A. Computational estimation of shear stress and flow rates in GLA-MPS

The FSS was one of the most important physical factors affecting the cell function and morphology,18,2618. Y. Du, N. Li, H. Yang, C. Luo, Y. Gong, C. Tong, Y. Gao, S. Lü, and M. Long, Lab Chip 17, 782 (2017). https://doi.org/10.1039/C6LC01374K26. R. G. Lentle and P. W. M. Janssen, J. Comp. Physiol. B 178, 673 (2008). https://doi.org/10.1007/s00360-008-0264-x but the previous GLA-MPSs faced challenges in the control. The flow perfusion conditions should be investigated for each organ cell. In this device, we estimated the FSS and flow rates using COMSOL simulation to investigate whether the GLA-MPS provides proper FSS on cells (Fig. 3). When the medium perfusion in the gut chamber was performed from 2 to 5 μl min−1, the surface FSS on the apical side of cells had a linear relationship with the external pumping flow rate [Figs. 3(a) and 3(b)]. The mean FSS for the intestinal cells was in the range of 8 × 10−3–2 × 10−2 dyn cm−2, which was in the physiologically relevant range2626. R. G. Lentle and P. W. M. Janssen, J. Comp. Physiol. B 178, 673 (2008). https://doi.org/10.1007/s00360-008-0264-x,2727. T. Ishikawa, T. Sato, G. Mohit, Y. Imai, and T. Yamaguchi, J. Theor. Biol. 279, 63 (2011). https://doi.org/10.1016/j.jtbi.2011.03.026 and could promote gut cell barrier formation and differentiation processes.2828. H. J. Kim, D. Huh, G. Hamilton, and D. E. Ingber, Lab Chip 12, 2165 (2012). https://doi.org/10.1039/c2lc40074j,2929. L. C. Delon, Z. Guo, A. Oszmiana, C.-C. Chien, R. Gibson, C. Prestidge, and B. Thierry, Biomaterials 225, 119521 (2019). https://doi.org/10.1016/j.biomaterials.2019.119521The FSS applied to cells from the basal side through the holes of the membrane was determined using the membrane thickness (h) and circulation flow rate [Figs. 3(c) and 3(d)]. The FSS was reduced dramatically with an increase in membrane thickness. In our setup parameters (20-μm-thick membrane), we found that the surface FSS (1 × 10−8–1.2 × 10−7 dyn cm−2) on the basal sides of cells by the circulation flow was considerably smaller than the FSS on the apical side of gut epithelial cells. Thus, in the effect of circulation flow, we not only considered the effects of FSSs of basal sides for the later cell experiments, but we also recognized that circulation flow would mediate communication between the gut and liver cells.In recent results for the gut on a chip,1717. W. Shin, C. D. Hinojosa, D. E. Ingber, and H. J. Kim, IScience 15, 391 (2019). https://doi.org/10.1016/j.isci.2019.04.037 the Caco-2 cells on the porous membrane experienced flow perfusion in the apical side or basal side, which showed an improvement in cell functions. In our device, by using the simulation model, the FSS on cells is well-designed and can be clearly used for investigating their effect on cells from both sides.

B. Evaluation of circulation flow by the micropump

The micropump for circulation flow was controlled using the developed pneumatic actuating method to induce the flow circulation between two organ chambers. We investigated the circulation flow rates controlled by an on-chip micropump after the fabrication of the GLA-MPS (Fig. 4). The mean flow rate increased with driving pressure (0–150 kPa) and pumping frequency (0–1.5 Hz). Working frequencies over 1 Hz exhibited no significant differences in the generated flow rates; in contrast, the GLA-MPS had disassembled by applying over 150 kPa in the control layer for 14 days (data not shown). Therefore, the working pressure was set from 50 to 150 kPa to carry out cell culture and assay in the GLA-MPS; this gave flow rates from 22 to 61 nl min−1 for circulation at a 1-Hz working frequency.The flow rate requirement for inducing two organ interactions could be estimated from the exchange ratio of circulation flow rate (q) and flow volume (Q), i.e., q/Q. In this device, the microfluidic system has a circulation flow volume of 1.5 μl. The exchange ratio is 1.5%–9% in 1 min. In other GLA-MPSs,3,73. J. M. Prot, L. Maciel, T. Bricks, F. Merlier, J. Cotton, P. Paullier, F. Y. Bois, and E. Leclerc, Biotechnol. Bioeng. 111, 2027 (2014). https://doi.org/10.1002/bit.252327. M. Trapecar, C. Communal, J. Velazquez, C. A. Maass, Y.-J. Huang, K. Schneider, C. W. Wright, V. Butty, G. Eng, O. Yilmaz, D. Trumper, and L. G. Griffith, Cell Syst. 10, 223 (2020). https://doi.org/10.1016/j.cels.2020.02.008 the exchange ratio is approximately 0.25%–1% in 1 min. Thus, our device realized a more intensified circulation flow between the gut and liver. For 9-day cell culture, this flow is sufficient to induce the interaction between two organs in the GLA-MPS device. Besides, the on-chip micropump could realize a fine range of flow rate adjustment, which is consistent with the aforementioned FSS simulation circulation flow rate range. Therefore, we could couple the circulation flow with gut perfusion flow to improve the cell function and induce the organ to organ interaction for cell-culture experiments.

