Interconnected multi-well device with three-dimensional shaker–driven culture medium circulation for use as a multi-organ microphysiological system

Interconnected multi-well device with three-dimensional shaker–driven culture medium circulation for use as a multi-organ microphysiological system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Interconnected multi-well device with three-dimensional shaker–driven culture medium circulation for use as a multi-organ microphysiological system Ren Yoshitomi, Shinji Sugiura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6713236/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multi-organ microphysiological systems (multi-organ MPSs) are in vitro platforms that simulate interactions between multiple organs in the body. However, most of existing multi-organ MPSs often suffer from high complexity and low-throughput due to their reliance on pumps for medium circulation. We have developed a simple multi-organ MPS device, namely interconnected multi-well device, that utilizes a three-dimensional shaker for medium circulation, eliminating the need for pumps. The device consists of two components: a set of four connected cell-culture cups and a series of four interconnected wells. Up to six devices can fit in a standard 24-well plate. The device supports various culture methodologies, including conventional two-dimensional culture and spheroid culture, and can accommodate cell culture inserts. Here, we demonstrate medium and immune cell circulation through the entirety of the device. Then, as a representative use-case scenario, we demonstrate using the device to evaluate the anticancer effects of the prodrug capecitabine, whose metabolite exhibit anticancer effect, in a two-organ system composed of liver and cancer. In short, our interconnected multi-well device is user-friendly, adaptable to various culture methods, and multi-throughput, and it shows promise for becoming a valuable tool for in vitro organ interaction research. Biological sciences/Biotechnology/Assay systems Biological sciences/Biological techniques/Biophysical methods Biological sciences/Biological techniques/Cytological techniques Biological sciences/Biological techniques/Lab on a chip Multi-organ microphysiological system 3D shaker cell circulation organ-organ interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Animal testing has long been a standard practice for assessing the in vivo effects of foods and pharmaceutical ingredients ahead of clinical trials. However, ethical concerns and the recognition of interspecies differences that limit the extrapolation of animal results to humans have led to a shift toward reducing animal testing 1 , 2 , as evidenced by the European Union's 2013 ban on the sale of cosmetics tested on animals, and the U.S. Food and Drug Administration's recent elimination of animal testing as a requirement for new drug development 3 . This shift has necessitated the establishment of alternative processes for pre-clinical testing that do not rely on animal models. Microphysiological systems (MPSs), including co-culture and three-dimensional (3D) cell culture, have emerged as promising approaches to study human organ and tissue function. These systems offer a more sophisticated and physiologically relevant platform compared to traditional cell culture methods 4 . Among the various types of MPSs, fluidic systems have shown promise in their ability to reflect complex biological phenomena. The value of fluidic MPSs in drug development has been demonstrated recently by the progression of a compound for the treatment of demyelinating neuropathies to phase II clinical trials after its efficacy was evaluated using a fluidic MPS 5 , 6 . This success highlights the potential of fluidic MPSs to accelerate drug development pipelines. Multi-organ MPSs, a type of fluidic MPS comprising several interconnected culture cells with each cell representing a different organ or cell type, offers several key advantages over the other MPSs. These systems enable the observation of multi-organ interactions mediated by factors like cytokines and exosomes 7 , 8 . They also enable the evaluation of drug effects, including those of prodrugs and side effects that occur through metabolic processes 9 – 11 . Furthermore, multi-organ MPSs allow for the assessment of functions that may be overlooked in conventional cell-based experiments without using living organisms. For example, several studies have indicated that multi-organ MPSs can be used for toxicological, drug metabolism, pharmacokinetic/pharmacodynamic, and absorption/distribution/metabolism/excretion/toxicity studies 12 – 19 . Despite these advantages, many multi-organ MPSs require complex tube connections and pumps for circulation of the culture medium, making their operation difficult 20 , 21 , 22 . In addition, most multi-organ MPSs are often only capable of single-culture throughput, which is also problematic for use in drug screening, where the effects of many compounds at different concentrations need to be evaluated. This complexity and low-throughput have created a demand for simpler culture systems that can operate without pump connections, are easy to handle, and allow for multi-throughput culture. While some pump-less MPSs exist, such as those using simple flow path connections or seesaw-type shakers 9 , 23 – 25 , they often fail to achieve adequate medium circulation, with medium either not flowing or moving back and forth ineffectively. To address these challenges, we have developed a novel multi-organ MPS device, namely interconnected multi-well device, that utilizes a 3D shaker for culture medium circulation. In the present study, we verified that complete cell and medium circulation can be achieved within the device. Then, as a representative use-case scenario, we demonstrate using the device to evaluate the anticancer effects of the prodrug capecitabine in a two-organ system. Our findings confirm that our device can be used for the observation of inter-organ interactions. Results Design and Operation of t he interconnected multi-well device The device comprises two components: a set of four cell-culture cups arranged in a square and connected by tabs (Figs. 1a, b, 2a) and a series of four interconnected wells (Figs. 1c, d, 2b). The device was basically designed based on the 24 well plate format considering SBS Recommended Microplate Specifications provided by Society for Laboratory Automation and Screening. The inner diameter of cell culture cups and wells were about 14 mm and the pitch was 18.6 mm. The cell-culture cups can be inserted in a commercially available 24-well plate and are used for culturing cells and are joined via easily detachable interconnected tabs. The interconnected wells are connected by 5.8-mm-wide channels through which the culture medium is able to circulate between wells. The device is fully assembled by inserting the cell-culture cups into the interconnected wells (Fig. 1e). In this study, the wells were numbered 1–4 clockwise starting with the upper-left well (Fig. 1f). The device is used in three steps. First, the cell-culture cups are placed in a 24-well plate (TPP, Trasadingen, Switzerland) and up to four different types of cells are cultured in the individual cups (Fig. 2c, Supplemental