<ol>
	<li>Van Norman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+animal+studies+for+predicting+toxicity+in+clinical+trials:+Is+it+time+to+rethink+our+current+approach.">Limitations of animal studies for predicting toxicity in clinical trials: Is it time to rethink our current approach.</a> JACC: Basic to Translational Science. 4 (7), 845-854 (2019).</li><li>Wange, R. L., Brown, P. C., Davis-Bruno, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+the+principles+of+the+3Rs+of+animal+testing+at+CDER:+Past,+present+and+future.">Implementation of the principles of the 3Rs of animal testing at CDER: Past, present and future.</a> Regulatory Toxicology and Pharmacology. 123, 104953 (2021).</li><li>Mosig, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-on-chip+models:+New+opportunities+for+biomedical+research.">Organ-on-chip models: New opportunities for biomedical research.</a> Future Science OA. 3 (2), (2017).</li><li>Al&#233;p&#233;e, N., 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=State-of-the-art+of+3D+cultures+(organs-on-a-chip)+in+safety+testing+and+pathophysiology.">State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.</a> Altex. 31 (4), 441-477 (2014).</li><li>MacArron, 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=Impact+of+high-throughput+screening+in+biomedical+research.">Impact of high-throughput screening in biomedical research.</a> Nature Reviews Drug Discovery. 10 (3), 188-195 (2011).</li><li>Hughes, J. P., Rees, S. S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> British Journal of Pharmacology. 162 (6), 1239-1249 (2011).</li><li>Kitaeva, K. V., Rutland, C. S., Rizvanov, A. A., Solovyeva, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+based+in+vitro+test+systems+for+anticancer+drug+screening.">Cell culture based in vitro test systems for anticancer drug screening.</a> Frontiers in Bioengineering and Biotechnology. 8, 322 (2020).</li><li>Mao, P., 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=Human+alveolar+epithelial+type+II+cells+in+primary+culture.">Human alveolar epithelial type II cells in primary culture.</a> Physiological Reports. 3 (2), 12288 (2015).</li><li>Zaitseva, M., Vollenhoven, B. J., Rogers, P. A. W. <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+culture+significantly+alters+gene+expression+profiles+and+reduces+differences+between+myometrial+and+fibroid+smooth+muscle+cells.">In vitro culture significantly alters gene expression profiles and reduces differences between myometrial and fibroid smooth muscle cells.</a> Molecular Human Reproduction. 12 (3), 187-207 (2006).</li><li>Singh, A., Brito, I., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+tissue+stiffness+and+bioadhesivity:+Advanced+biomaterials+to+model+tumor+microenvironments+and+drug+resistance.">Beyond tissue stiffness and bioadhesivity: Advanced biomaterials to model tumor microenvironments and drug resistance.</a> Trends in Cancer. 4 (4), 281-291 (2018).</li><li>Nawroth, J. 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=Stem+cell-based+Lung-on-Chips:+The+best+of+both+worlds.">Stem cell-based Lung-on-Chips: The best of both worlds.</a> Advanced Drug Delivery Reviews. 140, 12-32 (2019).</li><li>Jensen, C., Teng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+time+to+start+transitioning+from+2d+to+3d+cell+culture.">Is it time to start transitioning from 2d to 3d cell culture.</a> Frontiers in Molecular Biosciences. 7, 33 (2020).</li><li>Kapa&#322;czy&#324;ska, 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=2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures.">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li><li>Sutherland, R. M., Inch, W. R., McCredie, J. A., Kruuv, 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+multi-component+radiation+survival+curve+using+an+in+vitro+tumour+model.">A multi-component radiation survival curve using an in vitro tumour model.</a> International Journal of Radiation Biology. 18 (5), 491-495 (1970).</li><li>Chandra, P., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+extracellular+microenvironment+for+modulating+stem+cell+behaviors.">Synthetic extracellular microenvironment for modulating stem cell behaviors.</a> Biomarker Insights. 10, 105-116 (2015).</li><li>Nicolas, 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=3D+extracellular+matrix+mimics:+Fundamental+concepts+and+role+of+materials+chemistry+to+influence+stem+cell+fate.">3D extracellular matrix mimics: Fundamental concepts and role of materials chemistry to influence stem cell fate.