The overall goal of this procedure is to manufacture and characterize polymeric thin film composite structures for advanced cell culture studies or as skin adhesives. This method can help answer key questions about how elastomeric composites can be used to achieve optimal sticking action, even on irregular surfaces. The main advantages of these techniques are that they are simple and universally applicable.
This allows large scale manufacturing with a very high precision. Generally individuals new to this method will struggle, because the adhesion measurements require intensive training and practice to achieve the necessary precision. Demonstrating the cell culture procedures and plasma application will be Angela Rutz, a technician from our lab.
To prepare the PDMS, mix and degas 1.1 grams of the prepolymer mixture in a speed mixer at 2350 RPM under vacuum for three minutes. To prepare the PDMS backing layer, use an automatically controlled doctor blade application machine. Place the doctor blade on top of a 100%isopropanol cleaned piece of PET foil, and use the micropositioning screws to adjust the thickness of the backing layer.
Load the PDMS into a single-use 10 milliliter syringe. Fill the reservoir of the doctor blade with the polymer, and start the movement of the blade with a velocity of approximately 2 millimeters per second. When the piece of PET film has been coated, transfer the film into a 95 degree Celsius oven for one hour.
To prepare the top layer of the composite film, use a blade to remove thin strips from the long sides of the backing layer. To allow the doctor blade to be slid on the PET foil, and apply the second layer of PDMS as described before. Then, place the foil into the oven.
To prepare the samples for adhesion measurements, use a razor blade to cut the films on the PET foil into four square centimeter area pieces and use UV glue to secure the pieces onto a glass slide. Illuminate the pieces with UV light for three minutes, and mount the first polymeric sample onto the sample holder. Use the goniometer to adjust the tilt angle, until the substrate is in contact with the polymeric film at a completely parallel alignment of both surfaces, as visualized by the camera images.
Optical alignment of the substrate to the sample is highly critical for interpreting the measurement results. Therefore, tilt angle adjustment need to be performed as precisely as possible using the inclinometer. Move the aligned substrate to the polymeric film surface until a pre-load stress of 13 plus or minus five kilopascals is achieved, and start the custom programed software package written in LabView.
Then, set the required measurement parameters to measure the adhesion properties of three independent manufactured samples at six different locations on each film surface. To prepare the films for optical microscopy, use a razor blade to cut the polymeric sample into 0.25 square centimeter pieces and attach the pieces to the edge of a glass slide. Then, place the slide in the vertical orientation under an upright microscope and measure the thickness of the film cross section.
To prepare a cell culture experiment, first use a scalpel to cut approximately five by five millimeter pieces from the PET supportive layer, and use tweezers to transfer the pieces onto individual 12 millimeter glass cover slips. Transfer the cover slips inside the reaction chamber of a plasma device, and close the device lid. Evacuate the chamber until a 1.6 times 10 to the minus second millibar pressure is reached, and treat the films with plasma for three minutes.
Then, ventilate the reaction chamber and transfer the samples into individual wells of a 24-well plate. Next, wash a 70 to 80%confluent L929 cell culture with calcium and magnesium free DPBS for 30 seconds in a laminar flow cabinet. Followed by a five minute incubation with two milliliters of an appropriate proteolytic and collagenolytic solution at 37 degrees Celsius, and 5%CO2.
When the cells have detached, add eight milliliters of serum supplemented medium and transfer the cells into 15 milliliter conical tube. Then, after counting, seed 60, 000 cells per milliliter of medium per well of the 24 well plate containing the polymeric samples, and place the plate in the cell culture incubator for three days before phase contrast image capture and fixation. Depending on the thickness of the top film, a decrease in the pull-off stress is observed with an increasing film thickness, regardless of the texture of the substrate surface.
The work of separation, however, is generally slightly lower for rough substrate surfaces compared to smooth ones. When the detachment mechanism is recorded, little cavitation is observed on the thinnest films, while the appearance of finger-like cracks is observable on the thicker films. Cell culture experiments reveal that cells seeded on pristine polymers display poor attachment and cellular spreading behavior, while the confluent monolayer is observed for cells cultured on plasma treated surfaces.
In general, lactate dehydrogiease levels are comparable for cells cultured on both polymeric materials, with less than 5%cytotoxicity. Once mastered, the production of the polymeric film composites can be completed in approximately three hours. Although subsequent analysis with a normal tack test requires training, it is a very powerful tool for investigating the adhesive properties of the films.
After watching this video, you should have a good understanding of how to manufacture thin polymeric composite films and to analyze them with specific adhesion tests and cell biological tools. Don't forget that working with chemicals and biological materials can be extremely hazardous. Therefore, take appropriate precautions, such as following mandatory safety procedures, wearing personal safety protection equipment, and handling these materials in a safety cabinet.
