acid whose plasma level decrease following intestinal radiation-induced damage, was also quantified. This study is an ongoing project and the injection of immune cells will be performed in the future to assess if the HuMiX can recapitulate the expression change of well-known radiation dosimetry biomarkers in blood. Overall, our data suggest that the HuMiX offers a promising tool to study radiation effect on human gut and test radiation countermeasure approaches.

What Can Spheroids Do for You? Exploring the Fine Balance Between Throughput and Physiological
Relevance – Madhu Nag, M.B.S., Ph.D. (InSphero)

“Context of Use”. A little-known phrase but one that has profound implications. This phrase underscores the importance of considering the practical and real-world aspects of how something will be used or applied. This understanding helps guide decision-making, assessment, and optimization to ensure that products and processes are safe, effective, and fit for their intended purpose. Understanding the context of use has profound implications across the drug discovery continuum that focusses on developing the right in vitro model to answer a specific biological question while maintaining the desired balance between physiological relevance and throughput. The InSphero 3D Insight (TM) platform delivers robust, reproducible, scalable and actionable data across different disease pathologies to provide solutions for specific contexts of use.

Using the LumeNEXT Platform to Create Complex Microphysiological Systems of Cancer and Infectious
Disease – Sheena Kerr, Ph.D. (University of Wisconsin-Madison)

Microphysiological systems (MPS) have strong potential for use in elucidation of biological mechanisms, in addition to clinical utility such as MPS in precision medicine applications. The LumeNEXT platform permits molding of lumens from a hydrogel matrix that when seeded with cells can form microvasculature, ductal structures or recapitulate the gut. We can use this platform to build complex MPS that represent the tumor microenvironment (TME). The tissue architecture is recapitulated with blood and lymphatic vessel mimics that are surrounded with stromal and immune cells while tumor cell spheroids represent the solid tumor. Due to the low cell number requirements, these MPS can be built using multiple cell types isolated from a single piece of tumor tissue to create patient-specific models. We are currently investigating the utility of patient-specific MPS of the head and neck cancer TME for predicting treatment efficacy. MPS can also be used to investigate highly challenging microenvironments to study such as the metastatic bone microenvironment. Population of the MPS with multiple bone stromal cell types in addition to tumor and immune cells and vasculature can provide insights into treatment responses or resistance. MPS also have utility in studying infectious diseases including parasitic infection, such as Toxoplasma Gondii. Extensive in vivo studies have elucidated the immune response to this parasite in mouse, however, these findings do not translate into human and very little is known about the human immune response to T. Gondii. Our MPS of the gut, which comprises an intestinal epithelial lumen and endothelial vasculature permits investigation of human immune responses to acute T. Gondii infection. We will present our ongoing work on LumeNEXT MPS systems that recapitulate the TME and parasitic infection in the gut.