Category: Preclinical Development
Purpose: Drug development is currently a long and costly process and the majority of drug candidates fail to reach clinical trials. The field still lacks proper human intestinal models that could guarantee drug safety and efficacy [1,2]. Additionally, the presence of the microbiota is lacking in current predictive models despite having an important role in the metabolism of xenobiotics. Systems with ex vivo intestinal tissue add more micro-environmental context present in human intestine. However, current ex vivo models are often difficult to operate, limited to maintain the tissue viability and not suitable for small tissue samples . Moreover, conventional intestinal models do not provide enough flexibility to gain a mechanistic view behind the intestinal processes. We have tackled these limitations and developed a 3D printed organ-on-a-chip device that integrates small sizes of intestinal tissues between two microchannels, allowing us to study processes that determine (human) intestinal permeability as well as (anaerobic) host-microbe-immune responses.
Methods: Fresh intestinal tissue of human origin was applied in the 3D printed microfluidic chip that realizes apical and basolateral flow on both sides of the tissue (Figure 1). Using two different flow rates of 2 ml/hr and 20 ml/hr the effect of flow rate on the tissue integrity and drug absorption was studied. To study host-microbe interaction, an in vitro fermentation screening platform (i-screen)  was inoculated with adult fecal microbiota of healthy subject or subjects with inflammatory bowel disease (IBD) and i-screen supernatant was applied to the apical side of the microfluid device. Furthermore, Salmonella bacteria and Clostridium Difficile toxin were used to study specific host-microbe interactions. Throughout the experiment, lactate dehydrogenase (LDH) and FITC-Dextran (4kDa) leakage were determined as measures of tissue viability and barrier integrity, respectively. Tissue absorption was studied using two model drugs, i.e. atenolol (paracellular transport) and antipyrine (transcellular transport). The innate immune response was studied via secretion of the pro-inflammatory cytokine IL-8.
Results: When applying human intestinal (colon) tissue, proper barrier function with low leakage of FITC dextran (4kDa, < 1%) up to 26 hours was shown for both flow rates. After 26 hours, lactate dehydrogenase (LDH) leakage from the tissue was 3-fold lower in the 2 ml/hr vs the 20 ml/hr flow rate (23% vs 77% of the initial LDH content) (Figure 2). As LDH levels within the applied tissue segments remained stable to values observed in control segments, tissue viability throughout the experiment was endured. Absorption of the tissue for atenolol and antipyrine was tested and we observed that the ratio of transcellular to paracellular permeability showed proper distinction of transcellular over paracellular transport (ratio ≥ 2), with a higher ratio for the lower flow rate (2.4 vs 2.2 after 26 hours). Exposure of human colon tissue to Salmonella bacteria or Clostridium difficile toxin significantly induced secretion of the pro-inflammatory cytokine Il-8. Furthermore, barrier properties were disrupted as was shown by increased permeability of paracellular transport markers. Human colon tissue exposed to IBD i-screen supernatant increased Il-8 secretion 10-fold compared to i-screen supernatant derived from healthy adults (Figure 3), demonstrating the unhealthy status of IBD patients microbiota.
Conclusion: In conclusion, using the novel ex vivo microfluidic model, we were able to fix human intestinal tissue in a microfluidic chip and maintain its functionality under physiological conditions for 26 hours. The developed model adds value to the conventional drug development methods as the gut-on-a-chip expands the time-frame to study intestinal absorption and enables the determination of local pro-inflammatory effects of microbial compounds on the intestine. Furthermore, the combination of the perfused human gut-on-a-chip with IBD i-screen supernatant demonstrates the potential of this platform as a physiologically relevant ex vivo IBD model.
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Guerra, Aurélie, et al. "Relevance and challenges in modeling human gastric and small intestinal digestion." Trends in biotechnology 30.11 (2012): 591-600.
Herrmann, Jeremy R., and Jerrold R. Turner. "Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport." American Journal of Physiology-Cell Physiology 310.6 (2016): C423-C431.
 Fehlbaum, Prudence, et al. “In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota.” International Journal of Molecular Sciences 19.10 (2018): 3097
Joanne Donkers– Zeist, Utrecht, Netherlands
Joanne Donkers– Zeist, Utrecht, Netherlands
Hossein Eslami Amirabadi– Zeist, Utrecht, Netherlands
Esmée Wierenga– Zeist, Utrecht, Netherlands
Tim Donkers– Zeist, Utrecht, Netherlands
Lianne Stevens– Zeist, Utrecht, Netherlands
Irene Nooijen– Zeist, Utrecht, Netherlands
Birol Usta– Zeist, Utrecht, Netherlands
Lisanne Pieters– Zeist, Utrecht, Netherlands
Bastiaan Ingenhut– Zeist, Utrecht, Netherlands
Frank Schuren– Zeist, Utrecht, Netherlands
Steven Erpelinck– Zeist, Utrecht, Netherlands
Evita van de Steeg– Zeist, Utrecht, Netherlands