movie 1). Once the cells in the cell-culture cups reach maturity, the cell-culture cups are inserted into the interconnected wells (Fig. 2d, Supplemental movie 2). Finally, the plate containing the assembled devices is placed on a 3D shaker for circulating culture (Fig. 2e, Supplemental movie 3). Cell culture insert (Corning, NY, USA) can be optionally used for cell culture model of barrier tissues, e.g. intestine model, in cell-culture cup in interconnected wells if necessary. Verification of complete medium circulation To confirm that complete medium circulation is achieved within the device, the assembled devices containing phosphate-buffered saline were treated with Gardenia Blue with or without shaking. When the 3D shaker was used, Gardinia Blue was observed in all four wells at only 9 s corresponding to the time for one rotation of the 3D shaker, after the start of 3D shaking (Fig. 3a, Supplemental movie 4), and complete mixing of the medium was confirmed at 60 s (Fig. 3b). In contrast, when the 3D shaker was not used in static test, very little of the Gardinia Blue solution had diffused into the adjacent cells at 60 s (Fig. 3b) or even 600 s (Fig. 3c), even though more the PBS containing the dye was used compared with the circulatory test. This study was conducted with a total medium volume of 2.5 mL during medium circulation using a 3D shaker. When the medium volume was as low as 1.5 mL, circulation is insufficient due to inadequate fluid coverage throughout the device (Supplemental movie 5). At 2.0 mL, medium circulation was inconsistent with successful circulation occurring about once in every three rotations. Based on these observations, we concluded that 2.5 mL of medium was necessary to realize sufficient medium circulation throughout the culture period. Applicability of the device to different cell culture models Cell culture models can be classified into two principal culture categories: two-dimensional and three-dimensional. We therefore verified whether our device can support both of these cell culture models. We confirmed that the device can be used not only for conventional two-dimensional cultures in the cell-culture cups (Fig. 4b) but also co-culture of cells on culture inserts (Fig. 4a) and Matrigel-embedded spheroid culture (Fig. 4c), demonstrating its wide applicability to couple of culture methods. Effect of 3D shaker rotation speed on circulation of immune cells To verify that cells are able to circulate through the whole of the device, we used human T lymphocyte Jurkat cells, which are a type of circulating immune cell. As in the experiment examining the circulation of the medium, cells were added to Well 1, the setup was shaken by a 3D shaker for 30 min and the number of cells in each well were counted. When the setup was not shaken, no migration of the cells from Well 1 to any of the other wells was observed (Fig. 5a). When the medium was circulated at low speed (12.5 rpm), some cell circulation was observed, but the measurement error was large (Fig. 5b). However, when the medium was circulated at high speed (52 rpm), the cells were uniformly distributed in all four wells (Fig. 5c). These results indicate that circulating the medium at high-speed causes floating cells to circulate through the device. We did not mix the medium prior to sampling by pipetting because the process of pipetting carries a potential risk of cell migration into adjacent wells during the cell sampling procedure. Therefore, we consider that the apparent decrease in total cell numbers in static and low speed culture condition was due to the settlement of seeded cells. At higher rotation speed, cells remain homogeneously suspended, allowing us to sample the consistent number of cells with the number of seeded cells. Applicability of the device as a two-organ system Finally, we verified the applicability of the device to examine the interactions between liver and cancer by using the anticancer prodrug capecitabine. In humans, capecitabine is metabolized by carboxylesterase in the liver to 5’-deoxy-5-fluorocytidine, which is then converted to 5’-deoxy-5-fluorouridine by cytidine deaminase primarily in liver and tumor tissue. Then, 5’-deoxy-5-fluorouridine is further convert to its active metabolite, 5-fluorouracil, by thymidine phosphorylase, which is present at high levels in tumor tissues where the metabolite exerts its antitumor effects. We therefore examined the effect of capecitabine on a co-culture of hepatocytes (HepaRG) and cancer cells (HCT-116), with reference to a previous study 10 . When HCT-116 cells were cultured alone, capecitabine was observed to have no effects on the viable cell count (Fig. 6a, b). Similarly, no effects were observed when the two cell lines were subjected to static culture in the presence of capecitabine. However, when the HCT-116 cells were subjected to circulatory culture together with HepaRG cells, capecitabine induced a significant reduction in the number of viable HCT-116 cells (Fig. 6c, d). These results indicate that the anticancer effect of capecitabine is only observable under co-culture with HepaRG with medium circulation. Discussion Current multi-organ MPSs face several critical limitations, such as their complexity due to numerous pumps, limited throughput capability, and operational difficulties during implementation 26 , 27 . To address these limitations, we have developed and validated a simplified, multi-throughput multi-organ MPS device. A key innovation of our system lies in its operational simplicity. By eliminating complex pump systems and simplifying assembly, we have developed a user-friendly platform that requires only basic consumables and a 3D shaker. This design directly addresses a fundamental challenge in current multi-organ MPS technology, where system complexity often compromises throughput efficiency. Our platform demonstrates a multi-throughput capacity, accommodating six devices per plate with the potential for multiple plates to be placed simultaneously on a single 3D shaker. Our system also significantly reduces medium consumption compared to conventional pump-based systems, which is crucial for maintaining optimal concentrations of soluble factors. As demonstrated by other groups 28 – 30 , even minor variations in soluble factors like cytokines and exosome can substantially impact cellular response patterns. Our device also exhibits remarkable versatility in culture methodology, supporting conventional sheet culture, cell culture inserts, and spheroid formation. This flexibility should allow for cultivation of diverse organ types, from polarized intestinal cells to complex 3D liver cultures, that more closely mimic in vivo conditions 31 – 34 . In addition, our device consists of four wells arranged in a square format (2×2 wells). When using a device with four wells arranged in a linear configuration (1×4 wells), significant differences in environmental conditions such as flow rate and flow velocity occur between the outer two wells and the inner two wells during circulation. On the contrary, in our square formatted device, flow rates and flow velocities are nearly uniform across all wells, indicating that environmental differences between wells are minimized. Our present findings emphasize the critical importance of medium circulation