STARTER : A Stand-Alone Reconfigurable and Translational OoC Platform based on Modularity and Open Design Principles

showed that transport in both setups was again comparable, with low transport of atenolol across the intes Ɵnal barrier, confirming intact Ɵght juncƟons. Notably, transport in both setups was significantly lower than that in control IEBCs without Ɵssue included. We, therefore, showcase comparable performances of established IEBCs on the plaƞorm, which features a much more compact footprint (Figure S5) and offers possibiliƟes for future sensor integraƟon and customizaƟon. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint 4 Conclusion and Outlook In this work we introduce STARTER, a stand-alone, modular reconfigurable plaƞorm based on TOP and demonstrate its versaƟlity for designing and carrying out OoC experiments. STARTER accommodates both Ɵssue culture insert-based and microfluidic channel-based OoC models, agnosƟc to the substrate material. Pumps, reservoirs and sensors are all inte grated within an ANSI/SLAS microplate footprint enabling dynamic monito ring of automated mul Ɵ-OoC experiments in a compact portable package. The fluidic circuits can be customized as per requirements, thus o ffering experimental freedom with the integrated modules. Demonstra Ɵons of mixing and metering, pump characteriza Ɵon and sensor characterizaƟon highlight the technical capabili Ɵes of our integrated system. To highlight the applicability of STARTER's versa Ɵlity in OoC experiments, both in-vitro and ex-vivo studies were Figure 4: a) i) SchemaƟc of the configuraƟon for the in-vitro experiments. ii) Fluorescence Microscopy images of VoC channels for 3 condiƟons. iii) pH measurement during the in-vitro experiment. Iv) Cell Number and cell coverage analysis comparing control, STARTER with two types of reservoirs. b) i) SchemaƟc of the configuraƟon for the ex-vivo experiments. This loop was repeated three Ɵmes on STARTER. ii) LDH secreƟon comparison between IEBC on STARTER and IEBC controls. iii) Atenolol transport in apical and basolateral sides. *(P<0.05), **(p<0.005) .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint performed. As an exemplar in-vitro experiment, HUVECs were cultured over the course of 3 days in a vessel-on-chip model with conƟnuous pH monitoring. The results confi rm similar cell numbers and coverage to controls while revealing di fference between 3D-printed and commercially available reservoirs. Furthermore, ex-vivo experiments were conducted in previously reported OoCs on STARTER over 24 hours. Cell viability and barrier func Ɵon of a porcine gut explant was assessed and shown to be comparable to controls in the tradiƟonal setup. These results showcase the advantage of STARTER in reducing experimental footprint, adding func Ɵonality and versa Ɵle integra Ɵon without compromising on biological performance. The design of STARTER is complaint with ISO 22916 and specifically the TOP Design rules (TDRs) which is a specific implementaƟon of ISO complaint footprints and port layouts. A wide variety of modules become eligible for integra Ɵon as long as the port loca Ɵons and footprints adhere to these standardized design guidelines. In this work, we showcase a few possible module combinaƟons along with a general strategy for the implementa Ɵon of the standardized designs in modular micro fluidic systems. The list of components used in this work as well as design files are all made freely available in an open source environment - GitHub - TOP-OoC/Starter-Kit. These openly accessible resources will serve as a ‘STARTER kit‘ for easier adopƟon and implementaƟon by developers at this nascent stage of standardizing modules. AddiƟonal resources such as ISO explainer documents, TDR guidelines and an automated fluidic rouƟng tool (MMFT Rou Ɵng Block Channel Router ) are also available. The fluidic rouƟng tool is developed by collaborators in Technical University of Munich (TUM) with renowned experience in