This work was supported by a grant from the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (HR16C0001); the Basic Science Research Capacity Enhancement Project through the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2019R1A6C1010003); and National Research Foundation of Korea (NRF) grants funded by the Korean Government (MSIT) (2022R1A4A5032702).

1. Fabrication of water-stable PVA nanofibers
<ol>
	<li>Weigh 0.02 g of poly(acrylic acid) (PAA) and 1 g of PVA, then add them into a clean glass bottle containing a magnetic bar. Add 7.8 mL of distilled water to the bottle, place the bottle on a plate stirrer, and set the temperature to 84 &#176;C, the speed to 500 rpm, and the time to 12 h.</li>
	<li>Let the bottle cool to room temperature before adding 0.2 mL of glutaraldehyde (GA). Place the bottle on the plate stirrer, and set the speed to 500 rpm, time to 30 min, and temperature to room temperature.</li>
	<li>Use a 5 mL syringe for electrospinning, separate the plungers from the barrels, and clean small plastic debris inside the barrels and the tip of plungers.</li>
	<li>Use needles to block the liquid flow, then pour PVA solution into each barrel (volume: 2.5-3 mL each). Assemble the plungers, then remove the needles. 
	NOTE: There might be a bubble in the solution, so wait until the bubble disappears. This takes 10-30 min.</li>
	<li>Assemble a new 27 G metal needle, then place it into the electrospinning machine.</li>
	<li>Wrap the collector well with foil.</li>
	<li>Let the injector panel return to the original position. Use the following settings for machine: (each pump) injection volume: 2 mL, injection rate: 6 &#181;L/min, roller speed: 100 rpm, tip to collector distance: 14 cm. Check the liquid flow for each pump.</li>
	<li>Run the machine with an electrical potential of approximately 12 kV.</li>
	<li>Ensure that the humidity is 40 - 50% in the beginning and 25 - 30% at the end of the process. Use a humidifier when the humidity decreases and place dishes containing distilled water inside the machine the day before electrospinning to increase humidity. 
	NOTE: This is a critical factor in the successful fabrication of PVA nanofibers.</li>
	<li>Check the flow regularly. If the needle is blocked, temporarily stop the machine. Use a laboratory wipe to remove the drop and wait until the new drop appears. If not, change the needle, then continue running the machine.</li>
	<li>The process of PVA nanofiber fabrication takes ~6 h to complete. After the electrospinning process finishes, detach the foil containing the fiber mat from the collector and store the nanofiber mat in desiccant under vacuum.</li>
</ol>
2. Treatment of a PVA nanofibrous membrane with hydrochloric acid (HCl) vapor and assessment of fiber quality
NOTE: The water stability of PVA NMs is well preserved after the post-crosslinking reaction by HCl vapor. This process is optimized for suitable thickness, pore size, and roughness of the NMs. PVA is a very hydrophilic polymer, and electrospun PVA nanofibers are unstable in water contact and humid conditions. PVA NMs should be treated with HCl vapor just after the fabrication of the PVA polymer.
<ol>
	<li>Cut a nanofiber mat into pieces of the desired size for the trans-well insert.</li>
	<li>Prepare a small dish containing 1.5 mL of pure HCl. Place the HCl-containing dish and nanofiber pieces inside a vacuum chamber and close the lid. 
	NOTE: HCl vapor is used as a postcrosslink agent to make the nanofiber water-stable.</li>
	<li>Open the valve connected to the vacuum pump, wait 10 s until the HCl fume rises, and then close the valve. Treat the membranes with HCl fume for 2 min.</li>
