This research was funded in part by the National Institute of Health under award numbers R43GM137651, R61HL154249, R16GM146687, and NSF grant CBET 2150798. The authors thank the RIT Machine Shop for aluminum mold fabrication. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This design can be used in various modes based on experimental requirements and the preferences of the end user. Prior to each experiment, consult the decision flow chart presented in Figure 2 to determine the necessary steps and modules for the protocol. For example, if the user intends to maintain the open-well format throughout an experiment to directly compare it with the Transwell-type system, the patterning stencil is not required for cell seeding. The core module is commercially available (see Table of Materials), and the ultrathin nanomembrane can be selected from a library of materials with different porosity and pore sizes to suit experimental needs.
1. Fabrication of the patterning stencil
NOTE: The patterning stencil serves to position cells exclusively on the porous region of the membrane chip, preventing cells from settling onto the surrounding silicon layer where they could potentially experience damage after the flow module is added16 (refer to Figure 3). Damage to the monolayer can adversely affect barrier integrity and compromise experimental outcomes. The stencil is unnecessary in an open, static culture, as there is no risk of damage.
<ol>
	<li>Purchase, machine, or 3D print a mold using the design provided in Supplementary Coding File 1 (Figure 4A). 
	NOTE: The stencil walls are tapered to increase media capacity and minimize bubble trapping compared to high aspect ratio straight-walled features.</li>
	<li>Attach a 130 &#181;m pressure-sensitive adhesive (PSA) film to a 3.2 mm acrylic sheet using a cold roller (see Table of Materials).</li>
	<li>Laser cut the acrylic sheet according to the design provided in Supplementary Coding File 2 to create an array of cavities (Figure 4B).</li>
	<li>Peel off the protective layer from the PSA film and attach the acrylic sheet to the mold. 
	NOTE: Ensure that the laser-cut sheet is aligned with the mold so that the mold features are centered within the cavity (Figure 4C). The accuracy of cell positioning on the membrane depends on this step.</li>
	<li>Thoroughly mix the PDMS prepolymer (using a 10:1 base to catalyst ratio) (see Table of Materials) and degas it in a vacuum chamber until no visible bubbles remain (5-15 min).</li>
	<li>Slowly pour the PDMS into the mold cavities, leveling it with a flat edge. 
	NOTE: To prevent bubble entrapment, pour the PDMS into each cavity slowly. If bubbles are present after pouring, place the mold in the vacuum chamber and degas it again.</li>
	<li>Cure the PDMS on a hotplate at 70 &#176;C for 1 h, and then allow it to cool to room temperature.</li>
	<li>Extract the stencils from the mold cavities using flat-tipped tweezers.</li>
</ol>
2. Fabrication of the flow module
NOTE: The flow module shares a similar footprint with the clover-shaped well of the core module and includes a molded microchannel (width = 1.5 mm, height = 0.2 mm, length = 5 mm). The clover shape aids in aligning the channel over the porous culture region (Figure 5).
<ol>
	<li>Use standard soft lithography techniques18 to create a silicon mold with features defined by the design provided in Supplementary Coding File 3 (Figure 4D).</li>
	<li>Attach a 130 &#181;m PSA film to a 3.2 mm thick acrylic sheet.</li>
	<li>Laser cut the acrylic sheet based on the design given in Supplementary Coding File 4 to create an array of clover-shaped cavities (Figure 4E). 
	NOTE: The height of the cavity determines the height of the flow module, which must be slightly taller (by 0-0.1 mm) than the height of the core module well to ensure proper sealing.</li>
	<li>Remove the protective layer from the PSA film and then attach the laser-cut sheet to the silicon mold by aligning the triangular alignment marks on the silicon mold with those on the laser-cut sheet. 
	NOTE: Similar to step 1.4, ensure that the laser-cut acrylic sheet is aligned with the silicon mold so that the mold features are centered within each cavity (Figure 4F). This step determines the position of the microchannel relative to the footprint.</li>
	<li>Slowly pour the degassed PDMS onto the mold cavities and level it with a flat edge. 
	NOTE: If bubbles are present after pouring, degas the mold until the bubbles are removed.</li>
	<li>Place the mold on a hotplate and bake the PDMS at 70 &#176;C for 1 h, then allow the mold to cool to room temperature.</li>
	<li>Carefully remove the flow modules from the mold cavities using flat-tipped tweezers.</li>
	<li>Orient the flow module with the microchannel features facing upward, and position the core inlet and outlet ports at the end of the channel using a biopsy punch (see Table of Materials).</li>
</ol>
3. Fabrication of lower and upper acrylic housings
NOTE: The core module fits into the lower housing. The attraction between embedded magnets in the housings compresses and seals the flow module to the core module (Figure 6).
<ol>
	<li>Laser-cut a 2 mm acrylic sheet using the design provided in Supplementary Coding File 5 to create the upper housing with holes for magnet insertion.</li>