design automaƟon[34] and is especially useful in designing rouƟng blocks for customized applicaƟons. These resources aim to lower the threshold of adop Ɵon of standardized designs, parƟcularly in the field of OoC. The architecture of STARTER can serve as a foundaƟon for generic plaƞorms aiming to add perfusion to exisƟng modules in a portable footprint. The ability to have a standalone and reuseable pla ƞorm makes it truly translaƟonal. However, implemenƟng STARTER in external lab seƫngs is a key next step in validaƟng the translatability, design choices, interfacing and robustness of the plaƞorm. A natural progression for wider adop Ɵon would involve simplifying the mechanical connec Ɵons to improve useability. This will parƟcularly benefit Ɵme constraining and space restricted workflows. Future work on reducing electrical connectors is also of vital importance. As the number of integrated sensors increase in a compact form factor, the web of cables running to these sensors can cause conges Ɵon and hamper useability. A method to further integr ate the electronics and sensor read-out on the plaƞorm will enhance ease-of-use while bene fiƟng from an already matured electronics industry. IntegraƟon of valves in the rou Ɵng block would enable ac Ɵve re-programmability of the pla ƞorm compared to the current method of replacing the block itself. This will allow further possibili Ɵes of mulƟplexing, pumping and fluidic operaƟons on the plaƞorm. DisseminaƟon of the bene fits of standardized designs also becomes vital to ignite adop Ɵon by component developers. The ISO 22916 standard could harmonize interna Ɵonal academic and commercial enƟƟes, iniƟaƟng an interoperable market. The benefits of the ISO standard is not limited to module developers alone, the growing ecosystem of complaint components can be used by system d esigners to realize custom plaƞorms for their specific applicaƟons. In a wider perspec Ɵve, a generic open-source pla ƞorm tackles fundamental obstacles in the industrial adop Ɵon of MPS systems, as emphasized by various global commiƩees.[35] Standardized design principles enable the development of tailored OoC models and plaƞorms with integrated automaƟon, facilitaƟng rapid iteraƟon of more novel and complex models. An OoC model developed for a standardized plaƞorm such as STARTER has the potenƟal to be adapted for high-throughput systems that follow the same standards in the future [36]. This transla Ɵonal capabili Ɵes not only strengthens collabora Ɵon but also supports con Ɵnuous .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint improvements informed by user feedback and preferences. This approach can bridge the gap between academia and the pharmaceu Ɵcal industry by first valida Ɵng experimental models on generic plaƞorms, then enabling their scaled automa Ɵon once validated. Therefore, embracing an open- source approach fosters collabora Ɵve development of solu Ɵons aimed at enhancing robustness, reproducibility, and technical maturity, ulƟmately facilitaƟng future regulatory approval processes. In conclusion, we introduce a novel generic plaƞorm designed for stand-alone mulƟ-OoC experiments, with in-line sensing and fluidic recon figurability capabili Ɵes. The versa Ɵlity of the architecture combined with a reversible and material agnos Ɵc integraƟon strategy makes STARTER suitable for a wide range of applica Ɵons. To the best of our knowledge, this represents the first applica Ɵon of standardized designs on a universal pla ƞorm capable of accommoda Ɵng diverse OoC models from mulƟple suppliers. The applicaƟons extend beyond OoC experiments demonstrated in this work and can include module tes Ɵng, quality control, benchmarking, integra Ɵon tests, and automated microfluidics, all within a portable form factor . The open access disseminaƟon of resources will foster broader collaboraƟon and contribu Ɵon of new designs from the community. Eventually, we expect that development, tesƟng and implementaƟon of new OoC applica Ɵons will be strongly accelerated, both in the se ƫng of early R&D and in commercial product development. The paradigm of open- source design represents a signi ficant breakthrough in e fficiency and innova Ɵon by crea Ɵng a precompeƟƟve domain in a field that has been converging towards ‘point solu Ɵons’ and proprietary plaƞo rms. The boom in development, valida Ɵon and tesƟng of OoCs will, in-turn stride towards the ulƟmate objecƟve of wider adopƟon of micro-physiological systems. 