when examining the effects of drugs and cellular interactions. Static culture conditions, as observed in traditional systems, can limit drug distribution and potentially lead to inaccurate pharmacological assessments. In contrast, our device enables observation of prodrug responses under conditions that more closely approximate in vivo environments 35 , 36 . Furthermore, the adjustable rotation speed feature of our 3D shaker–based system presents a notable advancement, particularly with respect to the analysis of circulating immune cells. Previous multi-organ MPS studies have demonstrated that cytokine concentrations fluctuate markedly depending on immune cell presence 37 . Although we observed that low-speed circulating culture can result in biased cell distribution between wells, potentially due to cup depth, we hypothesize that gel implementation could resolve this issue by elevating the cell-culture section base. The enhanced prodrug and metabolic activity observed in our multi-organ MPS using a co-culture of liver and cancer cells, aligns with previous research 11 . Together, these results suggest our device's potential as a valuable tool for drug discovery applications, potentially reducing dependence on animal studies. In conclusion, our novel device represents a significant advancement in multi-organ culture technology, offering a simplified yet effective platform that affords appropriate medium circulation. This innovation provides researchers with a robust tool for use in detailed cell–cell interaction studies and drug–response assessments. Future development will focus on expanding the range of compatible organ types and establishing standardized protocols for various experimental configurations, further enhancing the devices utility in drug discovery and development. Limitations of the study The cell-culture cups showed a slight, though not statistically significant, difference in viable cell count compared to the wells of a 24-well plate, even without circulation. (Supplemental Fig. 1). We attribute this minor variation to the small difference in culture area between the cell-culture cup and the 24-well plate. Furthermore, cell counts were significantly lower in cultures circulated through our device compared to the non-circulating culture group. Since previous reports have suggested that flow velocity affects cell proliferation, it is possible that it influences cell proliferation within our device 38 – 40 . Circulation at slower speeds may eliminate these observed differences in viable cell counts; however, most 3D shakers are not compatible with CO 2 incubators, meaning we were unable to validate shaker speeds slower than 12.5 rpm using the 3D shakers that were available for the present study. Methods Design and fabrication of the device The cell-culture cups and culture medium wells were designed in the Autodesk Fusion software Ver.2.0.19440 (Autodesk, Inc., CA, USA). Both components were fabricated from polystyrene by injection molding. To facilitate cell attachment and reduce the effect of surface tension along the flow path, the components were oxidized at 100 W for 80 s (40s × 2) (cell-culture cups) or 60 s (interconnected wells) using an oxygen plasma reactor (PR500; Yamato Scientific Co., Tokyo, Japan). Culture of spheroids Spheroids of human colon cancer HCT-116 cells (REKEN BRC, Ibaraki, Japan) were cultured in 10% fetal bovine serum (FBS) (Cytiva, Tokyo, Japan)-Dulbecco's Modified Eagle Medium (DMEM) (041-29775, FUJIFILM Wako Pure Chemical, Osaka, Japan) using TASCL-600 3D culture microplates (Cymss-bio Co., Ltd, Nagoya, Japan). The spheroids were embedded in Matrigel (Corning, NY, USA) for avoiding spheroid migration. Microscopy was performed after placing the cell-culture cups into a 24 well TPP Tissue Culture Test Plate (TPP, Trasadingen, Switzerland). Verification of complete culture medium circulation To confirm that the device setup on the 3D shaker resulted in complete circulation of the culture medium, the interconnected wells were placed in 24-well plates and 2.3 mL (circulatory test) or 2.8 mL (static test) phosphate-buffered saline was added to each well. The devices were then placed on the table of a 3D shaker (Multi-bio 3D; BS-010125-AAK, Kenis, Osaka, Japan) at a 7-degree angle of inclination with Well 1 at the lowest position (circulatory test) or in the horizontal plane (static test). Well 1 was treated with 0.2 mL phosphate-buffered saline containing Gardenia Blue (FUJIFILM Wako Pure Chemical, Osaka, Japan) and the rotation of the 3D shaker was started at a speed of 5 rpm for the circulatory test. The 3D shaker (Multi-bio 3D; BS-010125-AAK) was exclusively used for this experiment. Verification of complete cell circulation To confirm that the device setup on the 3D shaker resulted in complete circulation of cells within the interconnected wells, the wells were each filled with 2.0 mL of 10%FBS-Roswell Park Memorial Institute 1640 medium (FUJIFILM Wako Pure Chemical, Osaka, Japan). Then, 0.5 mL of 5.0 × 10 5 cells/mL human T lymphocyte Jurkat cells were added to Well 1 and allowed to settle for 10 min (final concentration of Jurkat cells was 1.0 × 10 5 cells/mL). The device was then shaken with a 3D shaker (KFS-410; Kenis, Osaka, Japan) at a 7-degree angle of inclination for 30 min. The 3D shaker was stopped at the point when the cell suspension was most concentrated in Well 3 (i.e., Well 3 was at the lowest position on the 3D shaker), and 100 µL of cell suspension was collected from each well and the cells were counted with a TC-20 automated cell counter (Bio-Rad, CA, USA). Evaluation of using the device for cell monoculture HCT-116 cells were seeded in 24-well plates containing cell-culture cups with 0.5 mL of 10% FBS-DMEM medium at a density of 1.0 × 10⁴ cells/mL and incubated in a CO 2 incubator. After 1 day, the cell culture-cups were placed in interconnected wells with each well containing 2.5 mL HepaRG Thawing/Plating/General medium (KAC Co., Ltd, Kyoto, Japan), again in 24-well plates. Once assembled, the plates were placed on a 3D shaker at a 7-degree angle of inclination and shaken at 12.5 rpm in the CO 2 incubator. After 3 days, viable cells were counted by using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Evaluation of cell–cell interactions in the presence of capecitabin This experiment was conducted as previously reported 10 . Immortalized human HepaRG hepatocytes (KAC Co., Ltd, Kyoto, Japan) were seeded at 9.6 × 10 5 cells/mL (Day 0) in cell-culture cups 1 and 2 using 0.5 mL of HepaRG Thawing/Plating/General medium and were replaced with 0.5 mL of HepaRG metabolic/maintenance medium (KAC Co., Ltd, Kyoto, Japan) (Day 1). The medium was changed every two days. On Day 6, HCT-116 cells were cultured in 0.5 mL of 10%FBS-DMEM and were seeded at 1.0 × 10 4 cells/mL in cell-culture cups 3 and 4. On Day 7, the cell-culture cups were transferred to interconnected wells containing 2.5 mL HepaRG Thawing/Plating/General medium with or without 100 µM of the anticancer prodrug capecitabin (TCI, Tokyo, Japan) and shaken on a 3D shaker at a 7-degree angle of inclination and shaken at 12.5 rpm in the CO 2 incubator. On Day 10, viable cells were counted by using a Cell Counting Kit-8. Statistical analysis All data are displayed as mean ± SE. The Tukey–Kramer test was used to determine whether differences between groups were statistically significant. The statistical analyses were performed using the GraphPad Prism 10 software (GraphPad Software, Inc., CA, USA). Statistical significance was defined as P < 0.05. Declarations Data availability statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Conflict of Interest The authors declare no competing financial interest. Acknowledgements We thank ELSS (https://www.elss.co.jp) for the English language review. Author contributions The author responsibilities were as follows: R.Y. and S.S: designed the research; R.Y.: analyzed the data; R.Y. and S.S. drafted the manuscript; and all authors: conducted the research, and read, revised, and approved the final manuscript. 