</a> Biomacromolecules. 21 (6), 1968-1994 (2020).</li><li>Brassard, J. A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+stem+cell+self-organization+to+build+better+organoids.">Engineering stem cell self-organization to build better organoids.</a> Cell Stem Cell. 24 (6), 860-876 (2019).</li><li>Lutolf, M. P., Gilbert, P. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+materials+to+direct+stem-cell+fate.">Designing materials to direct stem-cell fate.</a> Nature. 462 (7272), 433-441 (2009).</li><li>Mantha, S., 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=Smart+hydrogels+in+tissue+engineering+and+regenerative+medicine.">Smart hydrogels in tissue engineering and regenerative medicine.</a> Materials. 12 (20), 3323 (2019).</li><li>Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+culture+models+in+drug+discovery+and+drug+repositioning.">Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.</a> Frontiers in Pharmacology. 9, 6 (2018).</li><li>Li, H., 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=Biomechanical+cues+as+master+regulators+of+hematopoietic+stem+cell+fate.">Biomechanical cues as master regulators of hematopoietic stem cell fate.</a> Cellular and Molecular Life Sciences. 78 (16), 5881-5902 (2021).</li><li>Donoghue, L., Nguyen, K. T., Graham, C., Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+chips+and+microphysiological+systems+for+disease+modeling+and+drug+testing.">Tissue chips and microphysiological systems for disease modeling and drug testing.</a> Micromachines. 12 (2), 139 (2021).</li><li>Ma, C., Peng, Y., Li, H., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-on-a-chip:+A+new+paradigm+for+drug+development.">Organ-on-a-chip: A new paradigm for drug development.</a> Trends in Pharmacological Sciences. 42 (2), 119-133 (2021).</li><li>Varone, 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=A+novel+organ-chip+system+emulates+three-dimensional+architecture+of+the+human+epithelia+and+the+mechanical+forces+acting+on+it.">A novel organ-chip system emulates three-dimensional architecture of the human epithelia and the mechanical forces acting on it.</a> Biomaterials. 275, 120957 (2021).</li><li>Hassell, B. 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=Human+organ+chip+models+recapitulate+orthotopic+lung+cancer+growth,+therapeutic+responses,+and+tumor+dormancy+in+vitro.">Human organ chip models recapitulate orthotopic lung cancer growth, therapeutic responses, and tumor dormancy in vitro.</a> Cell Reports. 21 (2), 508-516 (2017).</li><li>Sadeghipour, A., Babaheidarian, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+formalin-fixed,+paraffin+embedded+blocks.">Making formalin-fixed, paraffin embedded blocks.</a> Biobanking. 1897, 253-268 (2019).</li><li>Grant, 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=Simulating+drug+concentrations+in+PDMS+microfluidic+organ+chips.">Simulating drug concentrations in PDMS microfluidic organ chips.</a> Lab on a Chip. 21 (18), 3509-3519 (2021).</li><li>Barrile, 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=Organ-on-chip+recapitulates+thrombosis+Induced+by+an+anti-CD154+monoclonal+antibody:+Translational+potential+of+advanced+microengineered+systems.">Organ-on-chip recapitulates thrombosis Induced by an anti-CD154 monoclonal antibody: Translational potential of advanced microengineered systems.</a> Clinical Pharmacology &amp; Therapeutics. 104 (6), 1240-1248 (2018).</li><li>Jain, 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=Primary+human+lung+alveolus-on-a-chip+model+of+intravascular+thrombosis+for+assessment+of+therapeutics.">Primary human lung alveolus-on-a-chip model of intravascular thrombosis for assessment of therapeutics.</a> Clinical Pharmacology &amp; Therapeutics. 103 (2), 332-340 (2018).</li><li>Campbell, S. B., Wu, Q., Yazbeck, J., Liu, C., Okhovatian, S., Radisic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+polydimethylsiloxane:+Alternative+materials+for+fabrication+of+organ-on-a-chip+devices+and+microphysiological+systems.">Beyond polydimethylsiloxane: Alternative materials for fabrication of organ-on-a-chip devices and microphysiological systems.</a> ACS Biomaterials Science and Engineering. 7 (7), 2880-2899 (2021).</li><li>Pun, S., Haney, L. C., Barrile, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+human+physiology+on-chip:+Historical+perspectives+and+future+directions.">Modelling human physiology on-chip: Historical perspectives and future directions.</a> Micromachines. 12 (10), 1250 (2021).</li></ol>