C. GLA culture on a chip

Caco-2 gut intestinal3030. Y. Sambuy, I. De Angelis, G. Ranaldi, M. L. Scarino, A. Stammati, and F. Zucco, Cell Biol. Toxicol. 21, 1 (2005). https://doi.org/10.1007/s10565-005-0085-6 and HepG2 hepatocyte-like cells3131. S. Lasli, H. J. Kim, K. J. Lee, C. A. E. Suurmond, M. Goudie, P. Bandaru, W. Sun, S. Zhang, N. Zhang, S. Ahadian, M. R. Dokmeci, J. Lee, and A. Khademhosseini, Adv. Biosyst. 3, e1900104 (2019). https://doi.org/10.1002/adbi.201900104 were used and cultured in GLA-MPS to establish in vitro GLA. Prior to the cell culture, the surface of the porous membrane should be coated with Matrigel for cell attachment. By applying the pressure barriers of caplillary,3232. N. R. Wevers, R. Van Vught, K. J. Wilschut, A. Nicolas, C. Chiang, H. L. Lanz, S. J. Trietsch, J. Joore, and P. Vulto, Sci. Rep. 6, 38856 (2016). https://doi.org/10.1038/srep38856 Matrigel was only coated on the surface of the cell-culture chamber side (upper side), but not the surface of holes and circulation channel side (bottom side). This is to reduce cell attaching and migrating through the hole of the porous membrane (Fig. S3 in the supplementary material).After culturing for 1 day under the static conditions for the cell attachment, only Caco-2 cells, but not HepG2 cells, were subjected to the perfusion flow, since the apical side of hepatocytes in the physiological situation is not subjected to the perfusion flow [Fig. 5(a)]. Both Caco-2 and HepG2 cells were maintained for eight days with the circulation flow in the circulation channel. After the culture, Caco-2 and HepG2 cells showed 95% and 92% cell viability, respectively [Fig. 5(b)]. It demonstrated that the cells could be maintained with relatively high cell viability in this device.The expression of ZO-1 and albumin (ALB), as functional markers of the gut and liver, respectively, was observed by immunocytochemistry to confirm the functions of cells cultured in the GLA-MPS (Fig. 6). Caco-2 cells expressed ZO-1 tight-junction protein to form the gut barrier [Fig. 6(a)]; the increase in flow rates of perfusion flows in the gut cell-culture chamber induced the ZO-1 expression in the Caco-2 cells at 5 μl min−1 (FSS = 2 × 10−2 dyn cm−2), as shown in Fig. 6(b) and Table S1 in the supplementary material. Furthermore, the circulation flow did not change the ZO-1 expression in the Caco-2 cells without perfusion flow; instead, it induced the ZO-1 expression in addition to the perfusion flow [Fig. 6(b)]. In light of the simulation results, FSS by circulation flow was considerably smaller than that by perfusion flows; however, the cultured cells could receive the FSS of basal sides. The cell-culture medium in the circulation channel could accumulate molecules (i.e., growth factors, cytokines, metabolites, and lipids) and exosomes secreted from Caco-2 and HepG2 cells. Thus, the medium contained both autocrine and paracrine factors and facilitated the interactions of Caco-2 and HepG2 cells by the circulation flow. Thus, the induction of the ZO-1 expression could be the outcome of the combinations of FSS by perfusion/circulation flows and molecular transport.In the case of HepG2 cells, the HepG2 cells were not directly subjected to perfusion flow in the cell-culture chamber, since we focus to evaluate the effects of LPS from the circulation channel and there are numbers of reports describing the effects of perfusion flow on hepatocytes;3333. S. Y. Lee, D. Kim, S. H. Lee, and J. H. Sung, APL Bioeng. 5, 041505 (2021). https://doi.org/10.1063/5.0061896 also, increasing the gut cell-culture chamber perfusion flow rates did not improve the ALB expression in HepG2 cells [Fig. 6(c)], while the increase in circulation flow rates from 0 to 61 nl min−1 significantly induced the ALB expression in HepG2 cells [Fig. 6(d) and Table S2 in the supplementary material]. These results suggest that HepG2 cells facilitated hepatic functions such as ALB production by FSS and as a molecular transport in the circulation channel.Thus, we selected the flow rates (gut perfusion flow: 2 μl min−1; circulation flow: 61 nl min−1) to establish in vitro GLA, which provided the highest expression of functional proteins in both Caco-2 and HepG2 cells among the tested conditions. Using this optimal condition, we tested the permeability of Caco-2 cells after the 9-day culture, and we confirmed the cell barrier function (Fig. 7). It indicated that Caco-2 cells formed an intact cell barrier to protect the transportation of 4-kDa FITC-dextran across the porous membrane.