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Fluorescence-based multifunctional light sheet imaging flow cytometry for high-throughput optical interrogation of live cells. Communications Physics 7 , 25 (2024). https://doi.org/10.1038/s42005-024-01522-y Zhan, C. et al. Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells. iScience 23 , 101653 (2020). https://doi.org/https://doi.org/10.1016/j.isci.2020.101653 Ilvesroiha, E. et al. Establishing a simple perfusion cell culture system for light-activated liposomes. Scientific Reports 13 , 2050 (2023). https://doi.org/10.1038/s41598-023-29215-6 Additional Declarations No competing interests reported. Supplementary Files Sup.mov.1IndividualCulturePhase.mp4 Sup.mov.2CellInteractionPhase.mp4 SupplementalMovie3Circulatingculturemethodology.mp4 SupplementalMovie4Circulationofculturemedium2.5mL.mp4 SupplementalMovie5Circulationofculturemedium1.5mL.mp4 SupplementalMovieLegend.docx supplementaryfigure.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6713236","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469849068,"identity":"8d2b3686-bc3c-472a-b0f3-b4ec0f13ad6a","order_by":0,"name":"Ren Yoshitomi","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Yoshitomi","suffix":""},{"id":469849069,"identity":"2798befc-72d7-48d5-a64b-569509f6028b","order_by":1,"name":"Shinji Sugiura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYDACCcYGBgkDEAvIYGCwYWCDSjATqyUNqIWZkBZU7mG8isFAfnZz2wOLAgZ7g/OL26Qras7L8/GfP8D4pYKB3RyHFoM7B9sNgA5L3HDjYZvkmWO3DdskkhmYZc4wMFs24NAikdgmAdSSYHDjYJtkA9ttxjYJZgZmyTYGZoMDOBw2A6LFHqLl3zn7Nv7D+LUw3IBoYdxwvrFNsrHtQGIbQzID40c8WgwgWiQSZ95gbLZs7EtOBvrF4DDDGQmcfpGfkf5MWuKPjT3f+eMPbzZ8s7Od33/w4cMfFTbJuEIMBJglQLEjkYAQOczDALQLjxbGDyCSH8npjD8YGOzwaRkFo2AUjIIRBQDRFVXBjuG2vwAAAABJRU5ErkJggg==","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Shinji","middleName":"","lastName":"Sugiura","suffix":""}],"badges":[],"createdAt":"2025-05-21 06:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6713236/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6713236/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84545589,"identity":"7bc575e3-2498-4bd4-ab86-539ff294577a","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustrations of the device components and assembly\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a-d) Side view and overhead view of \u0026nbsp;the cell-culture cups (a, b), interconnected wells (c, d).\u003c/p\u003e\n\u003cp\u003e(e-f) Illustration of assembled device (e, f). Wells are numbered 1–4 clockwise, starting from the upper left. The red arrow indicates the direction of medium circulation.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/4b333e4ebbccb1fea118f8cb.png"},{"id":84545597,"identity":"6166497d-9c59-4e2c-882c-50de6989b21c","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":472553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFabricated components and use of the device\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotographs of the fabricated cell-culture cups (a) and interconnected wells (b). Up to six assembled devices can be inserted into a commercial 24-well plate. (c) Individual cell-culture phase: different types of cells can be cultured in the individual cell-culture cups in the 24-well plate. (d) Cell interaction phase: the cell-culture cups are inserted into the interconnected wells and the cells in the cell-culture cups are allowed to interact with each other. (e) Assembled multi-throughput plate on a three-dimensional shaker for circulation of the culture medium.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/a96e98e6f835182ffb812779.png"},{"id":84545592,"identity":"f846b12f-8409-4e9e-8020-b3edccfdaf6d","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of circulatory and static cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Images of the culture medium at 0, 3, 6, and 9 s after addition of Gardenia Blue dye. (b) Comparison of the circulatory and static cultures at 60 s after Gardenia Blue addition. (c) Distribution of the culture medium in the static culture at 600 s after Gardenia Blue addition.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/c0a4eff352a6389d4ec12d03.png"},{"id":84545593,"identity":"4406de11-4577-4ddd-8215-97db1d236f15","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApplicability of the device to different culture models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Photograph of the assembled device with optional cell culture insert. Inset: Magnified view of the insert attachment area. (b) Photomicrograph of human colon cancer HCT-116 cells in conventional two-dimensional culture within the cell-culture cup. (c) Photomicrograph of HCT-116 cells in Matrigel-embedded spheroid culture within the cell-culture cup. Scale bars: 500 μm.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/4f4d935bcab83fcb43022ea6.png"},{"id":84545595,"identity":"2f089ef8-758e-4ed3-a7e0-435be96d8fc3","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":464253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell distribution in static and circulating cultures of floating cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman T lymphocyte Jurkat cells were added to Well 1 and allowed to settle for 10 min. Then, the devices were left stationary (a) or shaken on the 3D shaker at the indicated circulation rates (b, c) for 30 min, after which the cells in each well were counted. Data are presented as means ± SE (n = 3). n.d., not detected; ns, not statistically different.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/0e1d7e09930d09911ab7fc95.png"},{"id":84547258,"identity":"0784c60c-24f7-45e4-b66f-050dab2d5cb9","added_by":"auto","created_at":"2025-06-13 09:24:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":111838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of device use for the study of a two-organ systems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Experimental design for the examination of a monoculture of human colon cancer HCT-116 cells. (b) Viable cell counts in HCT-116 monocultures with and without circulation or capecitabine treatment. Cell counts are compared with that without capecitabine treatment. (c) Experimental design for the examination of a two-organ system comprising HCT-116 cells and immortalized human HepaRG hepatocytes. (d) Viable cell counts in the co-culture with and without circulation or capecitabine treatment. Cell counts are compared with that without capecitabine treatment. Data are presented as means ± SE (n = 4). ns, not statistically different; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/090c53a9b41f547b09fe5b03.png"},{"id":85929581,"identity":"1ae926cb-b908-49eb-869d-64d13920950a","added_by":"auto","created_at":"2025-07-03 09:09:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2655230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/9c365e1a-8033-45dc-82e9-bb7f1bcbad86.pdf"},{"id":84545624,"identity":"17346b3e-d8c0-41a6-afd0-b2d8f2dff1ee","added_by":"auto","created_at":"2025-06-13 09:16:32","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41831564,"visible":true,"origin":"","legend":"","description":"","filename":"Sup.mov.1IndividualCulturePhase.