The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Varone Antonio is a former employee of Emulate Inc. and may hold equity interest in Emulate.

The Open-Top Chip represents an enabling platform for investigating the complex cellular interplay occurring between endothelium, stroma, and epithelium in a controlled microenvironment, in real time. This technology offers critical advantages over conventional organotypic and organoid cultures, such as the integration of physical and biochemical cues that are relevant to reconstitute the human tissue microenvironment, including fluidic shear (flow), cyclic stretching, and reconstruction of the epithelial surface topogra...

Historically, scientists have relied on preclinical animal testing for drug discovery, but a growing number of these methods have been questioned because of poor correlation with human outcome1. The implementation of the &#34;3Rs&#34; principles to Replace, Reduce, and Refine animal experimentation urges scientists to find new in vitro alternative methods to support preclinical drug and chemical toxicology risk assessment2. However, many in vitro models developed to date lack the biological architecture, cellular complexity, and mechanical environment necessary to recapitulate the dynamic nature of human living organs3,4.
Conventional in vitro preclinical systems typically employ 2D monocultures of human cells grown on a rigid plastic surface. These methods provide a tool for conducting simple mechanistic studies and enable rapid screening of drug candidates. Owing to their relatively low cost and high robustness, 2D models are often paired with automatic high-throughput systems and used for the rapid identification of potential drug candidates during the early stage of the drug development process5,6. However, such 2D models do not provide a translational approach for modeling tissue-level, organ-level, or systemic responses to therapeutic candidates, which is needed for accurate predictions of drug safety and efficacy during the preclinical stage of their development. Flat cell cultures do not recapitulate the native tissue microenvironment, including the complex multicellular interplay, biomechanical properties, and three-dimensional (3D) architecture of human tissues7. Cells growing on a flat surface often do not acquire a mature phenotype and, therefore, cannot respond to pharmacological stimuli as they would in the native tissue. For example, primary human alveolar epithelial cells grown in vitro exhibit a squamous phenotype and lose key phenotypic markers, including surfactant proteins C and B (SP-C and SP-B)8. In addition to insufficient differentiation, primary cells frequently become insensitive to biological stressors in vitro, as certain biochemical pathways associated with tissue inflammation become non-functional9. Such loss of cell function seems to be primarily associated with the use of stiff substrates as well as the lack of soluble factors naturally released by tissue-specific stromal cells such as lung fibroblasts and smooth muscle cells10,11.
Understanding that the lack of chemo-physical and biological complexity limits the physiological behavior of cells in vitro has fostered the development of more sophisticated multicellular models, which have proven to better capture the complexity of human tissues outside the body12,13. Since the creation of the first co-culture models in the early 1970s14, the introduction of synthetic and natural hydrogels has significantly improved the ability to mimic native tissue microenvironments and has become an invaluable tool for driving cellular differentiation, guiding the self-organization of cells into tissue-like structures, and restoration of native tissue functions15,16. For instance, when grown in the appropriate 3D scaffold, human cells can self-arrange into functional structures such as spheroids or organoids, expressing stem cell markers, and are capable of self-renewal17. In contrast, human cells (including stem cells), when grown on traditional 2D substrates, rapidly age and undergo senescence after a few passages18. In addition, hydrogels can be &#34;tailored&#34; to match specific tissue properties such as porosity, pore size, fiber thickness, viscoelasticity, topography, and stiffness or further engineered with tissue-derived cellular components and/or bioactive molecules enabling emulation of the physiological or pathological conditions19,20. Despite their enormous potential for drug testing, 3D hydrogel-based models used in pharmaceutical research do not fully recapitulate the complex cytoarchitecture of the in vivo tissues and lack important hemodynamic and mechanical stimuli normally present in the human body, including hydrostatic pressure, cyclic stretch, and fluid shear21.