D. In vitro recapitulation of the inflammatory bowel disease in GLA-MPS

To demonstrate the applicability of the GLA-MPS for disease modeling, we selected to recapitulate inflammatory bowel disease (IBD)77. M. Trapecar, C. Communal, J. Velazquez, C. A. Maass, Y.-J. Huang, K. Schneider, C. W. Wright, V. Butty, G. Eng, O. Yilmaz, D. Trumper, and L. G. Griffith, Cell Syst. 10, 223 (2020). https://doi.org/10.1016/j.cels.2020.02.008in vitro (Fig. 8). IBD is one of the chronic inflammatory intestinal disorders and is often caused by genetics, immune systems, microbiome, and environmental exposure, and their combinations. Importantly, IBD patients have a high risk of autoimmune liver disease via GLA,3434. E. V. Loftus, G. C. Harewood, C. G. Loftus, W. J. Tremaine, W. S. Harmsen, A. R. Zinsmeister, D. A. Jewell, and W. J. Sandborn, Gut 54, 91 (2005). https://doi.org/10.1136/gut.2004.046615 but the underlying mechanisms of IBD progression are not fully understood yet. The feature of the presented GLA-MPS has an individual addressability for each cell-culture chamber. This feature is advantageous for modeling and investigating multiple organ disease interactions such as IBD and liver disease.To recapitulate IBD using the GLA-MPS, we utilized the bacterial endotoxin, LPS,3535. X.-X. Wu, X.-L. Huang, R.-R. Chen, T. Li, H.-J. Ye, W. Xie, Z.-M. Huang, and G.-Z. Cao, Inflammation 42, 2215 (2019). https://doi.org/10.1007/s10753-019-01085-z administrated in the Caco-2 culture chamber to induce gut inflammation. After the treatment with 1 mg ml−1 of LPS on Caco-2 cells for 4 days [Fig. 8(a)], most of the Caco-2 samples expressed iNOS, which is often observed in IBD;3636. M. M. Fort, A. Mozaffarian, A. G. Stöver, J. d. S. Correia, D. A. Johnson, R. T. Crane, R. J. Ulevitch, D. H. Persing, H. Bielefeldt-Ohmann, P. Probst, E. Jeffery, S. P. Fling, and R. M. Hershberg, J. Immunol. 174, 6416 (2005). https://doi.org/10.4049/jimmunol.174.10.6416 the comparison with non-treated Caco-2 cells is shown in Figs. 8(b) and 8(c), Tables S3 and S4 in the supplementary material. Following the inflammatory induction of Caco-2 cells, the iNOS expression was observed in four out of nine samples of HepG2 cells, while no change in the iNOS expression was observed in the other five samples. Despite the use of established cell lines, such as Caco-2 and HepG2 cell lines, the complexity of the experimental setup could have caused the observed individual differences in the LPS stimulation. In contrast, the treatment of 200 μM of dexamethasone (Dex), an anti-inflammation agent, on the Caco-2 cells during 1 mg ml−1 lPS treatment suppressed the iNOS expression in both Caco-2 and HepG2 cells. Thus, inflammatory responses of HepG2 cells were induced by the LPS-treated Caco-2 cells.To identify the inflammatory mediators that induced inflammatory responses of HepG2 cells via the circulation channel, ELISA was performed for TNF-α, which was elevated in the gut of IBD models.3636. M. M. Fort, A. Mozaffarian, A. G. Stöver, J. d. S. Correia, D. A. Johnson, R. T. Crane, R. J. Ulevitch, D. H. Persing, H. Bielefeldt-Ohmann, P. Probst, E. Jeffery, S. P. Fling, and R. M. Hershberg, J. Immunol. 174, 6416 (2005). https://doi.org/10.4049/jimmunol.174.10.6416 However, TNF-α in the cultured medium was not observed because the amount of TNF-α within the circulation channel with a limited volume (1.5 μl) was lower than the detection limit of ELISA (Fig. S4 in the supplementary material). Therefore, highly sensitive methods are required to detect the mediators within such a small volume to identify the mediators that influence the interaction between Caco-2 and HepG2 cells via the circulation channel.Gut microbiome3737. S. L. Collins and A. D. Patterson, Acta Pharm. Sin. B 10, 19 (2020). https://doi.org/10.1016/j.apsb.2019.12.001 plays critical roles in the pathophysiological scenarios in the gut; they can influence other organs, including liver,3838. N. Ohtani and N. Kawada, Hepatol. Commun. 3, 456 (2019). https://doi.org/10.1002/hep4.1331,3939. S. L. Friedman, B. A. Neuschwander-Tetri, M. Rinella, and A. J. Sanyal, Nat. Med. 24, 908 (2018). https://doi.org/10.1038/s41591-018-0104-9 brain,4040. P. A. Muller, M. Schneeberger, F. Matheis, P. Wang, Z. Kerner, A. Ilanges, K. Pellegrino, J. del Mármol, T. B. R. Castro, M. Furuichi, M. Perkins, W. Han, A. Rao, A. J. Pickard, J. R. Cross, K. Honda, I. de Araujo, and D. Mucida, Nature 583, 441 (2020). https://doi.org/10.1038/s41586-020-2474-7 and the immune system.4141. J. Schluter, J. U. Peled, B. P. Taylor, K. A. Markey, M. Smith, Y. Taur, R. Niehus, A. Staffas, A. Dai, E. Fontana, L. A. Amoretti, R. J. Wright, S. Morjaria, M. Fenelus, M. S. Pessin, N. J. Chao, M. Lew, L. Bohannon, A. Bush, A. D. Sung, T. M. Hohl, M.-A. Perales, M. R. M. van den Brink, and J. B. Xavier, Nature 588, 303 (2020). https://doi.org/10.1038/s41586-020-2971-8 Instead of liver cells, our MPS device allows the introduction of other tissue cells such as neurons, and we can investigate the interactions with the gut microbiome. Thus, our MPS is not limited to recapitulating only GLA, but it is also applicable to other organs and the microbiome.

