The overall goal of this protocol is to describe a polyethylene glycol and zinc oxide templating method to create a porous, ultra-thin, biomimetic basement membrane that supports dual cell culture, and has tuneable mechanical and chemical properties. The main advantage of this technique is that we can create hydrogels that are ultra-thin, porated, mechanically and biochemically tuneable. They allow support of either single or dual cell culture.
This method can help answer key questions in the tissue engineering field, including how dual cell cultures react to, and interact with, microenvironments that mimic human basement membranes. The implications of this technique extend toward a wide variety of biological systems and tissues, ultimately allowing more biomimetic research with enhanced translation for human disease. Add 250 milliliters of zinc nitrate to a flask and begin stirring the reaction.
Then, add 150 milliliters of sodium hydroxide solution. A white precipitate will form briefly and then disappear as the two solutions continue to mix. Stir the mixture for two hours uncovered.
Heat the flask to 55 to 60 degrees celsius and continue stirring for 24 hours. Turn off the heat and allow solution to cool to room temperature while stirring. Filter the solution though a Buchner funnel with an 11 micron pore size and allow it to dry overnight, uncovered.
When the filter paper is no longer wet from the poured solution and a white powder has formed over the funnel holes, the particles are fully dried. After checking the needle leg morphology of zinc oxide needles, as described in the text protocol, clean three inch by one inch plain microscope slides with 70 percent ethanol and a disposable wipe. Hold the glass slide with tweezers and use a glass pipet with a pipet bulb to coat the slides with five drops of zinc acetate forming a thin layer dispersed on the slide.
Allow excess solution to drip back into the stock solution. Then, place the slide into a preheated Petri dish on a hot plate with the zinc acetate coated side facing up for 15 minutes. Remove the slide with tweezers and allow it to cool to room temperature.
The slide will appear to be coated with white streaks. Remove any excess zinc that is remaining on the slides, using a disposable wipe to remove permanent white streaks. The surface should be uniform.
Suspend 20 milligrams of zinc oxide needles in 270 microliters of ten percent FBS/PBS solution. Vortex to mix for approximately 15 seconds. Briefly spin the solution in a bench top mini centrifuge to bring any zinc oxide aggregates to the base of the tube.
Following centrifugation, collect 250 microliters from the top of the zinc oxide solution to ensure that aggregates remain at the bottom of the tube. Combine the collected zinc oxide solution with PEG diacrylate and PEG-RGD to create a PEG solution with approximately 2.5 millimolar RGD. Then vortex to mix for approximately 15 seconds.
Next, add two microliters of acetophenone and vinyl pyrrolidone photoinitiator and vortex briefly to mix. Add 20 microliters of the polymer solution along the center of the zinc oxide coated slides. Slowly lower a second slide on top, with the zinc oxide coating facing down so that the PEG solution is between two zinc oxide coated layers.
Try to prevent the formation of any air bubbles. Move the slides laterally along their axis to allow any bubbles to escape and to spread the solution into a thin layer. Create a slight overhang with the top slide to ensure that the slides can be pulled apart in their later steps.
Finally, cross link the slides under a 365 nanometer UV lamp for 15 minutes. Apply pressure to the overhanging slide and manually pull the two slides apart at a slow pace in order to avoid cracking the slides. Allow the gels to dry for at least five minutes or overnight.
Once the gels are dry, place the slide in a sterile 25 milliliter glass Petri dish and gently pour the one molar HCL solution onto the slide using only enough to cover the slide. Gently rock the Petri dish. The gel should begin to lift off the slide as the HCL dissolves the sacrificial zinc coating and needles.
Once the gel is free from the slide, pour the HCL back into the stock solution. Rinse the gel by gently pouring approximately 25 milliliters of 1x PBS into the Petri dish until the slide and gel are submerged. Carefully pour off PBS into a waste container.
Then, gently pour approximately 25 milliliters of 1x PBS into the Petri dish until the gel is floating in the solution. Use tweezers to carefully slide the silicone isolator under the gel and lift the gel onto it. Transfer the isolator and the gel into a six well dish filled with sterile 1x PBS.
Prepare A549 cells as detailed in the text protocol. While cells are spinning down, add a small drop of complete Dulbecco's Modified Eagle Medium to each well of a six well plate. Use tweezers to transfer the silicone isolators with the gels, into a new six well plate such that the gel is resting on the drop of media.
Now that the gels are spread into a thin layer, check to ensure that there are no large holes visible to the eye. Add the resuspended cell suspension drop-wise to the center of the gel, trying to maintain a meniscus in the punched out area of the silicone membrane. After allowing the cells to adhere for four hours, add 0.5 milliliters of Dulbecco's Modified Eagle Complete Medium to the well.
Incubate overnight at 37 degrees Celsius to allow for complete adhesion. The following day, prepare huvecs for cell seeding as described in the text protocol. Then, add a small drop of Complete Medium 199 to each well in a new six well plate.
Place a new silicone isolator in the well with a drop of media in the center of the silicone. Using tweezers, carefully flip the silicone isolators supporting the peg gel with A459 cells onto the isolator in the new six well plate. Add the huvec suspension to the center of the flipped gel drop-wise to maintain a meniscus on the gel within the punched out area of the silicone membrane.
After allowing the cells to adhere for two hours, gently add two milliliters of M199 Complete Media to each well. Use a manual pipet to carefully remove the media from the gels, and add approximately 500 microliters of four percent PFA to the center of the gel where the cells are seeded. After the PFA sits for 30 minutes at room temperature, carefully remove the PFA and add approximately 500 microliters of two percent BSA in PBS to each gel.
Following one hour incubation at room temperature, carefully remove the BSA. Add 500 microliters of prepared primary antibodies at a dilution of one to 100 in two percent BSA in PBS to each gel. Apply the secondary antibodies and phalloidin as detailed in the text protocol.
Then, using tweezers and silicone isolators, transfer one gel to a glass slide. Mount the gel using a DAPI mounting medium. Finally, seal the samples with nail polish and acquire the images.
Endothelial cells are able to form confluent monolayers on PEG hydrogels as demonstrated by fluorescent staining for PECAM-1 in red and DAPI in blue. Similarly, fluorescent staining for A549 in red demonstrates that epithelial cells also form confluent monolayers. Furthermore, endothelial cells can form intact cadherin junctions on PEG hydrogels as demonstrated by fluorescent staining for VE-cadherin in green and DAPI in blue.
Staining for E-cadherin in green reveals the epithelial cells also form intact cadherin junctions. Following this technique, other methods like migration assays or permeability assays can be performed to understand how dual cell cultures and different microenvironments maintain or attenuate barrier function. After watching this video, you should have a good understanding on how to create ultra-thin PEG gels and modulate their porosity in addition to their mechanical and chemical properties in order to mimic the basement membrane.
This technique will pave the way for researchers in the fields of biomaterials and biomedical engineering to explore and decouple mechanical, biochemical, and cell-mediated pathways that contribute to human disease. Though the system is widely applicable to biological applications, it can be applied to any system where cell-cell signaling and mechanical or biochemical extracellular cues can play a role.