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/f62a25c5761d3a46338ffad3.mp4"},{"id":84545625,"identity":"89b72518-d704-4239-a392-0f2215692e64","added_by":"auto","created_at":"2025-06-13 09:16:32","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":34299260,"visible":true,"origin":"","legend":"","description":"","filename":"Sup.mov.2CellInteractionPhase.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/651a1065d7b8fd8fe40b6c7c.mp4"},{"id":84545628,"identity":"b11933ca-3a3d-435e-9608-c57605b2f194","added_by":"auto","created_at":"2025-06-13 09:16:32","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":46874126,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMovie3Circulatingculturemethodology.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/78b99fb360d1a0ce92372169.mp4"},{"id":84545626,"identity":"652acefb-4144-4d5a-ba9c-c667ccb1bd49","added_by":"auto","created_at":"2025-06-13 09:16:32","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":39999760,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMovie4Circulationofculturemedium2.5mL.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/891455940f4c8b6f434f3dc0.mp4"},{"id":84547271,"identity":"ce5efae0-6982-4380-9b2e-3a3137972025","added_by":"auto","created_at":"2025-06-13 09:24:32","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":31612490,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMovie5Circulationofculturemedium1.5mL.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/2b2c3184d9c84f3a7d0e6413.mp4"},{"id":84545601,"identity":"afec68f5-b351-40b1-9dd0-14a181feb583","added_by":"auto","created_at":"2025-06-13 09:16:31","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMovieLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/4444948f0ba47915091031fb.docx"},{"id":84545590,"identity":"77883e4a-9fa0-4c31-933e-eac508b10caf","added_by":"auto","created_at":"2025-06-13 09:16:30","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":120986,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6713236/v1/37fa5a509e627bf8043c3be3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interconnected multi-well device with three-dimensional shaker–driven culture medium circulation for use as a multi-organ microphysiological system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnimal testing has long been a standard practice for assessing the in vivo effects of foods and pharmaceutical ingredients ahead of clinical trials. However, ethical concerns and the recognition of interspecies differences that limit the extrapolation of animal results to humans have led to a shift toward reducing animal testing \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, as evidenced by the European Union's 2013 ban on the sale of cosmetics tested on animals, and the U.S. Food and Drug Administration's recent elimination of animal testing as a requirement for new drug development\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This shift has necessitated the establishment of alternative processes for pre-clinical testing that do not rely on animal models.\u003c/p\u003e \u003cp\u003eMicrophysiological systems (MPSs), including co-culture and three-dimensional (3D) cell culture, have emerged as promising approaches to study human organ and tissue function. These systems offer a more sophisticated and physiologically relevant platform compared to traditional cell culture methods\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Among the various types of MPSs, fluidic systems have shown promise in their ability to reflect complex biological phenomena. The value of fluidic MPSs in drug development has been demonstrated recently by the progression of a compound for the treatment of demyelinating neuropathies to phase II clinical trials after its efficacy was evaluated using a fluidic MPS\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This success highlights the potential of fluidic MPSs to accelerate drug development pipelines.\u003c/p\u003e \u003cp\u003eMulti-organ MPSs, a type of fluidic MPS comprising several interconnected culture cells with each cell representing a different organ or cell type, offers several key advantages over the other MPSs. These systems enable the observation of multi-organ interactions mediated by factors like cytokines and exosomes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. They also enable the evaluation of drug effects, including those of prodrugs and side effects that occur through metabolic processes\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, multi-organ MPSs allow for the assessment of functions that may be overlooked in conventional cell-based experiments without using living organisms. For example, several studies have indicated that multi-organ MPSs can be used for toxicological, drug metabolism, pharmacokinetic/pharmacodynamic, and absorption/distribution/metabolism/excretion/toxicity studies\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite these advantages, many multi-organ MPSs require complex tube connections and pumps for circulation of the culture medium, making their operation difficult\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition, most multi-organ MPSs are often only capable of single-culture throughput, which is also problematic for use in drug screening, where the effects of many compounds at different concentrations need to be evaluated. This complexity and low-throughput have created a demand for simpler culture systems that can operate without pump connections, are easy to handle, and allow for multi-throughput culture. While some pump-less MPSs exist, such as those using simple flow path connections or seesaw-type shakers\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, they often fail to achieve adequate medium circulation, with medium either not flowing or moving back and forth ineffectively.