	<li>Attach a 130 &#181;m PSA film to a 3.5 mm acrylic sheet.</li>
	<li>Laser-cut the acrylic sheet based on the design given in Supplementary Coding File 6 to create the lower housing with holes for magnet insertion. 
	NOTE: The thickness of the lower housing is a critical parameter and must match the overall thickness of the core module to prevent leakage during flow.</li>
	<li>Remove the PSA protective layer and attach the lower housing to a glass coverslip.</li>
	<li>Press-fit 4.75 mm nickel-plated neodymium magnets (see Table of Materials) into the laser-cut holes of the housings. 
	NOTE: Ensure that magnets in the lower and upper housings are placed with opposite polarity to ensure magnetic attraction (Figure 6).</li>
</ol>
4. Fabrication of the flow circuit
NOTE: The closed-loop flow circuit contains two sample collection vials as reservoirs (Figure 7). The inlet reservoir has a polyvinylidene difluoride (PVDF) filter to allow the cell media to equilibrate with the CO2 concentration in the incubator.
<ol>
	<li>Use a 1 mm biopsy punch to create two holes in the cap of a sample collection vial.</li>
	<li>Use biopsy punches to create two holes (1 mm and 4 mm) in the cap of a separate sample collection vial, and create a 1 mm hole in the lower part of this vial.</li>
	<li>Insert 20 G dispensing tips into each of the 1 mm holes and seal them with hot glue.</li>
	<li>Place a 0.22 &#181;m PVDF filter (see Table of Materials) in the 4 mm hole and seal it with hot glue.</li>
	<li>Laser-cut a 1.5 mm acrylic sheet using the design provided in Supplementary Coding File 7 to create a sample holding stage.</li>
	<li>Position the reservoirs on the acrylic stage.</li>
	<li>Connect the dispensing tips using micro flow tubes (see Table of Materials).</li>
	<li>Link the tubes to the inlet and outlet of the flow module using 21 G dispensing tips.</li>
	<li>Utilize a peristaltic pump (see Table of Materials) for flow circulation. 
	&#8203;NOTE: Syringe or pneumatic pumps can also be used to introduce flow if desired. The flow rate should be determined based on experimental needs. A comprehensive table, derived from an experimentally validated COMSOL model, is provided in Supplementary Table 1 to assist users in selecting the necessary flow rate to achieve the desired shear stress at the cell monolayer16.</li>
</ol>
5. Cell seeding
NOTE: Similar to conventional membrane inserts, different cell types can be cultured on the nanomembrane. A secondary cell type can also be co-cultured on the other side of the membrane in the bottom channel15.
<ol>
	<li>Coat the membrane with 5 &#181;g/cm2 of fibronectin (see Table of Materials) at room temperature for 1 h to improve cell attachment. 
	NOTE: The chosen cell type may influence the required coating and cell density. The procedure described here is for Human umbilical vein endothelial cells (HUVECs, see Table of Materials).</li>
	<li>Aspirate the coating solution using a P200 pipette and place a patterning stencil in the core module well.</li>
	<li>Add cell media to the well and bottom channel of the device.</li>
	<li>Seed HUVECs at a density of 40,000 cells/cm2 into the well. 
	NOTE: HUVECs at this density typically form a confluent monolayer after 24 h of incubation16,19.</li>
	<li>Place the device in a Petri dish. Add a 15 mL conical tube cap with DI water to the Petri dish to maintain local humidity.</li>
	<li>Transfer the Petri dish to the incubator. 
	&#8203;NOTE: Cell media in the top well and bottom channel of the device should be changed every 24 h. To change the media in the bottom channel, gently inject fresh media into one of the ports to displace the old media from the other port.</li>
</ol>
6. Reconfiguration to microfluidic mode
<ol>
	<li>Sterilize the upper housing, lower housing, flow module, reservoirs, tubes, and dispensing tips by placing them in a UV sterilization chamber for 30 min prior to assembly. Circulate 70% ethanol and then sterile PBS in the flow circuit.</li>
	<li>Fill the reservoirs and tubes with Endothelial Cell Growth media (see Table of Materials).</li>
	<li>Place the lower housing on the sample stage. Insert the open-well core module into the lower housing.</li>
	<li>Aspirate cell media from the well of the module. Place the flow module in the well.</li>
	<li>Place the upper housing to seal the flow module in the core module well. Connect the inlet and outlet dispensing tips to the flow module ports.</li>
	<li>Start the peristaltic pump to introduce fluid flow into the system.</li>
</ol>
7. Conducting downstream analysis in open-well format after flow introduction
NOTE: The culture time here depends on the experimental goals. Users can conduct downstream analysis (e.g., Immunocytochemistry, RNA extraction) either in the open-well or microfluidic formats based on their preference. For instance, if an open-well format is preferred, the system should be reconfigured to conduct assays based on standard protocols16,19.
<ol>
	<li>Stop the pump when the desired time for shear flow exposure is reached. Remove the inlet and outlet dispensing tips.</li>
	<li>Remove the upper housing. Gently remove the flow module from the well using tweezers.</li>
	<li>Add 100 &#181;L of cell media to the well. Conduct desired assays based on standard protocols.</li>
</ol>

