Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

The main advantage of this technique is that it can produce gradient patterns with high reproducibility and allowed the screening of cell response to parasites observational structures. The response observes to physical stimulation is not yet fully discovered. We believe that our method will be an essential tool to uncover these unknown mechanisms.

To begin, connect a double jacket beaker containing the electropolishing solution to a circulator and set the temperature at seven degrees Celsius. Leave the solution under stirring for at least 30 minutes until the temperature drops to seven degrees Celsius. Immerse the ultra-pure aluminum plate and carbon counter electrode into the electropolishing solution using crocodile clips and copper wire.

Adjust the position of the aluminum plate in the carbon counter electrode to face each other. Connect the aluminum plate to the positive terminal and connect the carbon counter electrode to the negative terminal of a direct current power supply. After applying a voltage of 20 volts, the aluminum plate goes from having a rough lustreless surface to having a mirror-like surface silver in color.

To perform the primary anodization, pour one liter of the electrolyte into a two liter double jacket beaker. Immersed the polished aluminum plate and carbon counter electrode into the solution with crocodile clips in copper wire. Install a vertical shaft on a u-shaped pedestal.

Attach an overhead stirrer and an impeller using metal clamps. Position the propeller of the impeller near the lower end of both electrodes. Set the rotation speed of the overhead stir between 200 and 300 rpm and apply a voltage of 195 volts under stirring for 16 hours.

Following primary anodization, the color of the aluminum plate changes from silver to gray. To perform alumina itching, immerse the anodized aluminum plate in the etching solution. Sealed the top of the beaker with aluminum foil and allowed to stir for at least 10 hours.

During the etching step, the color of the aluminum plate changes from gray to white. For the secondary anodization, set up the same experimental conditions as the primary anodization and again apply a voltage of 195 volts for six hours. Following secondary anodization, the color of the aluminum plate changes from white back to gray.

To perform the gradient pore widening, first connect the double jacket beaker containing a phosphoric acid solution to the circulator and set the temperature at 30 degrees Celsius. Place a linear moving stage vertically next to the beaker and attach a bracket to the moving part of the linear stage. Set the moving part of the linear stage to the home position.

Attach the clip of the bracket and load the anodized aluminum plate. Manipulate the position of the aluminum plate manually so that the aluminum plate will come right above the surface of the solution. Gradually immerse the aluminum plate into the solution at a speed of four point eight six microns per second for 120 minutes to make a 35-millimeter AAO mold with a size gradient from 120 nanometers to 200 nanometers.

Start a stopwatch at the moment the aluminum plate touches the surface of the solution. After 100 minutes have passed, half of the AAO mold should be in the solution. To make GP 200 to 280 and GP 280 to 360 molds, Immersed the entire area of GP 120 to 200 mold in the pore widening solution for two to four hours respectively.

Immerse the AAO mold and piranha solution for 30 seconds using a non-metallic tweezer. Rinse the AAO mold several times with deionized water. Soak the mold in deionized water for 30 minutes and put in methyl alcohol for another 30 minutes.

Completely dried the AAO mold under vacuum for three hours. For self-assembled monolayers deposition, immerse the AAO mold in 57 milliliters of filtered and hexane and a 100 milliliter beaker. Then add three milliliters of HDFS stock solution to the N hexane to make a 2.9 millimolar HDFS solution and wait 10 minutes for the reaction to proceed.

After transferring the AAO mold to fresh and hexane, soaked the AAO mold in 60 milliliters of methoxy-nonafluorobutane. Ultrasonically clean the AAO mold in a beaker for 10 seconds per one time to remove physically adsorbed HDFS molecules. Repeat the cleaning procedure 10 times before drying the AAO mold under vacuum for 24 hours.

Cut a one point one millimeter thick polystyrene sheet to a size of two point nine millimeters wide by three point seven millimeters long using a printed circuit board cutter. Working in a clean room, cut the AAO mold 35 millimeters from the bottom using a nipper. Mark the sample name and data manufacturer on the back.

Clean an eight point zero inch wafer with the small dose of ethyl alcohol, then put the polystyrene sheets on the wafer and mount the AAO mold. Next, put the bottom film in the drawer of a thermal imprinter and attach the top film to the gasket, then put the wafer on the bottom film and place the gasket on the wafer. Make sure that there is no dust on the wafer.

After closing the drawer of the thermal and printer apply 165 degrees Celsius of heat and 620 point five/two kilopascal of pressure from 100 seconds. Then cool the sample to room temperature. Once cool, vent the chamber and the pressure will go down to zero kilo Pascal.

Gently twist the polystyrene sheet to release the AAO mold. Collect 50 milliliters of human blood using a heparin inhibitor tube. Put six milliliters of hydrophilic polysaccharide solution in a 15 milliliter tube and add eight milliliters of blood to the hydrophilic polysaccharide solution.

A blood needs to be collected immediately and slowly added on top of the hydrophilic polysaccharide solution. After centrifuging the tube at 1020 times G for 20 minutes to pellet the cells, harvest the opaque cell layer and transferred to a new 15 milliliter tube. At 10 milliliters of PBS containing 10%FBS in centrifuge the tube at 1020 times G for 10 minutes to pellet the cells.

Remove the supernatant and add red blood cell lysis buffer. After incubating on ice for five minutes at PBS containing 10%FBS and centrifuge the tube at 1020 times G for five minutes to pellet the cells. Remove the supernatant and add one milliliter of endothelial cell expansion medium supplemented with 10%FBS.

Seed eight point zero million cells per well for each collagen coded dish. Induce the human and ethereal colony forming cells as described in the text protocol and grow the cells to passage seven for further experiments. Next, coat the gradient nano pattern plates with one milliliter of zero point one percent protein coating solution in PBS for 10 minutes at room temperature.

Make a suspension of human ECFCs from the culture dish by a standard cell splitting method using trypsin. After diluting the cell suspension to achieve the desired confluence, seed the human ECFCs cease on to the gradient nano pattern plates in a flat control. Culture the cells on flat or gradient nano pattern plates for two days in the incubator.

These images show the AAO molds fabricated under three different pour widening conditions. One side with the shortest time immersed in the phosphoric acid etching solution has the smallest pore size, whereas the other side having the longest time in the etching solution has the largest pore size. Since the nano pattern was transferred to the polystyrene through the thermal imprinting method, nano pillar structures are generated on the polystyrene surface and the size gradient of the AAO mold is maintained in nano pattern plates.

The confluent human ECFCs showed typical cobblestone like morphology inform a colony which can be easily distinguished under a phase contrast microscope. After two days of cultivation of human ECFCs on the flat in gradient nano pattern plates, significant filopodia outgrowth was observed in cells cultured on GP 120 to 200. Conversely, no remarkable change was observed in GP 200 to 280 or GP 280 to 360 in comparison with the flat control.

This result indicates that the response of the cells is different depending on the size of the surface nano structure. There are many steps to fabricate a nano pattern plate. So it can be complicated at first, but once mastered, you will get down patterns with desires size gradient without any difficulties.

After watching this video, you should have a great understanding of how to produce gradient nano pattern plates and the cultivate to human and the sillier colony-forming series on the nano pattern plate. Also this method is available to various types of series. This method can provide a bridge between material engineering and biotechnology that will help to understand the injections of cells and surface Nano topography.