\u003c/p\u003e \u003cp\u003eTo address these challenges, we have developed a novel multi-organ MPS device, namely interconnected multi-well device, that utilizes a 3D shaker for culture medium circulation. In the present study, we verified that complete cell and medium circulation can be achieved within the device. Then, as a representative use-case scenario, we demonstrate using the device to evaluate the anticancer effects of the prodrug capecitabine in a two-organ system. Our findings confirm that our device can be used for the observation of inter-organ interactions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDesign and Operation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003ehe\u003c/strong\u003e \u003cstrong\u003einterconnected multi-well device\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe device comprises two components: a set of four cell-culture cups arranged in a square and connected by tabs (Figs. 1a, b, 2a) and a series of four interconnected wells (Figs. 1c, d, 2b). The device was basically designed based on the 24 well plate format considering SBS Recommended Microplate Specifications provided by Society for Laboratory Automation and Screening. The inner diameter of cell culture cups and wells were about 14 mm and the pitch was 18.6 mm. The cell-culture cups can be inserted in a commercially available 24-well plate and are used for culturing cells and are joined via easily detachable interconnected tabs. The interconnected wells are connected by 5.8-mm-wide channels through which the culture medium is able to circulate between wells. The device is fully assembled by inserting the cell-culture cups into the interconnected wells (Fig. 1e). In this study, the wells were numbered 1\u0026ndash;4 clockwise starting with the upper-left well (Fig. 1f). The device is used in three steps. First, the cell-culture cups are placed in a 24-well plate (TPP, Trasadingen, Switzerland) and up to four different types of cells are cultured in the individual cups (Fig. 2c, Supplemental movie 1). Once the cells in the cell-culture cups reach maturity, the cell-culture cups are inserted into the interconnected wells (Fig. 2d, Supplemental movie 2). Finally, the plate containing the assembled devices is placed on a 3D shaker for circulating culture (Fig. 2e, Supplemental movie 3). Cell culture insert (Corning, NY, USA) can be optionally used for cell culture model of barrier tissues, e.g. intestine model, in cell-culture cup in interconnected wells if necessary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification of complete medium circulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm that complete medium circulation is achieved within the device, the assembled devices containing phosphate-buffered saline were treated with Gardenia Blue with or without shaking. When the 3D shaker was used, Gardinia Blue was observed in all four wells at only 9 s corresponding to the time for one rotation of the 3D shaker, after the start of 3D shaking (Fig. 3a, Supplemental movie 4), and complete mixing of the medium was confirmed at 60 s (Fig. 3b). In contrast, when the 3D shaker was not used in static test, very little of the Gardinia Blue solution had diffused into the adjacent cells at 60 s (Fig. 3b) or even 600 s (Fig. 3c), even though more the PBS containing the dye was used compared with the circulatory test. This study was conducted with a total medium volume of 2.5 mL during medium circulation using a 3D shaker. When the medium volume was as low as 1.5 mL, circulation is insufficient due to inadequate fluid coverage throughout the device (Supplemental movie 5). At 2.0 mL, medium circulation was inconsistent with successful circulation occurring about once in every three rotations. Based on these observations, we concluded that 2.5 mL of medium was necessary to realize sufficient medium circulation throughout the culture period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplicability of the device to different cell culture models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell culture models can be classified into two principal culture categories: two-dimensional and three-dimensional. We therefore verified whether our device can support both of these cell culture models. We confirmed that the device can be used not only for conventional two-dimensional cultures in the cell-culture cups (Fig. 4b) but also co-culture of cells on culture inserts (Fig. 4a) and Matrigel-embedded spheroid culture (Fig. 4c), demonstrating its wide applicability to couple of culture methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of 3D shaker rotation speed on circulation of immune cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify that cells are able to circulate through the whole of the device, we used human T lymphocyte Jurkat cells, which are a type of circulating immune cell. As in the experiment examining the circulation of the medium, cells were added to Well 1, the setup was shaken by a 3D shaker for 30 min and the number of cells in each well were counted. When the setup was not shaken, no migration of the cells from Well 1 to any of the other wells was observed (Fig. 5a). When the medium was circulated at low speed (12.5 rpm), some cell circulation was observed, but the measurement error was large (Fig. 5b). However, when the medium was circulated at high speed (52 rpm), the cells were uniformly distributed in all four wells (Fig. 5c). These results indicate that circulating the medium at high-speed causes floating cells to circulate through the device. We did not mix the medium prior to sampling by pipetting because the process of pipetting carries a potential risk of cell migration into adjacent wells during the cell sampling procedure. Therefore, we consider that the apparent decrease in total cell numbers in static and low speed culture condition was due to the settlement of seeded cells. At higher rotation speed, cells remain homogeneously suspended, allowing us to sample the consistent number of cells with the number of seeded cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplicability of the device as a two-organ system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we verified the applicability of the device to examine the interactions between liver and cancer by using the anticancer prodrug capecitabine. In humans, capecitabine is metabolized by carboxylesterase in the liver to 5\u0026rsquo;-deoxy-5-fluorocytidine, which is then converted to 5\u0026rsquo;-deoxy-5-fluorouridine by cytidine deaminase primarily in liver and tumor tissue. Then, 5\u0026rsquo;-deoxy-5-fluorouridine is further convert to its active metabolite, 5-fluorouracil, by thymidine phosphorylase, which is present at high levels in tumor tissues where the metabolite exerts its antitumor effects. We therefore examined the effect of capecitabine on a co-culture of hepatocytes (HepaRG) and cancer cells (HCT-116), with reference to a previous study\u003csup\u003e10\u003c/sup\u003e. When HCT-116 cells were cultured alone, capecitabine was observed to have no effects on the viable cell count (Fig. 6a, b). Similarly, no effects were observed when the two cell lines were subjected to static culture in the presence of capecitabine. However, when the HCT-116 cells were subjected to circulatory culture together with HepaRG cells, capecitabine induced a significant reduction in the number of viable HCT-116 cells (Fig. 6c, d). These results indicate that the anticancer effect of capecitabine is only observable under co-culture with HepaRG with medium circulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrent multi-organ MPSs face several critical limitations, such as their complexity due to numerous pumps, limited throughput capability, and operational difficulties during implementation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To address these limitations, we have developed and validated a simplified, multi-throughput multi-organ MPS device. A key innovation of our system lies in its operational simplicity. By eliminating complex pump