Furthermore, the presented GLA-MPS allows a collection of conditioned cell-culture media from two cell-culture chambers and circulation channels separately for investigating the underlying molecular mechanisms of the GLA by biochemical assays such as ELISA and mass spectroscopy. Since these methods for the cell-culture medium are non-invasive for cultured cells, we will be able to determine the transitions of molecular contents in the medium during the experimental period to explore interesting molecular biomarkers. At the end of the experiments, GLA-MPS allows performing invasive cell-analytical methods such as immunocytochemistry, as shown in this paper, as well as multi-omics analysis (i.e., genomics, transcriptomics, proteomics, and metabolomics) to obtain deeper insights into normal and diseased GLA.

One advantage of using microfluidic technology for disease modeling is the minimization of the number of cells, such as the primary cells obtained from the patients. These cells are limited in their ability to recapitulate the patients' pathological conditions in vitro. The presented MPS requires only 1.5 × 104 cells for each cell-culture chamber, which is considerably smaller than those of the 24-well cell-culture inserts (3 × 105 cells). Therefore, we efficiently use precious patient samples, such as biopsy specimens. In the present study, we used widely used cell lines (Caco-2 and HepG2) that can provide the standard performance of the presented GLA-MPS. Since patient-derived primary cells could have large patient-to-patient variations, it would be beneficial to obtain such standard data sets with the use of commonly used cell lines.

The device is fabricated using the microfabrication method with a four-layered planer microfluidic structure. Owing to this feature, another advantage is the capability to integrate electrical read-out sensors in cell-culture chambers and circulation channels, which allows for monitoring cell conditions and behaviors during cell culture and treatments in a real-time and non-invasive fashion.4242. J. R. Soucy, A. J. Bindas, A. N. Koppes, and R. A. Koppes, IScience 21, 521 (2019). https://doi.org/10.1016/j.isci.2019.10.052 Such sensors will provide temporal information on tissue formation, disease progression, and drug treatments. In the case of the GLA, the sensor for measuring trans-epithelial electrical resistance (TEER) was beneficial for evaluating gut barrier formation and causing damage during disease progression. In this study, we used a fluorescent dye to investigate gut barrier function; however, the dye might also influence cellular functions. Until now, gut-on-a-chips integrated with TEER measurements have been reported.4343. T. Miyazaki, Y. Hirai, K. Kamei, T. Tsuchiya, and O. Tabata, Electron. Commun. Jpn. 104, 285 (2021). https://doi.org/10.1002/ecj.12296,4444. O. Y. F. Henry, R. Villenave, M. J. Cronce, W. D. Leineweber, M. A. Benz, and D. E. Ingber, Lab Chip 17, 2264 (2017). https://doi.org/10.1039/C7LC00155J The designs and fabrication processes need to be further improved for better accuracy and reproducibility and for integration with multi-organ MPS. This integrative approach will lead to the advancement of MPS for a better understanding of disease mechanisms and applications in drug discovery.