	<li>After treatment with HCl vapor, add some drops of distilled water (DW) to the membranes, put them on a glass slide, and remove the membrane from the foil. After air drying for 20 min, the membranes naturally stick to the slide.</li>
	<li>Coat the membranes on carbon tape with a thin layer of gold using an autosputter coater. Obtain scanning electron microscope (SEM) images.</li>
	<li>After crosslinking PVA NMs with HCl, wash them 3x with 1x PBS, and check the pH after soaking them in culture media to confirm the absence of residual HCl.</li>
</ol>
3. Fabrication of PCL nanofibers and nanofiber quality assessment
<ol>
	<li>Weigh 1.5 g of PCL and add it to a clean glass bottle. Add 8.5 mL of chloroform to the bottle, place the bottle on a plate stirrer, and dissolve PCL in chloroform for 12 h at room temperature.</li>
	<li>Repeat steps 1.3 to 1.9, except for steps 1.7 and 1.8, set the injection rate at 8 &#181;L/min and the voltage at 17.5 kV. 
	NOTE: PCL fabrication takes ~3.5 h to finish.</li>
	<li>Cut a small piece of the membrane and coat the pieces by auto-sputter process for 5 min. Obtain SEM images.</li>
</ol>
4. Assembly of two layers of membranes in a trans-well
NOTE: In this protocol, we use a 24-well plate and its respective insert well. The PVA NM is attached to the insert well, while the PCL NM is attached to the bottom well.
<ol>
	<li>Cut a nanofiber mat into round pieces, with a diameter of 10 mm and 13 mm for PVA and PCL NMs, respectively.</li>
	<li>Use polydimethylsiloxane (PDMS) Sylgard 184 as an adhesive agent to attach the membranes to the insert and chamber well. Before using, mix Sylgard 184 Base (part A) and Curing Agent (part B) in a plastic cup in a 10:1 mass ratio. Use a clean glass slide to mix the PDMS solution vigorously for 2-3 min until the entire mixture is filled with bubbles. Let the bubble form and disappear for 10 min before using; meanwhile, let the slide warm to 100 &#176;C.</li>
	<li>Spread the PDMS on a glass slide, and dip the bottom edge of the insert well slightly in the PDMS. Keep the insert well upside down on the slide warmer for the PDMS to harden (~10-15 min). Carefully check with a 200 &#181;L tip to make sure the PDMS is not sticky.</li>
	<li>Treat the PVA NM pieces as described in steps 2.2 to 2.4.</li>
	<li>Gently press the insert well onto the PVA NM piece so that the bottom of the insert well faces the nanofiber side of the membrane piece. Add a drop of distilled water to the center of the well and use forceps to remove the foil. Put the insert well in a new 24-well plate, and let the membrane dry.</li>
	<li>When attaching the PCL NM piece to the bottom well, be sure to dip the 200 &#181;L tip in the PDMS mixture and dot it precisely at the four corners and in the center.</li>
	<li>Place the plate on the slide warmer and allow the PDMS to harden for 5-7 min to ensure the quality of the assembly.</li>
	<li>Put the PCL NM piece into the PDMS-containing well, with the nanofiber side of the membrane attached to the PDMS. Use a small clean brush to press the foil side of the membrane piece slightly for complete attachment. Add 0.2 mL of 70% ethanol to the well to easily remove the foil.</li>
	<li>Sterilize the insert and the bottom well in the trans-well setup under UV light overnight. 
	NOTE: The mixed PDMS can be stored at -20 &#176;C for further use. CAUTION: The PCL nanofibrous membrane-attached plate can be stored dry but must be wet with 70% ethanol before use.</li>
