Skip to main content
2023 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

    Nano and Micro Technologies

    (K-421) Optimization of organoid-derived epithelia for building enteric neuro-epithelial connections in a microfluidic device

    Friday, October 13, 2023
    3:30 PM – 4:30 PM PDT
    Location: Exhibit Hall - Row K - Poster # 421
    Introduction:: Neuro-epithelial sensory connections enable communication between external stimuli and internal organs while maintaining barrier integrity. In the gastrointestinal tract, enteroendocrine cells (EECs) sense stimuli via specialized receptors, including ion channels such as Piezo2 and G-protein couple receptors (GPCRs) [1,2,3]. The enteric nervous system uses intrinsic primary afferent neurons (IPANs) to detect stimuli, such as forces. The relationship of neuroepithelial connections between EECs and IPANs remains poorly defined [4,5]. Organoids are a powerful model for epithelial cells, but a differentiation process is necessary for them to contain the EEC population. Differentiation media contains factors such as insulin-like growth factor (IGF), fibroblast growth factor (FGF), and/or epidermal growth factor (EGF), called IFE. Removal of EGF (IF* media) drives cells towards secretory phenotype which includes EECs [6,7]. Enriching EECs in mouse colon organoids in microfluidic devices remains a critical knowledge gap. The goal of this study is to establish a differentiation pipeline to increase EEC density in microfluidic devices.

    Materials and Methods:: We've developed a microfluidic culture platform for epithelia and neurons. It has two chambers connected by 2.5µm tall microgrooves, allowing for neuronal processes to innervate epithelial cells in their preferred media. Chambers were coated with collagen 1 and poly-L-ornithine for epithelial and neuronal cells respectively. Colon organoids from transgenic Piezo2::GCaMP5 mice were fragmented using TrypLE to digest whole organoids for injection into the epithelial chamber of the device to produce monolayers. Monolayers were grown for 5 days, and confluency was calculated as percent monolayer coverage using ImageJ to calculate the area. In parallel, monolayers were seeded in 35mm MatTek dishes using 95% DMEM, and 5% Matrigel coating. After achieving confluency, organoids were incubated in IFE or IF* media with alteration of added factors and timing to determine the optimal differentiation process. Imaging of all microfluidic devices was done via a Zeiss LSM980 confocal microscope.  

    Results, Conclusions, and Discussions:: After 5 days, we found that mouse colon organoids covered 96±1% of the epithelial chamber area (n=4), while MatTek dish confluency was 37±10% (n=5). There were no fluorescent EECs without differentiation media. After incubation in the IF* media for 5 days, Microfluidic monolayers had 2±1 fluorescent cells (n=4), MatTek disheshad 4±2 fluorescent cells (n=5), and three-dimensional organoid cultures had 2±1 fluorescent cells (n=8). Monolayers for both microfluidic and MatTek dishes that were incubated in IFE yielded no fluorescent cells. 

    This work     establishes a foundation for enriching EECs in microfluidic devices toward the goal of creating and maintaining neuro-epithelial connections. Transgenic mouse organoids form highly confluent monolayers in microfluidic devices. Greater confluency in microchannels may be due to the microscale area the monolayers grow on. To normalize these differences between the two monolayer cultures, future experiments will assess EEC density. IF* drove the appearance of fluorescent EECs, indicating the differentiation. Future experiments will include adjustments to media factors and timing of the exposure to further enhance the differentiation of mouse colon organoids.


    Acknowledgements (Optional): :

    References (Optional): :

    1. Mercado-Perez, A. & Beyder, A. (2022) Gut feelings: mechanosensing in the gastrointestinal tract. Nature Reviews Gastroenterology & Hepatology 19, 283–296. 




    2. Moriez, R. et al. (2009) Neuroplasticity and neuroprotection in enteric neurons: Role of epithelial cells. Biochemical and Biophysical Research Communications 382, 577–582. 




    3. Alcaino, C., Knutson, K. R., Treichel, A. J., Yildiz, G., Strege, P. R., Linden, D. R., ... & Beyder, A. (2018). A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release. Proceedings of the National Academy of Sciences, 115(32), E7632-E7641. 




    4. Mackie, G. O. (2004) Epithelial conduction: recent findings, old questions, and where do we go from here?. Hydrobiologia 530, 73-80. 




    5. Fung, C., Vanden Berghe, P. (2020) Functional circuits and signal processing in the enteric nervous system. Cell. Mol. Life Sci. 77, 4505–4522. 




    6. Hanyu, H., Sugimoto, S., & Sato, T. (2023). Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures. Intestinal Differentiated Cells: Methods and Protocols (pp. 141-153).  




    7. Goldspink, D. A., Lu, V. B., Miedzybrodzka, E. L., Smith, C. A., Foreman, R. E., Billing, L. J., ... & Gribble, F. M. (2020). Labeling and characterization of human GLP-1-secreting L-cells in primary ileal organoid culture. Cell reports, 31(13).