Microphysiological systems (MPSs) such as Organs-on-chips (OOCs) have recently emerged as tools that are capable of capturing complex physiological responses&#8239;in vitro22,23. These models often employ the use of microfluidic platforms, which enable the modelling of the dynamic microenvironment of living organs.
We have combined the principles of 3D tissue bioengineering and mechanobiology to create an Open-Top Chip model of complex human epithelial tissue. This allowed us to closely recapitulate the multicellular and dynamic microenvironment of epithelial tissues. This includes tissue-specific biochemical and biomechanical cues naturally present in living organs but often neglected by traditional in vitro models24. The Open-Top Chip incorporates two compartments: a vascular compartment (Figure 1A) and a stromal compartment (Figure 1B) separated by a porous membrane, allowing for the diffusion of nutrients between the two chambers (Figure 1C). The vascular compartment is exposed to continuous fluid flow to recapitulate physiological shear stress, while the stretchable design of the stromal chamber allows for the modeling of the mechanical strain associated with breathing motions or intestinal peristalsis. The stromal compartment houses the tunable 3D hydrogel scaffold designed to support the physiological growth of tissue-specific fibroblasts. It possesses a removable lid that facilitates the establishment of an air-liquid interface, a condition that allows greater emulation of human physiology of mucosal tissues as well as direct access to the tissue for administrating drugs directly onto the epithelial layer. Supplementary Figure 1 captures some of the key components of the Open-Top Chip design including dimensions and biological compartments (Supplementary Figure 1A-D) as well as the main technical steps described in this protocol (Supplementary Figure 1E).
Perfusion of the Open-Top Chip is achieved with a programmable peristaltic pump (Figure 1D). The peristaltic pump setup allows 12 Open-Top Chips to be perfused simultaneously. Most incubators can house two setups enabling the culture of up to 24 chips per incubator. Mechanical stretching is achieved using a custom-made programmable vacuum pressure regulator (Figure 1E). It consists of an electro-pneumatic vacuum regulator controlled electronically by a digital-to-analog converter. In other words, the electro-pneumatic vacuum regulator generates a sinusoidal vacuum profile with an amplitude and frequency that is determined by the user. Cyclic strain ranging from 0% to 15% is generated by applying negative pressure to the vacuum channel of the Open-Top Chip at an amplitude ranging from 0 to -90 kPa and a frequency of 0.2 Hz. It is a custom-made system equivalent to the commercially available Flexcell Strain Unit previously adopted and described in other papers25. To mimic the mechanical tissue deformation associated, for example, with the breathing motion of the lung or the peristalsis of the intestine, the pneumatic actuator applies sinusoidal vacuum/strain waves whose magnitude and amplitude can be adjusted to match the physiological level of strain and frequency that human cells experience in their native tissue.
Here, we describe an efficient and reproducible method for engineering and culturing organotypic epithelium equivalents on a prototype Open-Top Chip platform. It allows the generation of complex organ models such as skin, alveolus, airway, and colon while integrating a vascular fluid flow and mechanical stretching. We will outline key technical aspects that must be considered while implementing principles of tissue engineering for generating complex epithelial models. We will discuss the advantages and possible limitations of the current design.
An overview of the main steps used to achieve tissue and organ maturation, including flow and stretch parameters, is reported in: Figure 2 for the skin, Figure 3 for the alveolus, Figure 4 for the airway, and Figure 5 for the intestine. Additional information concerning media composition and reagents used for culturing the different organ models are included in the supplementary tables (Supplementary Table 1 for the skin; Supplementary Table 2 for the alveolus; Supplementary Table 3 for the airway, and Supplementary Table 4 for the intestine).