5 Materials and Methods 5.1 Assembly The MFFBs and clamps were connected to the FCB using M2 nuts and bolts. 40° shore-A silicone O- rings (Gteek) with dimensions 1.02 mm × 0.74 mm were recessed at the locaƟons of the fluidic ports. The O-rings were manually placed into the recesses in the FCB before connec Ɵng the MFBBs with screws onto the FCB. The compression of the so Ō O-rings with the screws enabled leak-free integraƟon. Sufficient O-ring compression was veri fied for O-ring pocket depths varying from 0.8 µm to 0.9 µm and a pocket diameter of 1.35 mm. 5.2 FCB, MFBBs and Clamps The OoCs were placed inside the clamp with ports facing toward the FCB. The clamp was then screwed onto the FCB similar to other MFBBs The FCB, MFBBs and clamps were designed in SolidWorks© (2022). The FCB was made of COC and manufactured by Micronit B.V. (The Netherlands). The clamps, and auxiliary MFBB parts were made by micromilling (Datron Neo, Germany) PMMA. The rouƟng block was micromilled similarly and closed off with medical grade double sided pressure sensiƟve adhesive tape (PSA, ARcare 90445Q). The bases for the pump block, sensor blocks and commercial reservoirs were manufactured similarly out of PMMA. It is also possible to fabricate the FCB in-house by micromilling PMMA and bonding with PSA tape as described above. All designs are shown in SI. 5.3 Organ-on-Chips PDMS (Sylgard 184 elastomer kit) was mixed (10:1 base polymer to curing agent (w/w)) and then degassed for 1 h. Degassed PDMS was then cast into a micromilled double sided PMMA mold and .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint degassed again for 1h. The basepl ate provides the features for the fluidic pathing as well as cu ƫng lines for 6 devices, while the top controls chip thickness and levelness. The degassed mold was placed at 60 °C for at least 3 h. The mold was then disassembled, and the individual microfluidic devices were cut out according to the cu ƫng guides, resul Ɵng in uniform 30×30 mm PDMS slabs. Subsequently, inlets and outlets were punched out using 1 mm biopsy punch (Ted Pella, Inc., USA). A clamp orients a single PDMS slab while a second micromilled punching guide was aligned to the PDMS using M2 bolts as guide pins. Inlet and outlet locaƟons were punched according to the TDRs and the PDMS slabs were plasma bonded to 24x24 mm coverslips (VWR). The final devices were placed at 60 °C for at least 1 h. 5.4 Pump Block This module uses six stepper-motor driven peristal Ɵc pumps, manufactured by Takasago Fluidic Systems. The stepper motors have a rated pump rate of 0.18 -180 µL/min. A custom pump driver PCB was used which was controlled by an Arduino Nano 33 IoT. Six unpopulated spaces for through-hole resistors were leŌ in the PCB for pump-specific feedback resistors to be added. These resistors control the current output by the driver and must be added during or a Ōer manufacturing. For the pumps listed here, a resistor value of 56 kOhm was used. A custom firmware for the pump driver PCB allowed individual control of the pumps via BLE. The pumps were a Ʃached to a micromilled PMMA base plate that connected the array of pumps to the FCB. The PCB was connected to the Pump Block Base with M2 PCB stando ffs. The PCB was conformal coated with epoxy before use in high humidity se ƫngs like incubators. All design files are available in the previously menƟoned Github page. 5.5 Reservoirs The reservoirs were either 3D printed or converted using commercially