systems and simplifying assembly, we have developed a user-friendly platform that requires only basic consumables and a 3D shaker. This design directly addresses a fundamental challenge in current multi-organ MPS technology, where system complexity often compromises throughput efficiency. Our platform demonstrates a multi-throughput capacity, accommodating six devices per plate with the potential for multiple plates to be placed simultaneously on a single 3D shaker. Our system also significantly reduces medium consumption compared to conventional pump-based systems, which is crucial for maintaining optimal concentrations of soluble factors. As demonstrated by other groups \u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, even minor variations in soluble factors like cytokines and exosome can substantially impact cellular response patterns. Our device also exhibits remarkable versatility in culture methodology, supporting conventional sheet culture, cell culture inserts, and spheroid formation. This flexibility should allow for cultivation of diverse organ types, from polarized intestinal cells to complex 3D liver cultures, that more closely mimic in vivo conditions\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In addition, our device consists of four wells arranged in a square format (2\u0026times;2 wells). When using a device with four wells arranged in a linear configuration (1\u0026times;4 wells), significant differences in environmental conditions such as flow rate and flow velocity occur between the outer two wells and the inner two wells during circulation. On the contrary, in our square formatted device, flow rates and flow velocities are nearly uniform across all wells, indicating that environmental differences between wells are minimized.\u003c/p\u003e \u003cp\u003eOur present findings emphasize the critical importance of medium circulation when examining the effects of drugs and cellular interactions. Static culture conditions, as observed in traditional systems, can limit drug distribution and potentially lead to inaccurate pharmacological assessments. In contrast, our device enables observation of prodrug responses under conditions that more closely approximate in vivo environments\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, the adjustable rotation speed feature of our 3D shaker\u0026ndash;based system presents a notable advancement, particularly with respect to the analysis of circulating immune cells. Previous multi-organ MPS studies have demonstrated that cytokine concentrations fluctuate markedly depending on immune cell presence\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Although we observed that low-speed circulating culture can result in biased cell distribution between wells, potentially due to cup depth, we hypothesize that gel implementation could resolve this issue by elevating the cell-culture section base. The enhanced prodrug and metabolic activity observed in our multi-organ MPS using a co-culture of liver and cancer cells, aligns with previous research\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Together, these results suggest our device's potential as a valuable tool for drug discovery applications, potentially reducing dependence on animal studies.\u003c/p\u003e \u003cp\u003eIn conclusion, our novel device represents a significant advancement in multi-organ culture technology, offering a simplified yet effective platform that affords appropriate medium circulation. This innovation provides researchers with a robust tool for use in detailed cell\u0026ndash;cell interaction studies and drug\u0026ndash;response assessments. Future development will focus on expanding the range of compatible organ types and establishing standardized protocols for various experimental configurations, further enhancing the devices utility in drug discovery and development.\u003c/p\u003e\n\u003ch3\u003eLimitations of the study\u003c/h3\u003e\n\u003cp\u003eThe cell-culture cups showed a slight, though not statistically significant, difference in viable cell count compared to the wells of a 24-well plate, even without circulation. (Supplemental Fig.\u0026nbsp;1). We attribute this minor variation to the small difference in culture area between the cell-culture cup and the 24-well plate. Furthermore, cell counts were significantly lower in cultures circulated through our device compared to the non-circulating culture group. Since previous reports have suggested that flow velocity affects cell proliferation, it is possible that it influences cell proliferation within our device\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Circulation at slower speeds may eliminate these observed differences in viable cell counts; however, most 3D shakers are not compatible with CO\u003csub\u003e2\u003c/sub\u003e incubators, meaning we were unable to validate shaker speeds slower than 12.5 rpm using the 3D shakers that were available for the present study.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDesign and fabrication of the device\u003c/h2\u003e \u003cp\u003eThe cell-culture cups and culture medium wells were designed in the Autodesk Fusion software Ver.2.0.19440 (Autodesk, Inc., CA, USA). Both components were fabricated from polystyrene by injection molding. To facilitate cell attachment and reduce the effect of surface tension along the flow path, the components were oxidized at 100 W for 80 s (40s \u0026times; 2) (cell-culture cups) or 60 s (interconnected wells) using an oxygen plasma reactor (PR500; Yamato Scientific Co., Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCulture of spheroids\u003c/h2\u003e \u003cp\u003eSpheroids of human colon cancer HCT-116 cells (REKEN BRC, Ibaraki, Japan) were cultured in 10% fetal bovine serum (FBS) (Cytiva, Tokyo, Japan)-Dulbecco's Modified Eagle Medium (DMEM) (041-29775, FUJIFILM Wako Pure Chemical, Osaka, Japan) using TASCL-600 3D culture microplates (Cymss-bio Co., Ltd, Nagoya, Japan). The spheroids were embedded in Matrigel (Corning, NY, USA) for avoiding spheroid migration. Microscopy was performed after placing the cell-culture cups into a 24 well TPP Tissue Culture Test Plate (TPP, Trasadingen, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eVerification of complete culture medium circulation\u003c/h2\u003e \u003cp\u003eTo confirm that the device setup on the 3D shaker resulted in complete circulation of the culture medium, the interconnected wells were placed in 24-well plates and 2.3 mL (circulatory test) or 2.8 mL (static test) phosphate-buffered saline was added to each well. The devices were then placed on the table of a 3D shaker (Multi-bio 3D; BS-010125-AAK, Kenis, Osaka, Japan) at a 7-degree angle of inclination with Well 1 at the lowest position (circulatory test) or in the horizontal plane (static test). Well 1 was treated with 0.2 mL phosphate-buffered saline containing Gardenia Blue (FUJIFILM Wako Pure Chemical, Osaka, Japan) and the rotation of the 3D shaker was started at a speed of 5 rpm for the circulatory test. The 3D shaker (Multi-bio 3D; BS-010125-AAK) was exclusively used for this experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVerification of complete cell circulation\u003c/h2\u003e \u003cp\u003eTo confirm that the device setup on the 3D shaker resulted in complete circulation of cells within the interconnected wells, the wells were each filled with 2.0 mL of 10%FBS-Roswell Park Memorial Institute 1640 medium (FUJIFILM Wako Pure Chemical, Osaka, Japan). Then, 0.5 mL of 5.