<table><tbody><tr><td>10x EMEM&amp;nbsp;</td><td>Lonza</td><td>12-684F</td><td>Medium; Stroma</td></tr><tr><td>18 Gauge needle</td><td>MicroGroup</td><td>316H18RW</td><td>Tube stainless steel 316 welded, 18RW Full Hard&amp;nbsp;</td></tr><tr><td>19 Gauge needle</td><td>MicroGroup</td><td>316H19RW</td><td>Tube stainless steel 316 welded, 19RW Full Hard</td></tr><tr><td>2-Stop PharMed BPT&amp;nbsp;</td><td>Cole-Palmer&amp;nbsp;</td><td>EW-95723-12</td><td>Tube, 0.25 mm, 12/pack</td></tr><tr><td>70% ethanol and wipes&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>For surface sterilization&amp;nbsp;</td></tr><tr><td>8-Bromoadenosine 3&amp;prime;,5&amp;prime;-cyclic monophosphate sodium salt (8-Br-cAMP)</td><td>Sigma</td><td>B7880</td><td>Medium supplement&amp;nbsp;</td></tr><tr><td>A-83-01&amp;nbsp;</td><td>Tocris&amp;nbsp;</td><td>2939</td><td></td></tr><tr><td>Adenine</td><td>Sigma</td><td>A9795</td><td></td></tr><tr><td>Advanced DMEM/F12&amp;nbsp;</td><td>Thermo</td><td>12634010</td><td></td></tr><tr><td>Airway Epithelial Cells</td><td>Lifeline Cell Technology</td><td>FC-0016</td><td></td></tr><tr><td>Aluminum foil&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-</td></tr><tr><td>Alveolar cells</td><td>Cell Biologics</td><td>H6621</td><td></td></tr><tr><td>Anti-ABCA3&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab24751&amp;nbsp;</td><td>Mouse monoclonal antibody [3C9]&amp;nbsp;</td></tr><tr><td>Anti-Aquaporin5 Alexa Fluor 647&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab215225&amp;nbsp;&amp;nbsp;</td><td>Rabbit monoclonal antibody [EPR3747]&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-Aquaporin5&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab92320&amp;nbsp;</td><td>Rabbit monoclonal antibody [EPR3747]&amp;nbsp;</td></tr><tr><td>Anti-beta IV Tubulin&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab11315&amp;nbsp;</td><td>Mouse monoclonal antibody [ONS.1A6]&amp;nbsp;</td></tr><tr><td>Anti-CD31 (PECAM-1)&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab9498&amp;nbsp;</td><td>Mouse monoclonal [JC/70A] antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-CK5&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab75869&amp;nbsp;</td><td>Rabbit recombinant monoclonal [AY1E6]&amp;nbsp;</td></tr><tr><td>Anti-Cytokeratin 10&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>MA5-13705&amp;nbsp;</td><td>Mouse monoclonal antibody (DE-K10)&amp;nbsp;</td></tr><tr><td>Anti-Cytokeratin 14&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab7800&amp;nbsp;</td><td>Mouse monoclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-E-Cadherin&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab1416&amp;nbsp;&amp;nbsp;</td><td>Mouse monoclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-Filaggrin&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>PA5-79267&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-HTI-56&amp;nbsp;</td><td>Terrace Biotech&amp;nbsp;</td><td>TB-29AHT1-56&amp;nbsp;&amp;nbsp;</td><td>Mouse monoclonal antibody (IgG1)&amp;nbsp;</td></tr><tr><td>Anti-HTII-280&amp;nbsp;</td><td>Terrace Biotech&amp;nbsp;</td><td>TB-27AHT2-280&amp;nbsp;</td><td>Mouse monoclonal antibody (IgM)&amp;nbsp;</td></tr><tr><td>Anti-Involucrin&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>MA5-11803&amp;nbsp;</td><td>Mouse monoclonal antibody (SY5)&amp;nbsp;</td></tr><tr><td>Anti-Isoforms TA p63-&amp;alpha;, -&amp;beta;, -&amp;gamma;&amp;nbsp;&amp;nbsp;</td><td>Biolengend&amp;nbsp;</td><td>618902&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-Ki67&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab8191&amp;nbsp;</td><td>Mouse monoclonal antibody [B126.1]&amp;nbsp;</td></tr><tr><td>Anti-LAMP3&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab111090&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-Mature SP-B&amp;nbsp;</td><td>Seven Hill&amp;nbsp;</td><td>WRAB-48604&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-MUC5AC&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>PA5-34612&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-Mucin-2&amp;nbsp;</td><td>SantaCruz Biotechnology</td><td>sc-7314</td><td>Mouse monoclonal antibody (IgG1)&amp;nbsp;</td></tr><tr><td>Anti-p63&amp;nbsp;&amp;nbsp;</td><td>Dako&amp;nbsp;</td><td>GA662&amp;nbsp;</td><td>Mouse monoclonal antibody p63 Protein (Dako Omnis)&amp;nbsp;Clone DAK-p63&amp;nbsp;</td></tr><tr><td>Anti-PCNA&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>PA5-32541&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-Podoplanin (AT-1&amp;alpha;)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab128994&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-Pro + Mature Surfactant Protein B&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab40876&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-Surfactant C&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Seven