available components (4.5mL InteracƟon Tanks Micro fluidic ChipShop). For the 3D printed reservoirs, designs were created in SolidWorks© 2022 and sliced in PreForm slicing soŌware; Formlabs BioMed Clear v1.0 resin was used i n a F o r m L a b s 3 B + p r i n t e r . T h e p r i n t e d r e s e r v o i r s w e r e t h e n w a s h e d i n 1 0 0 % I P A u n d e r a g i t aƟon (FormLab Form Wash) followed by a 2-hour UV cure (Form Cure) and a final overnight bake at 80 °C. The boƩom of the reservoirs (side interfacing with the FCB) was sanded to obtain a flat surface both in terms of roughness and curvature. In case of the commercial reservoirs, a 30mm x 15mm base plate with required ports were milled onto which the reservoirs were aƩached by a press fit. 5.6 Flow Sensor measurements The flow sensor used was the LPG10-1000 (Sensirion AG) and logged in the Sensirion Viewer soŌware. The data was logged as .csv files and ploƩed later using OriginPro (2024). The flow r ate was measured at different pump frequencies for a dura Ɵon of 5 min for each data point. The average flow rate was then taken over the 5 min duraƟon. 5.7 pH measurements The pH sensor spot used was a commercial product (PHSP5-PK6) from PyroScience GmBH and the measurements were conducted using SPFIB-BARE op Ɵcal fibers connected to a FiresƟng Pro. Prior to the measurements, a 2-point calibra Ɵon was performed as suggested by the supplier using the recommended pH 2 (PHCAL2) and pH 11 (PHCAL11) calibra Ɵon capsules. The calibraƟon was done in ambient condiƟons under a flow rate of 15 µL/min. For the pH measurements, bu ffer soluƟons of pH levels ranging from 6.0-8.0 were prepared in posphate bu ffered saline (PBS) using 0.1 M NaOH and flowed through the plaƞorm. Each buffer soluƟon was allowed to recirculate for 30 minutes followed .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint by a dry air run of 10 min to ensure complete removal of the previous soluƟon prior to introducing the next soluƟon. 5.8 Vessel-on-Chip Cell Seeding Green fluorescent protein-tagged human umbilical vein endothelial cells (GFP-HUVECs, ZHC-2402, Cellworks) were cultured as suggested by the supplier. In short, cells were expanded on 0.1 mg/ml collagen-1 (rat tail collagen-1, Gibco) coated T75 flasks in endothelial cell growth medium-2 (ECGM-2, PromoCell) supplemented with penicillin-streptomycin (50 U/mL, Gibco) at 37 °C in humidified air with 5% CO 2. To facilitate cell adhesion in the Vessel-on-Chip (VoC) channels of the PDMS chip, PDMS surface was funcƟonalized with 2 mg/mL dopamine (Sigma-Aldrich) in 10 mM Tris-HCl buffer (pH 8.5) for 1 hour at room temperature (RT), followed by 3 washes with sterile filtered deionized water and finalized with a 0.1 mg/mL collagen-1 coa Ɵng for 30 minutes at 37 °C. A Ōerwards, the channels were w a sh e d w i t h E C G M - 2 t o r e m o v e n o n - b ou n d c ol l a g en . H U VE C s w e r e th e n o b t a i n e d f r o m c o nfluent flasks using 0.05% trypsin-EDTA (Gibco), seeded at 2·10 6 cells/mL and incubated for 1 hour at 37 °C followed by a wash of fresh medium to remove non-adhered cells. To enable full a Ʃachment of the cells to the VoC channel walls, the chips were kept sta Ɵc in 37 °C for 4 hours. A Ōerwards, VoCs were transferred onto a rocking pla ƞorm providing bi-direcƟ onal flow to ensure frequent medium refreshment (OrganoFlow, Mimetas, set at 10°, 1-hour intervals). Medium was refreshed twice per day and cells were allowed to form a monolayer prior to start of the experiment on STARTER. 