	<li>The next day, wash the insert and bottom well by adding 200 &#181;L and 500 &#181;L of 1x phosphate-buffered saline (PBS), respectively, for 3 x 10 min. The insert and bottom well are ready to use.</li>
</ol>
5. Cell seeding and assembly of two layers for the coculture model
NOTE: MLE-12 cells were cultured in 1x Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotic. NIH3T3 cells were cultured in 1x DMEM high glucose with 10% FBS and 1% penicillin/streptomycin antibiotic.
<ol>
	<li>Maintain the cells in a 150 mm culture dish. When they are 70-80% confluent, wash the cells with 10 mL of 1x PBS and trypsinize them with 2 mL of prewarmed 0.05% trypsin/EDTA solution for 3 min at 37 &#176;C.</li>
	<li>Gently tap the dish to dislodge the cells from the bottom, and then add 8 mL of complete culture media to each type of cell to neutralize the trypsin enzyme. Transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 400 &#215; g for 5 min, and discard the supernatant. Stain the cells with Cell-Tracker to observe the distribution of seeded cells on NM (see section 6 for more details).</li>
	<li>Resuspend the cell pellet in 5 mL of complete DMEM/F12 media. Determine the cell number and adjust to 6 &#215; 106 MLE-12 cells/mL and 8 &#215; 105 NIH3T3 cells/mL. 
	NOTE: Complete DMEM/F12 media is used for coculture with MLE-12 and NIH3T3 cells.</li>
	<li>Fill the bottom well with 500 &#181;L of complete DMEM/F12 media (the well without PCL). Transfer the PVA nanofibrous membrane-attached insert to the trans-well, and add 50 &#181;L of MLE-12 cell suspension. Incubate the cells at 37 &#176;C&#160;and 5% CO2.</li>
	<li>Simultaneously, fill the PCL nanofibrous membrane-attached bottom well with 500 &#181;L of NIH3T3 cell suspension and incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>After 2 h, assemble the MLE-12 cell-containing insert and the NIH3T3 cell-containing bottom well in the trans-well setup. This is the coculture model with MLE-12 cells as the upper layer and NIH3T3 cells as the lower layer. Coculture the cells in the two-layered membranes for the desired time.</li>
</ol>
6. Cell distribution assay
<ol>
	<li>Prepare 5 &#181;M of Cell-Tracker red and green solution in a total volume of 2 mL of complete DMEM/F12 medium.</li>
	<li>After trypsinization, mix MLE-12 cells with previously prepared Cell-Tracker red solution and NIH3T3 with Cell-Tracker green solution. Incubate the cells at 37 &#176;C and 5% CO2 for 30 min to ensure optimal cell labeling. Add 8 mL of complete DMEM/F12 medium to the samples and centrifuge at 400 &#215; g for 5 min. 
	NOTE: Cell Tracker cannot affect cell viability and proliferation. Hence Cell-Tracker-stained cells can be cultured until confluence. The fluorescence intensity in the stained cells might reduce 4-5 days after culture.</li>
	<li>Repeat steps 5.3 to 5.6.</li>
	<li>While observing under a confocal microscope, transfer the insert well from the 24-well plate to a 12-well plate. 
	NOTE: This minimizes the distance between the objective lens and the cells, allowing for the observation of cell morphology.</li>
</ol>
7. Confocal observations of 3D cocultured cells
NOTE: Epithelial cell attachment to a PVA NM is weak compared to that of fibroblasts to a PCL NM. Discard old media manually by pipette aspiration and add new media or solution gently to the wall of wells. During the staining process, try not to let the membranes dry.
NOTE: Cell attachment can be improved by coating laminin or incorporating peptides of integrin-binding motifs in PVA nanofibers19.
<ol>
	<li>Transfer the insert to a new 24-well plate. Discard the old media and add 150 &#181;L of a 4% paraformaldehyde (PFA) solution. Incubate for 15 min at room temperature.</li>