Hill&amp;nbsp;&amp;nbsp;</td><td>WRAB-9337&amp;nbsp;&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;</td></tr><tr><td>Anti-Uteroglobin/SCGB1A1&amp;nbsp;</td><td>Hycult Biotech&amp;nbsp;</td><td>HM2178&amp;nbsp;</td><td>Mouse monoclonal antibody [AY1E6]&amp;nbsp;</td></tr><tr><td>Anti-VE-cadherin&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab33168&amp;nbsp;</td><td>Rabbit polyclonal antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Anti-ZO-1&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>33-9100&amp;nbsp;</td><td>Mouse monoclonal antibody [1A12]&amp;nbsp;</td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>Aspirating pipettes&amp;nbsp;</td><td>Corning / Falcon&amp;nbsp;</td><td>357558&amp;nbsp;</td><td>2 mL, polystyrene, individually wrapped&amp;nbsp;</td></tr><tr><td>Aspirating tips&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>Sterile (autoclaved)&amp;nbsp;</td></tr><tr><td>B27</td><td>Thermo</td><td>17504044</td><td></td></tr><tr><td>Blocker BSA (10X) in PBS solution&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>37525&amp;nbsp;</td><td>Blocker agent&amp;nbsp;</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>CHIR 99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>Collagen I</td><td>Advanced Biomatrix</td><td>5133</td><td>10 mg/mL (Stroma)</td></tr><tr><td>Collagen I&amp;nbsp;</td><td>Advanced BioMatrix</td><td>5005</td><td>3 mg/mL (Vascular ECM)</td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>&amp;nbsp;C5533</td><td></td></tr><tr><td>Collagen-IV</td><td>Sigma&amp;nbsp;</td><td>C5533-5MG&amp;nbsp;</td><td>Collagen from human placenta, 5 mg powder, reconstitute to 1 mg/mL&amp;nbsp;</td></tr><tr><td>Colonic Fibroblasts&amp;nbsp;</td><td>Cell Biologics</td><td>&amp;nbsp;H6231</td><td></td></tr><tr><td>Colonic microvascular endothelial cells</td><td>&amp;nbsp;Cell Biologics</td><td>H6203&amp;nbsp;</td><td></td></tr><tr><td>Conical tubes&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>15 mL and 50 mL&amp;nbsp;polypropylene, sterile&amp;nbsp;</td></tr><tr><td>Crosslinker (ER-1)&amp;nbsp;</td><td>Emulate&amp;nbsp;</td><td>10461</td><td>5 mg powder&amp;nbsp;</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dilactate)&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>D3571&amp;nbsp;</td><td>DNA probe&amp;nbsp;</td></tr><tr><td>Dermal fibroblasts</td><td>ATCC</td><td>PCS-201-010</td><td></td></tr><tr><td>Dermal microvascular endothelial cells</td><td>ATCC</td><td>CRL-3243</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>11054020</td><td></td></tr><tr><td>DMEM/F-12&amp;nbsp;</td><td>GIBCO&amp;nbsp;</td><td>11320082</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX&amp;nbsp;&amp;nbsp;</td><td>GIBCO&amp;nbsp;</td><td>10565-018&amp;nbsp;</td><td>Basal medium for ALI medium&amp;nbsp;</td></tr><tr><td>Donkey Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 488)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab150105&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Donkey Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 568)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab175472&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;</td></tr><tr><td>Donkey Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 647)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab150107&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;</td></tr><tr><td>Donkey Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 488)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>&amp;nbsp;ab150073&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;</td></tr><tr><td>Donkey Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 568)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab175470&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;</td></tr><tr><td>Donkey Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 647)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab150075&amp;nbsp;</td><td>Donkey Anti-Mouse secondary antibody&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (DPBS-/-) (without Ca2+, Mg2+)&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>21-031-CV&amp;nbsp;</td><td>1x&amp;nbsp;</td></tr><tr><td>Epidermal Growth Factor (EGF) human, recombinant in E. coli</td><td>PromoCell</td><td>C-60170</td><td>Medium supplement&amp;nbsp;</td></tr><tr><td>F-12 Ham&amp;rsquo;s</td><td>Invitrogen&amp;nbsp;</td><td>21700-108</td><td>For vascular ECM</td></tr><tr><td>FibriCol&amp;nbsp;</td><td>Advanced BioMatrix&amp;nbsp;</td><td>5133-20ML&amp;nbsp;</td><td>Collagen-I solution (10 mg/mL)</td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>Fibronectin, Human, Natural,&amp;nbsp;&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>47743-654&amp;nbsp;</td><td>human plasma fibronectin&amp;nbsp;</td></tr><tr><td>Fine-tip precision tweezers&amp;nbsp;</td><td>Aven</td><td>18056USA&amp;nbsp;</td><td>Technik Style 5B-SA Precision Stainless Steel Tweezers</td></tr><tr><td>Glutamax</td><td>Invitrogen&amp;nbsp;</td><td>21700-108</td><td></td></tr><tr><td>Glutamax&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>35050061</td><td></td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 