5.9 Vessel-on-chip Analysis The number of cells was monitored over the course of the 3-day experiment and compared between the VoCs connected to Starter with either commercial or 3D-printed reservoirs, as well as VoCs kept with bi-direcƟonal flow on a rocking plaƞorm in plain ECGM-2 or supplemented with pro-inflammatory cytokine TNF- α ( 2 n g / m l ) a s p o s iƟve and nega Ɵve control respec Ɵvely. For this, the GFP-tagged HUVECs were imaged daily starƟng directly aŌer connecƟng the VoCs to Starter using the EVOS M5000 Imaging System. Cell numbers were determined in CellProfiler (version 4.2.8), in which individual cells were segmented using three-class Otsu thresholding method based on the GFP-intensity images. VoC channels were excluded from analysis if the amount of cells at the start of the experiment was less than 75% of the ECGM-2 condiƟons, or if technical faults resulted in sudden loss of cells. AddiƟonally, cell morphology was assessed using immunostaining of endothelial marker vascular endothelial-cadherin (VE-cadherin), cytoskeleton and nuclei. Directly a Ōer comple Ɵon of the experiment, VoCs were removed from the Starterkit and HUVECs were fixed in 4% paraformaldehyde in PBS containing Ca 2+ and Mg2+ for 10 minutes at RT. AŌerwards, cells were permeabilized and blocked in permeabiliza Ɵon and blocking bu ffer (PBB) containing 0.1% Triton X-100 (Sigma-Aldrich) and 10 mg/mL bovine albumin serum (BSA, Sigma-Aldrich) in PBS for 60 minutes at RT. AŌerwards, HUVECs were incubated with 5 µg/mL goat an Ɵ-human VE-cadherin (R&D systems) in PBB overnight at 4 °C. Extensive washing was performed to remove primary an Ɵbodies using 3 rinses and 3 10-minute incubaƟons with PBS. AŌerwards, HUVECs were incubated with 10 µg/mL donkey anƟ-goat Alexa Fluor 546, 4 droplets/mL Ac ƟnGreen (ThermoFischer Scien Ɵfic) and 12.5 µg/mL 4’,6-diamidino-2- phenylindole (DAPI) in PBB for 4 hours at RT. A Ōer another set of extensive washing, samples were imaged using the EVOS FL Auto 2 Imaging System. VE-cadherin paƩern was used as input in CellProfiler to determine the percentage coverage of the HUVECs in the VoC channel. Percentage cell coverage was determined in CellProfiler, in which the total area of the sum of the individual cells was determined with respect to the channel area. For this, cells were segmented first using three-class Otsu thresholding of the DAPI images, followed by two-class Otsu thresholding of the VE-cadherin images. .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint 5.10 IEBC Ɵssue culture The STARTER plaƞorm and Explant Barrier Chips were prepared one day prior to an experiment. AŌer assembling STARTER, reservoirs were filled and the system was subsequently flushed with 20% biofilm (UmweltanalyƟk) and aŌerwards flushed with PBS. Next, Williams E supplemented with 1% BSA was added for overnight incubaƟon in a humidified incubator at 37 °C with 5% CO2. Flow rates of 33 µL/min were used for overnight incubaƟon and flushing, respecƟvely. The next day, systems were transferred to a working bench. Porcine intesƟnal colon Ɵssue from a healthy adult pig was obtained from a local slaughterhouse. No ethical approval was needed for the collecƟon of intesƟnal Ɵssue from these animals as the Ɵssue was redundant to the slaughter procedure. Procedures for handling and processing the Ɵssue was according to previously published methods [28,37]. In brief, intesƟnal Ɵssue was collected within 15 minutes upon death of the animal and immediately flushed with ice cold supplemented Williams E buffer to remove fecal content. During transportaƟon and preparaƟon in the lab, the Ɵssue was placed in ice cold supplemented Williams E buffer. At the laboratory, fat Ɵssue and the musculo- serosal layer of the mucosal layer was dissected off and round segments of 11.1 mm in diameter (area of 0.968 cm2) were punched. MounƟng of the segments into the IEBC occurred within 4 hours aŌer intesƟnal Ɵssue collecƟon. All experiments were performed in compliance with Dutch legislaƟon on the use of redundant human (AVG, WMO) and slaughterhouse porcine Ɵssue, and insƟtuƟonal guidelines on handling human and animal Ɵssue regarding safety and security. 