A Reconfigurable Barrier Tissue Platform with Open-Well and Microfluidic Modes

<ol>
	<li>Claesson-Welsh, L., Dejana, E., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+the+Endothelial+Barrier:+Identifying+and+Reconciling+Controversies.">Permeability of the Endothelial Barrier: Identifying and Reconciling Controversies.</a> Trends in Molecular Medicine. 27 (4), 314-331 (2021).</li><li>Vera, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+tissue+barrier+models+on+hydrogel+microfluidic+platforms.">Engineering tissue barrier models on hydrogel microfluidic platforms.</a> ACS Applied Materials &amp; Interfaces. 13 (12), 13920-13933 (2021).</li><li>Wang, Y. I., Abaci, H. E., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+blood-brain+barrier+model+provides+in+vivo-like+barrier+properties+for+drug+permeability+screening.">Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening.</a> Biotechnology and Bioengineering. 114 (1), 184-194 (2017).</li><li>Sakolish, C. M., Esch, M. B., Hickman, J. J., Shuler, M. L., Mahler, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+barrier+tissues+in+vitro:+methods,+achievements,+and+challenges.">Modeling barrier tissues in vitro: methods, achievements, and challenges.</a> eBioMedicine. 5, 30-39 (2016).</li><li>Kaisar, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+experimental+models+of+the+blood-brain+barrier+for+CNS+drug+discovery.">New experimental models of the blood-brain barrier for CNS drug discovery.</a> Expert Opinion on Drug Discovery. 12 (1), 89-103 (2017).</li><li>Tan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+microfluidic+microphysiological+system+(PREDICT-96)+to+recapitulate+hepatocyte+function+in+dynamic,+re-circulating+flow+conditions.">A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.</a> Lab on a Chip. 19 (9), 1556-1566 (2019).</li><li>Ayuso, J. M., Virumbrales-Mu&#241;oz, M., Lang, J. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+microfluidic+systems+in+precision+medicine.">A role for microfluidic systems in precision medicine.</a> Nature Communications. 13 (1), 3086 (2022).</li><li>Katt, M. E., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier:+building+in+physiological+complexity.">In vitro models of the blood-brain barrier: building in physiological complexity.</a> Current Opinion in Chemical Engineering. 30, 42-52 (2020).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organs-on-chips+for+disease+modelling,+drug+development+and+personalized+medicine.">Human organs-on-chips for disease modelling, drug development and personalized medicine.</a> Nature Reviews Genetics. 23 (8), 467-491 (2022).</li><li>Duncombe, T. A., Tentori, A. M., Herr, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+reframing+biological+enquiry.">Microfluidics: reframing biological enquiry.</a> Nature reviews. Molecular cell biology. 16 (9), 554-567 (2015).</li><li>Williams, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost,+rapidly+integrated+debubbler+(rid)+module+for+microfluidic+cell+culture+applications.">A low-cost, rapidly integrated debubbler (rid) module for microfluidic cell culture applications.</a> Micromachines. 10 (6), 360 (2019).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fiber+anisotropy+within+natural+collagen+hydrogels.">Engineering fiber anisotropy within natural collagen hydrogels.</a> American Journal of Physiology-Cell Physiology. 320 (6), C1112-C1124 (2021).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineered+3D+collagen+gels+with+independently+tunable+fiber+anisotropy+and+directionality.">Microengineered 3D collagen gels with independently tunable fiber anisotropy and directionality.</a> Advanced Materials Technologies. 6 (4), 2001186 (2021).</li><li>&#321;ach, A., Wnuk, A., W&#243;jtowicz, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+to+study+the+functions+of+the+blood-brain+barrier.">Experimental models to study the functions of the blood-brain barrier.</a> Bioengineering. 10 (5), 519 (2023).</li><li>McCloskey, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Modular+&#181;SiM:+A+mass+produced,+rapidly+assembled,+and+reconfigurable+platform+for+the+study+of+barrier+tissue+models+in+vitro.">The Modular &#181;SiM: A mass produced, rapidly assembled, and reconfigurable platform for the study of barrier tissue models in vitro.