594)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab150080&amp;nbsp;</td><td>Goat Anti-Mouse secondary antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alexa Fluor 647)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab150115&amp;nbsp;</td><td>Goat Anti-Mouse secondary antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (FITC)&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab6785&amp;nbsp;</td><td>Goat Anti-Mouse secondary antibody&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Goat Anti-Mouse IgG1 Alexa Fluor 568&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>A-21124&amp;nbsp;</td><td>Goat Anti-Mouse IgG1 secondary antibody&amp;nbsp;</td></tr><tr><td>Goat Anti-Mouse IgM Alexa Fluor 488&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>A-21042&amp;nbsp;</td><td>Goat Anti-Mouse IgM secondary antibody&amp;nbsp;</td></tr><tr><td>Handheld vacuum aspirator&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4930&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>Heat Inactivated HyClone FetalClone II Serum (FCS)&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30066.03</td><td></td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>HEPES</td><td>Thermo</td><td>15630080</td><td></td></tr><tr><td>Human [Leu15] - Gastrin&amp;nbsp;</td><td>Sigma</td><td>G9145</td><td></td></tr><tr><td>Human colonoids</td><td>Obtained from clinical resections</td><td></td><td>Obtained from clinical resections</td></tr><tr><td>Human EGF Recombinant Protein&amp;nbsp;</td><td>Thermo</td><td>PHG0311L</td><td></td></tr><tr><td>human epithelial growth factor&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>PHG0311</td><td></td></tr><tr><td>HyClone FetalClone II Serum (U.S.) &amp;nbsp;</td><td>GE Healthcare&amp;nbsp;</td><td>SH30066.02HI&amp;nbsp;&amp;nbsp;</td><td>Sterile FBS&amp;nbsp;heat-inactivated&amp;nbsp;</td></tr><tr><td>Hydrocortisone 21-hemisuccinate sodium salt</td><td>Sigma</td><td>H4881</td><td></td></tr><tr><td>Hydrocortisone&amp;nbsp;</td><td>PromoCell&amp;nbsp;&amp;nbsp;</td><td>C-64420&amp;nbsp;</td><td>Medium supplement&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Ice bucket&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>Ismatec IPC-N&amp;nbsp;</td><td>Cole-Palmer</td><td>EW-78000-41</td><td>Low-Speed Digital Peristaltic Pump; q24-Channel (1 per 12 Chips)</td></tr><tr><td>ITES</td><td>BioWhittaker</td><td>17-839Z</td><td></td></tr><tr><td>Keratinocyte Growth Factor (KGF), also known as Basic Fibroblast Growth Factor 7 (FGF-7), human, recombinant in HEK</td><td>PromoCell</td><td>C-63821</td><td></td></tr><tr><td>Keratinocytes</td><td>ATCC</td><td>PCS-200-010</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Biolamina</td><td>CT521-0501&amp;nbsp;</td><td></td></tr><tr><td>Laminin, 521 CTG (CT521)&amp;nbsp;</td><td>Biolamina&amp;nbsp;&amp;nbsp;</td><td>CT521-0501&amp;nbsp;</td><td>human recombinant laminin 521&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Lung Fibroblast</td><td>Cell Biologics</td><td>H6013</td><td></td></tr><tr><td>Lung Fibroblast</td><td>Lifeline Cell Technology</td><td>FC-0049</td><td></td></tr><tr><td>Lung microvascular endothelial cells</td><td>Lonza</td><td>CC-2527</td><td></td></tr><tr><td>Lung smooth muscle cells</td><td>Lifeline Cell Technology</td><td>FC-0046</td><td></td></tr><tr><td>Manual counter&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-</td></tr><tr><td>Masterflex (TPE) Transfer Tubing&amp;nbsp;</td><td>Cole-Palmer</td><td>FV-96880-02</td><td>PharMed BPT, 1/32&quot; ID x 5/32&quot; OD</td></tr><tr><td>Medium 199, no phenol red</td><td>Thermo&amp;nbsp;</td><td>11043023</td><td></td></tr><tr><td>Microcentrifuge tube&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;&amp;nbsp;</td><td>1.5 mL, sterile&amp;nbsp;</td></tr><tr><td>Microscope (with camera)&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>For bright-field imaging&amp;nbsp;</td></tr><tr><td>N2</td><td>Sigma</td><td>17502001</td><td></td></tr><tr><td>N-acetyl cysteine</td><td>Sigma</td><td>A5099</td><td></td></tr><tr><td>Noggin (HEK293T conditioned medium)</td><td>Sigma</td><td>N17001</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>50062Z&amp;nbsp;</td><td>Blocking solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>O-phosphosrylethanolamine&amp;nbsp;</td><td>Sigma</td><td>P0503</td><td></td></tr><tr><td>Paraformaldehyde (4% wt/vol)&amp;nbsp;&amp;nbsp;</td><td>EMS&amp;nbsp;</td><td>15710&amp;nbsp;</td><td>Fixing agent&amp;nbsp;</td></tr><tr><td>Penicillin Streptomycin</td><td>GIBCO</td><td>15140122</td><td></td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P4333&amp;nbsp;</td><td>10,000 U/mL; 10 mg/mL&amp;nbsp;</td></tr><tr><td>Pipette tips&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>P20, P200, and P1000&amp;nbsp;sterile, low adhesion</td></tr><tr><td>Pipette&amp;nbsp;</td><td>Gilson&amp;nbsp;</td><td>&amp;nbsp;F167380&amp;nbsp;</td><td>P20, P200, and