(M1530-10-66) Next Level Drug Research in an Ex Vivo Tissue Gut-on-a-Chip Model: Advanced Applications of the Intestinal Explant Barrier Chip (IEBC)

Purpose: Gut-on-a-chip systems are increasingly gaining more attention since they offer more translational value compared to conventional in vitro models. The Intestinal Explant Barrier Chip (IEBC) integrates small ex vivo intestinal tissue explants between two microchannels. By maintaining the in vivo distribution and properties of the different intestinal cell types in their correct structure and architecture, intestinal processes can be studied in an advanced physiological model. Previously, we have described the applicability of the IEBC to study intestinal permeability of small molecule drugs [1]. Here, we report on the added value of using the IEBC to study advanced research questions in the field of drug absorption, metabolism and host-microbe (immune) responses.

Methods: Human jejunum, ileum or colon tissue explants were fixed in the IEBC and apically exposed to drugs dissolved in standard cell culture medium or in fecal water containing microbial excretion products (iscreen technology [2]) for 24 hours (Figure 1). Additionally, the combination of human tissue explants with iscreen spent medium was used to map host-microbe interactions. The addition of an aerobic-anaerobic interface to the IEBC took host-microbe interaction studies one step further as the apical anaerobic environment, similar to the gut lumen, allowed inclusion of living gut microbiome.

Results: Phase 1 and phase 2 metabolism over a period of 24h was demonstrated in human jejunum tissue using coumarin and 7-OH coumarin (Figure 2). Coumarin was predominantly hydroxylated to 7-OH coumarin (via CYP2A6) at the apical side of the intestinal tissue and demonstrated an apparent permeability (P_app) rate of 2 x 10^-6 cm/s. Next, 7-OH coumarin was metabolized further into 7-OH coumarin glucuronide and 7-OH coumarin sulfate via phase 2 conjugating enzymes and appeared basolateral at high rates with P<sub>app values of 41.1 and 24.2 x 10^-6 cm/s, respectively. Effective and preserved phase 1 and 2 metabolism was also shown for midazolam (to OH midazolam via CYP3A4), diclofenac (to 4-OH diclofenac via CYP2C9), testosterone (to 6β-OH testosterone and androstenedione via CYP3A4 and 17β hydroxysteroid dehydrogenase) and irinotecan (to SN-38 via CES2). Human colon tissue (n=3 donors) was exposed to short chain fatty acids (SCFA) or iscreen spent medium collected under favorable conditions for microbial SCFA production. Average results showed improved tissue barrier integrity measured by a 1.7-3.5 fold reduction in FITC-dextran 4000 (FD4) leakage and 1.4-2.1 lower transcellular and paracellular transport of antipyrine and atenolol, respectively. Furthermore, the release of pro-inflammatory cytokine TNF-α was significantly reduced under these circumstances. Tissue explants cultured in the aerobic-anaerobic interface showed proper tissue functionality (transcellular/paracellular transport >2), intact tissue integrity (FD4 leakage < 0.5%/h) and significant differences in microbial beta diversity compared to aerobic tissue conditions, with a higher abundance of e.g. bifidobacterium and lactobacillus species. Tissue functionality and integrity remained intact upon co-culture with bifidobacterium animalis, for which growth and tissue-attachment were shown by targeted qPCR analysis (Figure 3).

Conclusion: In conclusion, we successfully demonstrate the use of the IEBC for advanced drug absorption and metabolism questions as well as presenting a novel way to understand host-microbe effects on tissue functionality and local inflammation in the human intestine.

References: 1. Eslami Amirabadi, H.; Donkers, J.; Wierenga, E.; Ingenhut, B.; Pieters, L.; Stevens, L.; Donkers, T.; Westerhout, J.; Masereeuw, R.; Bobeldijk, I.; et al. Intestinal Explant Barrier Chip: Long-Term Intestinal Absorption Screening in a Novel Microphysiological System Using Tissue Explants. Lab Chip 2021, doi:10.1039/d1lc00669j.
2. Fehlbaum, S.; Prudence, K.; Kieboom, J.; Heerikhuisen, M.; van den Broek, T.; Schuren, F.H.J.; Steinert, R.E.; Raederstorff, D. In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota. Int. J. Mol. Sci. 2018, 19, doi:10.3390/ijms19103097.

Acknowledgements: Nothing to disclose.

Schematic overview of the IEBC setup. Colon tissue of human or porcine origin is added in the IEBC. In host-microbe interaction studies microbiome is added at the apical side of the tissue.

Human jejunum tissue explants were mounted in the IEBC and exposed to coumarin (3 µM) or 7-OH coumarin (333 µM) for 24 hours (n = 4). Parent compound and metabolites were measured by LC-MS/MS. Data are presented as mean + SD.

Aerobic-anaerobic interface of the IEBC. Strict anaerobic Bifidobacterium Animalis was co-cultured with porcine colon tissue. Attachment of B. animals to the tissue was demonstrated by qPCR.