	<li>Remove the 4% PFA solution and wash the adhered cells with 150 &#181;L of 1x PBS.</li>
	<li>For blocking and permeabilization, add 150 &#181;L of 5% normal goat serum and 0.2% Triton X-100 diluted in 1x PBS and incubate for 30 min at room temperature.</li>
	<li>Prepare the Alexa Fluor 594-conjugated anti-zona occludens Ab (1:400) and Phalloidin-iFlour 488 (1:1,000) by diluting them in antibody diluent reagent. Remove the blocking solution, add 150 &#181;L of the fluorophore solution per well, and incubate for 1 h at room temperature. 
	NOTE: Prepare the antibody solutions with utmost precision, as it will directly impact the quality of the results. Avoid exposure to light.</li>
	<li>Discard the antibody solution and wash the cells with 150 &#181;L of 1x PBS for 10 min.</li>
	<li>Add 150 &#181;L of Hoechst 33342 diluted in 1x PBS (1:1,000) and incubate for 10 min; avoid exposure to light.</li>
	<li>Remove the Hoechst 33342 solution and wash the adhered cells with 150 &#181;L of 1x PBS for 10 min.</li>
	<li>Prepare a clean glass slide and add a precise amount of aqueous mounting gel. Use forceps with pointed tips to delicately detach the membranes from the surrounding edge. Place the membranes on the mounting gel, ensuring the side containing the cells is facing upward. Add a precise amount of mounting gel and cover with a cover slide. Seal the slides with nail polish, ensuring a precise and complete seal.</li>
	<li>Observe cells under a confocal microscope using the following imaging settings: Laser Power control: 405 Intensity 10; Laser Power control: 488 Intensity 10; Laser Power control: 561 Intensity 10; Detector sensitivity: 420LP (600 value); Detector sensitivity: 525/50 (700 value); Detector sensitivity: 561LP (750 value).</li>
</ol>
8. Assay of cell attachment to nanofibers
<ol>
	<li>Discard the old media and wash the cells with 150 &#181;L of 1x PBS.</li>
	<li>Fix the cells with 150 &#181;L of 3% GA diluted in 1x PBS for 1 h at room temperature.</li>
	<li>Wash the cells with 150 &#181;L of 1x PBS in 10 min.</li>
	<li>Fix the cells secondarily by adding 150 &#181;L of 1% osmium tetroxide (OsO4) diluted in 1x PBS for 1 h. 
	CAUTION: OsO4 and its vapor are harmful. It must be used in a fume hood.</li>
	<li>Discard the solution and wash the cells with 300 &#181;L of 1x PBS for 2 x 10 min.</li>
	<li>Dehydrate the cells with graded ethanol from 50%, 70%, 90%, and 95% to 100%, for 10 min in each step. Perform the last dehydration step by adding fresh 100% ethanol for 10 min.</li>
	<li>Air dry the cells and membranes completely. Use forceps with the pointed tips to detach the membranes, and place the membranes on the specimen holder using a two-side tap. Perform ion-sputtering for 5 min before observing the cells under SEM at 2,000x magnification.</li>
</ol>
9. Cell growth assay
NOTE: In this protocol, the CCK-8 assay is used to quantify cell growth rate following the manufacturer&#39;s instructions.
<ol>
	<li>Prepare the CCK-8 mixture by mixing 9 parts of prewarmed DMEM/F12 (by volume) with 1 part of the CCK-8 reagent. After removing the old culture media, add 150 &#181;L of the CCK-8 mixture to the cell-containing well.</li>
	<li>Transfer the insert to the new 24-well plate to separate it from the bottom well containing cells. Incubate the cells for 45 min at 37 &#176;C and 5% CO2.</li>
	<li>Collect the supernatant and transfer it to a 1.5 mL tube. Centrifuge at 1,800 &#215; g for 3 min.</li>
	<li>Add 100 &#181;L of the supernatant from the microcentrifuge tube to the 96-well plate. Read optical density (OD) at 450 nm.</li>
</ol>