1 mm thick EPDM rubber rings (Eriks), intesƟnal Ɵssue segments (mucosal side upwards) on a woven mesh of 170 μm in thickness and 50% open area (Nitex, Sefar) and a fixing insert were clicked in the snap fit mechanism, thereby separaƟng the apical and basolateral compartments of the microfluidic chip. Subsequently, the Williams E supplemented with 1% BSA was replaced by the apical and basolateral media: Williams E supplemented with FD4 (50μM) and atenolol (10 μM) and Williams E supplemented with 4% BSA, respecƟvely. ThereaŌer, the system was placed back in the incubator and perfused at 33 µL/min. Apical and basolateral samples were collected from the medium reservoirs at previously menƟoned Ɵme points. At the end of the experiment, Ɵssue segments were flushed with warm PBS and removed from the Explant Barrier Chips and collected for subsequent analyses. The whole STARTER plaƞorm, tubings, chips and reservoirs were flushed and washed with first with 20% biofilm and then with 70% ethanol. 5.11 IEBC Analysis [3H]Atenolol was applied as reference marker for paracellular transport and mixed with non- radiolabeled atenolol, to obtain final nominal concentraƟons of 10 μM in the apical soluƟon with an associated radioacƟvity of 10 kBq mL−1. Transport was measured by taking apical (100 μL) and basolateral (500 μL) samples at indicated Ɵmepoints. RadioacƟve labelled compounds were measured using the Tri-Carb 3100TR Liquid ScinƟllaƟon counter (LSC, Perkin Elmer, Boston MassachuseƩs, United States) aŌer adding scinƟllaƟon liquid (UlƟma Gold, Perkin Elmer Inc., Boston, MassachuseƩs, United States) to the apical and basolateral samples. To assess the viability of the ex vivo intesƟnal segments, the cytosolic enzyme lactate dehydrogenase (LDH) was measured in the apical and basolateral supernatants of the two-compartmental model using an LDH kit (Sigma-Aldrich). Intracellular LDH levels in control Ɵssue segments collected before incubaƟons were measured with the same kit, aŌer homogenizing the Ɵssue segments in ice-cold Williams E buffer using a PoƩer–Elvehjem type Teflon pestle Ɵssue grinder (Braun) for 5 min at 200 rpm. Excreted LDH levels were expressed as percentage leakage of the total intracellular LDH of .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint these blanc intesƟnal Ɵssue segments. Samples were analyzed using the BioTek Synergy HT microplate reader (BioTek Instruments Inc., Winooski, VT) with an excitaƟon/emission wavelength of 490 nm and 520 nm. 5.12 StaƟsƟcs Data are provided as the mean ± standard deviaƟon or standard error of the mean. Differences between 2 groups were analyzed using 2-tailed Student's t test; 1-way ANOVA with Tukey's or DunneƩ's post hoc analysis was used for comparisons of mulƟple groups. StaƟsƟcal significance was considered at p < 0.05, and calculaƟons and graphs were generated using GraphPad Prism 8.0 (GraphPad SoŌware Inc.) and Origin Pro (2024). Author ContribuƟons A.P . and E.R.S contributed equally to this work. A.P ., E.R.S., J.L.Z., M.O., and A.D.v.d.M. conceptualized the study. A.P . and E.R.S., designed and tested the plaƞorm. L.E.d.H conducted in-vitro experiments and analyzed the data. B.d.W. and H.E.A designed IEBC for the plaƞorm. E.v.d.S. and K.W. conducted ex-vivo experiments and analyzed data. A.D.v.d.M., M.O., J.L.Z., and A.R.V supervised the research. Conflict of Interest The authors declare no conflict of interest.

This work was supported by the NWO-TTW PerspecƟve Programme of the Dutch Research Council (NWO, project number P19-03) as part of the SMART Organ-on-Chip consorƟum. We acknowledge Henk Waayer for designing the pump block PCB and Sandro Meucci for assistance in fabricaƟng the FCBs at Micronit B.V. (The Netherlands). .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 17, 2025. ; https://doi.org/10.1101/2025.05.13.653800doi: bioRxiv preprint 6 References 1 Huh, D., MaƩhews, B. D., Mammoto, A., Montoya-Zavala, M., Hsin, H. Y ., & Ingber, D. E. (2010). ReconsƟtuƟng Organ-Level lung funcƟons on a chip. Science, 328(5986), 1662–1668. hƩps://doi.org/10.1126/science.1188302 2 Huh, D., Hamilton, G. A., & Ingber, D. E. (2011). From 3d cell culture to organs-on-chips. 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