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

Our research group uses poly vinyl alcohol, PVA, and poly epsilon-caprolactone, PCL, nanofibrous membranes to develop a 3D coculture system. It aims to answer how the membranes support epithelial and fibroblast cells mimics tissue environments and facilitate the study of patterning signalings and cellular interactions. We will show that porous electro spun nanofibrous membranes allow epithelial cells to form epithelial tissue, allowing multi-layers, and fibroblasts to grow in each additional matrix.

The methods highlight how an expanded 3D system can provide growing tissue with better environments compared to traditional to 2D cultures. We offer a coculture system employing differentiated epithelial cells. Organizing using stem cells requires complex differentiation procedure, but our model has advantages in simplicity and time savings in the development of a 3D coculture system.

We focus on overcoming the limitation of nanofibrous scaffolds by combining nanofibrous membranes with over layer hydrogels in the cocultures. To begin, add 0.02 grams of polyacrylic acid and one gram of PVA to a clean glass bottle containing a magnetic bar. Add 7.8 milliliters of distilled water to the bottle, then place the bottle on a plate stirrer.

Once the bottle cools to room temperature, add 0.2 milliliters of glutaraldehyde to it and place it back on the plate stirrer at 500 RPM for 30 minutes at room temperature. Use a five milliliter syringe for electro spinning. Separate the plungers from the barrels and clean small plastic debris inside the barrels and the tips of the plungers.

Next, use needles to block the liquid flow and pour the PVA solution into each syringe barrel. Assemble the plungers into the barrels and remove the needles. After assembling a new 27 gauge metal needle, place it into the electro spinning machine.

Wrap the collector tightly with aluminum foil. Once the injector panel returns to its original position, set the electro spinning machine to an injection volume of two milliliters, injection rate of six microliters per minute, roller speed of 100 RPM, and tip to collector distance of 14 centimeters. Check the liquid flow for each pump, then run the electro spinning machine with an electrical potential of approximately 12 kilovolts.

After the electro spinning process is complete, detach the foil containing the fiber mat from the collector. Store the nanofiber mat in a desiccant under vacuum. Add 1.5 grams of PCL and 8.5 milliliters of chloroform to a clean glass bottle.

Place the bottle on a plate stirrer and dissolve the PCL in chloroform for 12 hours at room temperature. Cut a small piece of the nanofiber membrane, coat the pieces with a thin gold layer using the auto sputter process for five minutes. To begin, cut the fabricated PVA nanofiber mat into pieces of the desired size for the transwell insert.

Prepare a small dish containing 1.5 milliliters of pure hydrochloric acid. Place the dish with hydrochloric acid and the nanofiber pieces inside a vacuum chamber and close the lid. Then open the valve connected to the vacuum pump and wait for 10 seconds until hydrochloric acid fumes rise.

Close the valve and treat the membranes with hydrochloric acid fumes for two minutes. After the hydrochloric acid treatment, add drops of distilled water to the membranes. Place the membranes on a glass slide and remove them from the foil.

Use an auto sputter coater to coat the membranes on carbon tape with a thin layer of gold. Then wash the PVA nano membranes cross-linked with hydrochloric acid three times with PBS and soak them in the culture media. After soaking, check the pH to confirm the absence of residual hydrochloric acid.

To begin, cut the nanofiber mats into round pieces with a diameter of 10 millimeters for PVA nano membranes and 13 millimeters for PCL nano membranes. Mix SYLGARD 184 base and curing agent in a 10 to one mass ratio in a plastic cup. Use a clean glass slide to vigorously mix the polydimethylsiloxane, or PDMS solution, for two to three minutes until bubbles form.

Spread the PDMS evenly on a glass slide. Dip the bottom edge of the insert well slightly into the PDMS. Then place the insert well upside down on the slide warmer for 10 to 15 minutes until the PDMS hardens.

Check the PDMS with a 200 microliter pipette tip to ensure it is no longer sticky. Now, gently press the insert well onto the PVA nano membrane piece, so that the bottom of the insert well faces the nanofiber side of the membrane. Add a drop of distilled water to the center of the well and use forceps to carefully remove the foil.

Then place the insert well into a new 24 well plate and allow the membrane to dry. When attaching the nano membrane piece, dip a 200 microliter pipette tip into the PDMS mixture and dot it precisely at the four corners and the center of the well. Place the plate on the slide warmer and allow the PDMS to harden for five to seven minutes.

Next, place the PCL nano membrane piece into the PDMS containing well with the nanofiber side attached to the PDMS. Use a small clean brush to press the foil side of the membrane slightly for complete attachment. Then add 0.2 milliliters of 70%ethanol to the well to facilitate foil removal.

Sterilize the insert and bottom well in the transwell setup under ultraviolet light overnight. On the next day, wash the insert and bottom well by adding 200 microliters of PBS to the insert and 500 microliters to the bottom well for three cycles of 10 minutes each. Once the cells attain 70 to 80%confluency, wash them with 10 milliliters of PBS and add two milliliters of 0.05%trypsin EDTA to trypsinise them.

Tap the dish gently to dislodge the cells from the bottom. Add eight milliliters of complete culture media to neutralize the trypsin enzyme, then transfer the cell suspension to a 50 milliliter centrifuge tube. Centrifuge the tube at 400G for five minutes.

After discarding the supra natant, re-suspend the cell pellet in five milliliters of complete DMEM with F12 media. Then fill the bottom well with 500 microliters of complete DMEM F12 media. Transfer the PVA nanofibrous membrane attached insert to the transwell and add 50 microliters of MLE-12 cell suspension.

Next, fill the PCL nanofibrous membrane attached bottom well with 500 microliters of NIH3T3 cell suspension. After incubating the cells for two hours, assemble the MLE-12 cell containing insert and the NIH3T3 cell containing bottom well in the transwell setup, The spatial distribution of NIH3T3 cells and MLE-12 cells in the coculture system was clearly distinguishable, with green NIH3T3 cells dispersed at varying depths and red MLE-12 cells forming layers. NIH3T3 cells infiltrate the PCL nanofibrous membrane and extend along the nanofiber axes, while MLE-12 cells adhere to the surface of the PDA nanofibrous membrane and exhibit an aggregation.