Advanced Two-Layered 3D Co-Culture System for Epithelial and Fibroblast Cells

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P., Martinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hope+and+the+hype+of+organoid+research.">The hope and the hype of organoid research.</a> Development. 144 (6), 938-941 (2017).</li><li>Luo, L., Liu, L., Ding, Y., Dong, Y., Ma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+biomimetic+hydrogels+for+organoid+culture.">Advances in biomimetic hydrogels for organoid culture.</a> Chem Commun. (Camb). 59 (64), 9675-9686 (2023).</li></ol>

The authors declare no conflicts of interest.

The transition from 2D culture to 3D culture models represents a significant advancement in developing in vitro coculture models. A 3D culture model must mimic the tissue microenvironment in which cells can proliferate, aggregate, and differentiate22. The use of scaffold-based 3D coculture models enhances the replication of tissue architecture. In this study, an NM-based trans-well system provides indirect 3D coculture of two types of cells. The PCL NM with large pores in the lower chambe...

Over the decades, the classic two-dimensional (2D) monolayer culture has been essential for understanding infections, pharmacology, and toxicology1,2. However, it is important to consider the complexity, dynamic interactions between multi-compositions, and three-dimensional (3D) architecture of the tissue microenvironment. Thus, the 2D cell culture has limitations in mimicking tissue-like structures3,4. For example, there is a lack of cell-to-cell and cell-to-extracellular matrix (ECM) signaling, which&#160;is essential in cell differentiation, proliferation, and cellular functions in the vivo microenvironment. For these reasons, the systems of 3D cell cultures have been developed5. The cells in 3D culture grow and interact with the surrounding extracellular matrix in three dimensions. Furthermore, a 3D coculture approach, composed of more than one cell type, has been developed to bridge the gap between simplistic single-cell type in vitro models and the dynamic cellular interactions that occur in vivo6. 3D coculture systems have gained popularity in the study of cell-cell communication and cell-extracellular matrix (ECM) interactions7,8,9,10. Current 3D coculture systems have several forms and can be separated into direct and indirect coculture8,9. In direct coculture, various cell functions in cultured cells can be mainly affected by direct contact rather than paracrine effects. In comparison, indirect coculture has a greater advantage in investigating paracrine interaction by using barriers or layers to separate different cell types.
Various materials and methods are utilized to fabricate biomimetic scaffolds mimicking the ECM using biocompatible and biodegradable substances10. The nanofibrous scaffold structure mimics the configuration of native ECM in biological tissues, providing a 3D architecture for cell culture11,12. Electrospun nanofibrous membranes (NMs) have been used in direct and indirect coculture of different types of cells13,14,15.
Epithelial cells form a physical barrier of the major organs, whereas fibroblasts are predominant stroma cells that mediate ECM remodeling and regulate the neighboring epithelium6,16,17. Epithelial cells and fibroblasts interact with laminin in the basement membrane and fibronectin in the interstitial matrix, respectively, through integrin receptors on the cell surface18. When epithelial cells are cultured on the poly(vinyl alcohol) (PVA) NMs with diameters of 150 - 250 nm and micropores, they form multi-layers instead of cell aggregates and spheroids, and their growth patterns are similar to those of the cells in epithelial tissue19. In comparison, the poly(caprolactone) (PCL) NMs with 400-1,500 nm diameters and 10-50 &#181;m micropores provide a similar spatial dimensionality to the interstitial matrix for the growth of cultured fibroblasts13,14. Due to their biocompatibility, PVA and PCL membranes are commonly used polymers in tissue engineering20,21.
This study introduces an advanced two-layer culture system using porous electrospun NMs to cultivate epithelial cells as tissue-like layers and fibroblasts within a 3D structure. The protocol can be divided into five key stages: nanofiber fabrication, fiber post-crosslink and quality assessment, preparation of membrane-attached culture well, cell seeding and 3D coculture, and assays of 3D cultured cells.