P1000&amp;nbsp;</td></tr><tr><td>PluriQ Serum Replacement (or alternatively KO Serum replacement)</td><td>AMSBIO (or Thermo)</td><td>N/A (or C1910828010)</td><td></td></tr><tr><td>Poly-L-Lysine coated microscope glass slides&amp;nbsp;&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P0425&amp;nbsp;</td><td>Glass slides&amp;nbsp;</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-1</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>ProLong Gold&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>P36931&amp;nbsp;</td><td>Antifade Mountant with DAPI&amp;nbsp;</td></tr><tr><td>Retinoic Acid&amp;nbsp;</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>ROCK inhibitor (Y27632)</td><td>Tocris</td><td>TB1254-GMP/10</td><td></td></tr><tr><td>R-spondin (HEK293T conditioned medium)</td><td>Sigma</td><td>SCC111</td><td></td></tr><tr><td>SAGM SingleQuots supplements&amp;nbsp;</td><td>Lonza</td><td>CC-4124</td><td></td></tr><tr><td>SAGMTM Small Airway Epithelial Cell Growth medium BulletKitTM&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>CC-4124&amp;nbsp;</td><td>Medium supplements&amp;nbsp;</td></tr><tr><td>SB2001190&amp;nbsp;</td><td>Tocris&amp;nbsp;</td><td>1264/10</td><td></td></tr><tr><td>Serological pipettes&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>2 mL, 5 mL, 10 mL, and 25 mL low endotoxin, sterile&amp;nbsp;</td></tr><tr><td>Small Airway Epithelial Cell Growth medium (SAGM)</td><td>Lonza&amp;nbsp;</td><td>CC-4124&amp;nbsp;</td><td></td></tr><tr><td>Solvent Buffer (ER-2)&amp;nbsp;</td><td>Emulate&amp;nbsp;</td><td>10462</td><td>25 mL bottle&amp;nbsp;</td></tr><tr><td>Steriflip-HV&amp;nbsp;</td><td>Millipore</td><td>SE1M003M00</td><td>Sterile filtering conical tube</td></tr><tr><td>Sterilin 100 mm Square Petri Dishes</td><td>Thermo</td><td>103</td><td>Sterile, 1 per 6 chips&amp;nbsp;</td></tr><tr><td>T25 flasks&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-</td></tr><tr><td>T75 flasks&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>Tri-iodothyronine</td><td>Sigma</td><td>T5516</td><td></td></tr><tr><td>Triton X-100 (0.3% (vol/vol)&amp;nbsp;&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>T8787&amp;nbsp;</td><td>Permeabilization agent&amp;nbsp;</td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>93595&amp;nbsp;</td><td>0.4% solution&amp;nbsp;</td></tr><tr><td>TrypEE solution&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>12604013&amp;nbsp;</td><td>Cell detaching solution&amp;nbsp;</td></tr><tr><td>TWEEN-20&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P2287&amp;nbsp;</td><td>Permeabilization agent&amp;nbsp;</td></tr><tr><td>UV Light Oven (peak frequency 365nm, intensity of 100 &amp;micro;J/cm&lt;font class=&quot;font6&quot;&gt;&lt;sup&gt;2&lt;/sup&gt;&lt;/font&gt;&lt;font class=&quot;font5&quot;&gt;)&lt;/font&gt;</td><td>VWR</td><td>21474-598</td><td>UVP, Long Range UV, 365 nm 60Hz Model CL-1000L</td></tr><tr><td>Vacuum set-up&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>Minimum pressure: -70 kPa&amp;nbsp;</td></tr><tr><td>Vascular Endothelial Growth Factor 165 (VEGF-165) human, recombinant in E. coli</td><td>PromoCell</td><td>C-64420</td><td></td></tr><tr><td>VEGF-165&amp;nbsp;&amp;nbsp;</td><td>PromoCell&amp;nbsp;&amp;nbsp;</td><td>C-64420&amp;nbsp;</td><td>Medium supplement&amp;nbsp;</td></tr><tr><td>Von Willebrand Factor conjugated FITC&amp;nbsp;&amp;nbsp;</td><td>ABCAM&amp;nbsp;</td><td>ab8822&amp;nbsp;</td><td>Sheep polyclonal antibody&amp;nbsp;</td></tr><tr><td>Water bath (or beads)&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td><td>Set to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Wnt3A (L-Wnt3A conditioned medium)</td><td>ATCC</td><td>CRL-2647</td><td></td></tr></tbody></table>

reconstituting cytoarchitecture and function of human epithelial tissues on an open-top organ-chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

Surface micropatterning 
Micropatterning of the extracellular matrix (ECM) can be used to replicate the spatial configuration of the intestinal crypt interface. The Open-Top Chip configuration can be modified to integrate micropatterned stamps specifically designed to mimic the natural topography of the colonic epithelium-stroma interface (Figure 6A,B) and the intestinal crypts at micrometer scale (Figure 6C-</stron...

Watch this Scientific Journal Video about Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip at JoVE.com

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

reconstituting-cytoarchitecture-function-human-epithelial-tissues-on

Research

JoVE Journal

Bioengineering

1.7K Views.  Merk Sharp & Dohme LLC. The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Video: Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biology

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Enhanced Intestine-on-Chip Platform for Immunological Insight

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...