</a> Advanced Healthcare Materials. 11 (18), 2200804 (2022).</li><li>Mansouri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+modular+&#181;sim+reconfigured:+integration+of+microfluidic+capabilities+to+study+in+vitro+barrier+tissue+models+under+flow.">The modular &#181;sim reconfigured: integration of microfluidic capabilities to study in vitro barrier tissue models under flow.</a> Advanced Healthcare Materials. 11 (21), 2200802 (2022).</li><li>Hudecz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+a+human+blood-brain+barrier+co-culture+using+an+ultrathin+silicon+nitride+membrane-based+microfluidic+device.">Modelling a human blood-brain barrier co-culture using an ultrathin silicon nitride membrane-based microfluidic device.</a> International Journal of Molecular Sciences. 24 (6), 5624 (2023).</li><li>Joshi, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineering+3D+Collagen+Matrices+with+Tumor-Mimetic+Gradients+in+Fiber+Alignment.">Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.</a> bioRxiv. , (2023).</li><li>Hsu, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniaturized+3D+printed+pressure+regulator+(&#181;PR)+for+microfluidic+cell+culture+applications.">A miniaturized 3D printed pressure regulator (&#181;PR) for microfluidic cell culture applications.</a> Scientific Reports. 12 (1), 10769 (2022).</li><li>Rogers, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+microfluidic+bilayer+co-culture+platform+to+study+endothelial-pericyte+interactions.">A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions.</a> Scientific reports. 11 (1), 12225 (2021).</li><li>Wettschureck, N., Strilic, B., Offermanns, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passing+the+vascular+barrier:+endothelial+signaling+processes+controlling+extravasation.">Passing the vascular barrier: endothelial signaling processes controlling extravasation.</a> Physiological Reviews. 99 (3), 1467-1525 (2019).</li><li>Wang, Y. I., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniChip+enables+long-term+recirculating+unidirectional+perfusion+with+gravity-driven+flow+for+microphysiological+systems.">UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.</a> Lab on a Chip. 18 (17), 2563-2574 (2018).</li><li>Nayak, L., Lin, Z., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Go+with+the+flow&amp;#34;:+how+Kr&#252;ppel-like+factor+2+regulates+the+vasoprotective+effects+of+shear+stress.">34;Go with the flow&amp;#34;: how Kr&#252;ppel-like factor 2 regulates the vasoprotective effects of shear stress.</a> Antioxidants &amp; Redox Signaling. 15 (5), 1449-1461 (2011).</li><li>Satoh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatic+pressure-driven+multi-throughput+microfluidic+circulation+culture+system.">A pneumatic pressure-driven multi-throughput microfluidic circulation culture system.</a> Lab on a chip. 16 (12), 2339-2348 (2016).</li><li>Abhyankar, V. V., Wu, M., Koh, C. Y., Hatch, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Reversibly+sealed,+easy+access,+modular+(seam)+microfluidic+architecture+to+establish+in+vitro+tissue+interfaces.">A Reversibly sealed, easy access, modular (seam) microfluidic architecture to establish in vitro tissue interfaces.</a> PLOS ONE. 11 (5), e0156341 (2016).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+extensional+flows+promote+long-range+fiber+alignment+in+3D+collagen+hydrogels.">Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.</a> Biofabrication. 14 (3), 035019 (2022).</li><li>Hasan, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+fabrication+of+flexible+nanotextured+PDMS+as+a+substrate+for+selective+cell+capture.">One-step fabrication of flexible nanotextured PDMS as a substrate for selective cell capture.</a> Biomedical Physics &amp; Engineering Express. 4 (2), 025015 (2018).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineering+3D+collagen+hydrogels+with+long-range+fiber+alignment.">Microengineering 3D collagen hydrogels with long-range fiber alignment.</a> Journal of Visualized Experiments. 187, e64457 (2022).</li></ol>