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL human T lymphocyte Jurkat cells were added to Well 1 and allowed to settle for 10 min (final concentration of Jurkat cells was 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL). The device was then shaken with a 3D shaker (KFS-410; Kenis, Osaka, Japan) at a 7-degree angle of inclination for 30 min. The 3D shaker was stopped at the point when the cell suspension was most concentrated in Well 3 (i.e., Well 3 was at the lowest position on the 3D shaker), and 100 \u0026micro;L of cell suspension was collected from each well and the cells were counted with a TC-20 automated cell counter (Bio-Rad, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of using the device for cell monoculture\u003c/h2\u003e \u003cp\u003eHCT-116 cells were seeded in 24-well plates containing cell-culture cups with 0.5 mL of 10% FBS-DMEM medium at a density of 1.0 \u0026times; 10⁴ cells/mL and incubated in a CO\u003csub\u003e2\u003c/sub\u003e incubator. After 1 day, the cell culture-cups were placed in interconnected wells with each well containing 2.5 mL HepaRG Thawing/Plating/General medium (KAC Co., Ltd, Kyoto, Japan), again in 24-well plates. Once assembled, the plates were placed on a 3D shaker at a 7-degree angle of inclination and shaken at 12.5 rpm in the CO\u003csub\u003e2\u003c/sub\u003e incubator. After 3 days, viable cells were counted by using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of cell\u0026ndash;cell interactions in the presence of capecitabin\u003c/h2\u003e \u003cp\u003eThis experiment was conducted as previously reported\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Immortalized human HepaRG hepatocytes (KAC Co., Ltd, Kyoto, Japan) were seeded at 9.6 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL (Day 0) in cell-culture cups 1 and 2 using 0.5 mL of HepaRG Thawing/Plating/General medium and were replaced with 0.5 mL of HepaRG metabolic/maintenance medium (KAC Co., Ltd, Kyoto, Japan) (Day 1). The medium was changed every two days. On Day 6, HCT-116 cells were cultured in 0.5 mL of 10%FBS-DMEM and were seeded at 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL in cell-culture cups 3 and 4. On Day 7, the cell-culture cups were transferred to interconnected wells containing 2.5 mL HepaRG Thawing/Plating/General medium with or without 100 \u0026micro;M of the anticancer prodrug capecitabin (TCI, Tokyo, Japan) and shaken on a 3D shaker at a 7-degree angle of inclination and shaken at 12.5 rpm in the CO\u003csub\u003e2\u003c/sub\u003e incubator. On Day 10, viable cells were counted by using a Cell Counting Kit-8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. The Tukey\u0026ndash;Kramer test was used to determine whether differences between groups were statistically significant. The statistical analyses were performed using the GraphPad Prism 10 software (GraphPad Software, Inc., CA, USA). Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank ELSS (https://www.elss.co.jp) for the English language review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author responsibilities were as follows: R.Y. and S.S: designed the research; R.Y.: analyzed the data; R.Y. and S.S. drafted the manuscript; and all authors: conducted the research, and read, revised, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that financial support was received for the research, authorship, and publication of this article. 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M. \u0026amp; Mondal, P. P. Fluorescence-based multifunctional light sheet imaging flow cytometry for high-throughput optical interrogation of live cells. \u003cem\u003eCommunications Physics\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 25 (2024). https://doi.org/10.1038/s42005-024-01522-y\u003c/li\u003e\n\u003cli\u003eZhan, C.\u003cem\u003e et al.\u003c/em\u003e Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells. \u003cem\u003eiScience\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 101653 (2020). https://doi.org/https://doi.org/10.1016/j.isci.2020.101653\u003c/li\u003e\n\u003cli\u003eIlvesroiha, E.\u003cem\u003e et al.\u003c/em\u003e Establishing a simple perfusion cell culture system for light-activated liposomes. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2050 (2023). https://doi.org/10.1038/s41598-023-29215-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multi-organ microphysiological system, 3D shaker, cell circulation, organ-organ interaction","lastPublishedDoi":"10.21203/rs.3.rs-6713236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6713236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMulti-organ microphysiological systems (multi-organ MPSs) are \u003cem\u003ein vitro\u003c/em\u003e platforms that simulate interactions between multiple organs in the body. However, most of existing multi-organ MPSs often suffer from high complexity and low-throughput due to their reliance on pumps for medium circulation. We have developed a simple multi-organ MPS device, namely interconnected multi-well device, that utilizes a three-dimensional shaker for medium circulation, eliminating the need for pumps. The device consists of two components: a set of four connected cell-culture cups and a series of four interconnected wells. Up to six devices can fit in a standard 24-well plate. The device supports various culture methodologies, including conventional two-dimensional culture and spheroid culture, and can accommodate cell culture inserts. Here, we demonstrate medium and immune cell circulation through the entirety of the device. Then, as a representative use-case scenario, we demonstrate using the device to evaluate the anticancer effects of the prodrug capecitabine, whose metabolite exhibit anticancer effect, in a two-organ system composed of liver and cancer. In short, our interconnected multi-well device is user-friendly, adaptable to various culture methods, and multi-throughput, and it shows promise for becoming a valuable tool for \u003cem\u003ein vitro\u003c/em\u003e organ interaction research.\u003c/p\u003e","manuscriptTitle":"Interconnected multi-well device with three-dimensional shaker–driven culture medium circulation for use as a multi-organ microphysiological system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 09:16:25","doi":"10.21203/rs.3.rs-6713236/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"01a46896-472d-423b-baed-547d3274b8e0","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49891369,"name":"Biological sciences/Biotechnology/Assay systems"},{"id":49891370,"name":"Biological sciences/Biological techniques/Biophysical methods"},{"id":49891371,"name":"Biological sciences/Biological techniques/Cytological techniques"},{"id":49891372,"name":"Biological sciences/Biological techniques/Lab on a chip"}],"tags":[],"updatedAt":"2025-07-03T09:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-13 09:16:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6713236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6713236","identity":"rs-6713236","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}