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.

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			<td>0.05% Trypsin/EDTA</td>
			<td>Welgene</td>
			<td>LS015-01</td>
			<td></td>
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			<td>100x Penicilin/Streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
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		<tr>
			<td>20x PBS</td>
			<td>LPS solution</td>
			<td>CBP007A</td>
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			<td>4% Paraformaldehyde</td>
			<td>Biosesang</td>
			<td>PC2031-050-00</td>
			<td></td>
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		<tr>
			<td>Alexa Fluor-594 conjugated ZO-1 antibody</td>
			<td>Invitrogen</td>
			<td>339194</td>
			<td></td>
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			<td>Antibody diluent OP Quanto</td>
			<td>Epredia</td>
			<td>10129-576</td>
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			<td>CCK-8 assay kit</td>
			<td>Donginbio</td>
			<td>CCK-3000</td>
			<td></td>
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		<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>
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			<td>Conical tube</td>
			<td>SPL</td>
			<td>50050</td>
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			<td>Cover glass</td>
			<td>Corning</td>
			<td>CLS2980245</td>
			<td></td>
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		<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>
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		<tr>
			<td>DURAN Desiccator bases with plane flange, screw thread</td>
			<td>DWK Life Sciences&#160;</td>
			<td>7.022 260</td>
			<td></td>
		</tr>
		<tr>
			<td>DURAN Glass Desiccator Lid &#38; 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&#160;</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&#160;</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&#160;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>
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		<tr>
			<td>Slide glass</td>
			<td>Marienfeld Superior</td>
			<td>K15663717</td>
			<td></td>
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			<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>
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</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

133 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 

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Methods Article

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