J.L.M. is a co-founder of SiMPore, Inc. and holds an equity interest in the company. SiMPore is commercializing the ultrathin silicon-based technologies, including the membranes used in this study.

The aim of this protocol is to develop a practical method for incorporating flow capabilities into an open-well platform featuring an ultrathin nanomembrane. In this design, a magnetic latching approach is utilized, allowing switching between open-well and fluidic modes during experiments and combining the advantages of both approaches. Unlike conventional permanently bonded platforms, magnetic latching allows the platform to be disassembled at convenient points during the experimental workflow16<...

Vascular barriers serve as a critical interface that separates the blood compartment from the surrounding tissue. They play a critical role in preserving homeostasis by attracting immune cells, controlling molecular permeability, and shielding against the intrusion of pathogens into the tissue1,2. In vitro culture models have been developed to mimic the in vivo microenvironment, enabling systematic investigations into the factors and conditions that impact barrier properties in both healthy and diseased states3,4.
The most widely used approach for such culture models is the Transwell-like &#34;open-well&#34; configuration5, where a porous, track-etched culture membrane separates media-filled compartments (Figure 1A). In this format, cells can be seeded on either side of the membrane, and a wide range of experimental protocols has been developed. However, these systems are limited in their ability to provide the fluid flows essential for supporting barrier maturation and mimicking immune cell circulation seen in vivo5,6. Consequently, they cannot be used for studies requiring dynamic flows that introduce drug doses, mechanical stimulation, or fluid-induced shear stresses6,7,8.
To overcome the limitations of open-well systems, microfluidic platforms that combine porous culture membranes with individually addressable fluidic channels have been developed9. These platforms offer precise control over fluid routing, perfusion, and the introduction of chemical compounds, controlled shear stimulation, and dynamic cell addition capabilities7,10,11,12,13. Despite the advanced capabilities provided by microfluidic platforms, they have not seen widespread adoption in bioscience laboratories due to complex microfluidic protocols and their incompatibility with established experimental workflows4,10,14.
To bridge the gap between these technologies, we present a protocol that employs a magnetically reconfigurable, module-based system. This system can be easily switched between open-well and microfluidic modes based on the specific needs of the experiment. The platform features an open-well device, known as m-&#181;SiM (modular microphysiological system enabled by a silicon membrane), with a 100 nm thick culture membrane (nanomembrane). This nanomembrane possesses high porosity (15%) and glass-like transparency, as illustrated in Figure 1B. It physically separates the top compartment from a bottom channel, allowing for molecular transport across physiological length scales15. Unlike conventional track-etched membranes, which have known challenges in imaging live cells with bright-field imaging, the nanomembrane&#39;s favorable optical and physical properties enable clear visualization of cells on either side of the membrane surface15,16,17.
The present protocol outlines the fabrication of specialized seeding and flow modules and explains the magnetic reconfiguration of the platform. It demonstrates how the platform can be employed to establish endothelial barriers under both static and dynamic conditions. This demonstration reveals that endothelial cells align along the flow direction, with an upregulation of shear-sensitive gene targets under shear stimulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 x 0.86 Micro Flow tubes</td>
			<td>Langer Instruments</td>
			<td>WX10-14 &#38; DG Series</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm Disposable Biopsy Punches, Integra Miltex</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS 7.4 pH</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>20 GAUGE IT SERIES DISPENSING TIP</td>
			<td>Jensen Global</td>
			<td>JG20-1.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>21 GAUGE NT PREMIUM SERIES ANGLED DISPENSING TIP</td>
			<td>Jensen Global</td>
			<td>JG21-1.0HPX-90</td>
			<td></td>
		</tr>
		<tr>