<table><tbody><tr><td>0.05% Trypsin/EDTA</td><td>Welgene</td><td>LS015-01</td><td></td></tr><tr><td>100x Penicilin/Streptomycin</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>20x PBS</td><td>LPS solution</td><td>CBP007A</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Biosesang</td><td>PC2031-050-00</td><td></td></tr><tr><td>Alexa Fluor-594 conjugated ZO-1 antibody</td><td>Invitrogen</td><td>339194</td><td></td></tr><tr><td>Antibody diluent OP Quanto</td><td>Epredia</td><td>10129-576</td><td></td></tr><tr><td>CCK-8 assay kit</td><td>Donginbio</td><td>CCK-3000</td><td></td></tr><tr><td>Cell culture dish (150x20)</td><td>SPL</td><td>20151</td><td></td></tr><tr><td>CellTracker Green CMFDA</td><td>Invitrogen</td><td>C7025</td><td></td></tr><tr><td>CellTracker Red CMTPX</td><td>Invitrogen</td><td>C34552</td><td></td></tr><tr><td>Chloroform</td><td>Samchun</td><td>C0584</td><td></td></tr><tr><td>Conical tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>Cover glass</td><td>Corning</td><td>CLS2980245</td><td></td></tr><tr><td>DMEM high glucose</td><td>Welgene</td><td>LM001-05</td><td></td></tr><tr><td>DMEM/F12</td><td>Welgene</td><td>LM 002-05</td><td></td></tr><tr><td>DURAN Desiccator bases with plane flange, screw thread</td><td>DWK Life Sciences&amp;nbsp;</td><td>7.022 260</td><td></td></tr><tr><td>DURAN Glass Desiccator Lid &amp;amp; Stopcock</td><td>Daihan Science</td><td>SM.2444061</td><td></td></tr><tr><td>DURAN Stopcock, with PTFE Spindle</td><td>DWK Life Sciences&amp;nbsp;</td><td>10322671</td><td></td></tr><tr><td>Electrospinning machine</td><td>NanoNC</td><td>ESR200RD</td><td></td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma Aldrich</td><td>459844</td><td></td></tr><tr><td>FBS</td><td>Sigma Aldrich</td><td>TMS-013-BKR&amp;nbsp;</td><td></td></tr><tr><td>Fluorescence Laser Confocal Scanner Module K1-fluo</td><td>Nanoscope Systems</td><td></td><td></td></tr><tr><td>Gel/mount</td><td>Biomeda&amp;nbsp;Corp.</td><td>M01</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma Aldrich</td><td>340855</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H1399</td><td></td></tr><tr><td>Metal nozzle 27G</td><td>NanoNC</td><td></td><td></td></tr><tr><td>Nail polish</td><td>Nature republic</td><td></td><td></td></tr><tr><td>Normal goat serum</td><td>Vector Laboratories</td><td>S-1000</td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma Aldrich</td><td>201030</td><td></td></tr><tr><td>Phalloidin-iFlour 488</td><td>abcam</td><td>ab176753</td><td></td></tr><tr><td>Plastic Syringe_Lure Lock (10 mL)</td><td>HENKE SASS WOLF</td><td>AL10</td><td></td></tr><tr><td>Poly(acrylic acid)</td><td>Sigma Aldrich</td><td>323667</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma Aldrich</td><td>341584</td><td></td></tr><tr><td>Polycaprolactone</td><td>Sigma Aldrich</td><td>440744</td><td></td></tr><tr><td>Pure HCL</td><td>Duksan</td><td>1129</td><td></td></tr><tr><td>Scanning Electron Microscopy</td><td>SEC</td><td>SNE-4500M</td><td></td></tr><tr><td>Slide glass</td><td>Marienfeld Superior</td><td>K15663717</td><td></td></tr><tr><td>Sylgard 184 set</td><td>omniscience</td><td>OMNI.05255</td><td></td></tr><tr><td>Synergy H1 Multimode Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td></td></tr></tbody></table>

establishing a three-dimensional coculture module of epithelial cells using nanofibrous membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

This protocol outlines the critical steps for coculturing MLE-12 lung epithelial cells and NIH3T3 fibroblasts by constructing a nanofibrous membrane-based two-layer model (Figure 1). This model suits live cell imaging, immunohistochemistry, and endpoint quantitative analysis of cocultured cells. Other cell types may also be utilized by optimizing cell density and growth conditions.
SEM shows the uniform distribution of fabricated PVA nanofibers without any bead fo...

Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

establishing-three-dimensional-coculture-module-epithelial-cells

Research

JoVE Journal

Bioengineering

207 Views.  Ajou University. Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane. 

Video: Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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, ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

Developmental Biology

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Cancer Research

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Biology

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...