			<td>3M 467 MP Pressure senstitive adhesive (PSA)</td>
			<td>DigiKey</td>
			<td>3M9726-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>3M 468 MP Pressure senstitive adhesive (PSA)</td>
			<td>DigiKey</td>
			<td>3M9720-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488 conjugated phalloidin</td>
			<td>ThermoFisher Scientific</td>
			<td>A12379&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#160;TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444556</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent grade, Alfa Aesar, Size = 10 g</td>
			<td>VWR</td>
			<td>AAJ64100-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K171</td>
			<td>12&#34; x 12&#34; x 1/16&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8589K31</td>
			<td>12&#34; x 12&#34; x 3/32&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K191</td>
			<td>12&#34; x 12&#34; x 7.64&#34;</td>
		</tr>
		<tr>
			<td>Corning Fibronectin, Human, 1 mg</td>
			<td>Corning</td>
			<td>47743-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses, Globe Scientific, L x W = 24 x 60 mm</td>
			<td>VWR</td>
			<td>10118-677</td>
			<td></td>
		</tr>
		<tr>
			<td>DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT</td>
			<td>Ellsworth Adhesives</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixture A1&#38;A2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixture B1&#38;B2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Thermo Fisher Scientific</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells (HUVEC)</td>
			<td>ThermoFisher Scientific</td>
			<td>C0035C</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Cell Imaging Kit (488/570)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Probes Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel-plated magnets (4.75 mm diameter, 0.34 kg pull force)</td>
			<td>K&#38;J Magnetics</td>
			<td>D31</td>
			<td>3/16&#34; dia.&#160;x 1/16&#34; thick</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4% w/v aq. soln., methanol free, Alfa Aesar</td>
			<td>Fisher Scientific</td>
			<td>aa47392-9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Langer Instruments</td>
			<td>BQ50-1J-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist SU-8 developer solution</td>
			<td>Fisher Scientific</td>
			<td>NC9901158</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF syringe filters</td>
			<td>PerkinElmer</td>
			<td>2542913</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University wafer,USA</td>
			<td>1196</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>Fisher Scientific</td>
			<td>NC0702369</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: eNOS (Hs01574659_m1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: GAPDH (Hs02786624_g1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: KLF2 (Hs00360439_g1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Pierce 20x PBS Tween 20</td>
			<td>Thermo Fisher Scientific</td>
			<td>28352</td>
			<td></td>
		</tr>
		<tr>
			<td>Transport Tube Sample White caps, 5 mL, Sterile</td>
			<td>VWR</td>
			<td>100500-422</td>
			<td></td>
		</tr>
		<tr>
			<td>TRI-reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrathin Nanoporous Membrane Chip</td>
			<td>SiMPore Inc.</td>
			<td>NPSN100-1L</td>
			<td>The design is&#160; compatible with all of SiMPore membranes</td>
		</tr>
		<tr>
			<td>uSiM component 1</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>uSiM component 2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

transforming static barrier tissue models into dynamic microphysiological systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

The open-well core module is initially positioned within a specific cavity created by a lower housing and a coverslip, as illustrated in Figure 6A. Subsequently, the flow module, which includes a microchannel and access ports, is inserted into the well of the core module. The flow module is securely sealed against the silicon support layer of the membrane due to the magnetic attraction force between magnets embedded in the lower and upper housings, as depicted in Figure ...

Watch this Scientific Journal Video about Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue at JoVE.com

Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...

transforming-static-barrier-tissue-models-into-dynamic

Research

JoVE Journal

Bioengineering

878 Views.  Rochester Institute of Technology. This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Video: Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...