The overall goal of this procedure is to pattern cells on silicon dioxide services according to a predefined underlying design of paraline. This is accomplished by first creating a photo mask of the desired pattern. The second step is to oxidize and then coat a silicon wafer with paraline.
Next photolithographic processes performed in a microelectronics clean room facility are used to selectively etch the paraline coating to reveal the desired design. The final step is to activate the chips using first piranha acid and then incubate in fetal calf serum for three to 12 hours after which a cell suspension can be applied for culture. Ultimately, cell patterning can be assessed either by live cell imaging using a dissecting microscope or after fixation using confocal fluorescence microscopy.
The main advantage of this technique over several existing cell patterning platforms is that there's no need to bring biological agents into the clean room. Also, pattern chips can be kept indefinitely until they're activated with first par acid and then serum. To design the desired paraline C configuration, use a layout editor software package that is capable of reading, writing CIF or GDS two files.
Outsource photo mask manufacturer to an appropriate microelectronics facility or make in-house. If facilities exist, then oxidize a silicon wafer in an atmospheric horizontal furnace at 950 degrees Celsius for 40 minutes to produce a 200 nanometer silicon dioxide layer. After that, confirm the thickness with a small spot.
Spectroscopic reflectometer. Place wafers in vacuum deposition system apply sine adhesion promoter to the inside of the chamber lid. During the pump down cycle, the sine adhesion promoter coats the oxidized wafer load paraline into the furnace deposit paraline C at ambient temperature at a rate of 1.3 nanometers per milligram of dimer using a vacuum deposition system.
After that deposit HMDS adhesion promoter on the paraline coated wafer. Using a suitable photo resist coating system, spin the wafer at 4, 000 RPM for 30 seconds whilst applying positive photo resist in order to obtain a thickness of one micron using the photo resist coating system, then soft bake the wafer for 60 seconds at 90 degrees Celsius. Now insert both the wafer and pre-manufactured photo mask into a mask aligner.
Expose the photoresist coated wafer with UV light so as to transfer the desired pattern into the photoresist. Afterward, bake the exposed wafer for 60 seconds at 110 degrees Celsius. Then remove all exposed photoresist from the wafer by developing it in an appropriate developer solution at Hoff the unprotected paraline by using an oxygen plasma etch system to reveal the underlying silicon dioxide.
Then store the chips after dicing in dust-free boxes until required. In this procedure, remove the residual photoresist from chips by washing an acetone for 10 seconds. Then rinse them in deionized distilled H2O three times, make the fresh piranha acid in an acid fume hood afterward, clean and etch the chips by immersion in piana acid for 10 minutes.
Then rinse the chips three times in deionized H2O and transfer to a sterile culture dish in a laminar flow tissue culture hood. Activate the chips for cell patterning by placing two chips per well into a six well plate. Next, add two milliliters of fetal bovine serum so as to fully immerse all chips.
Incubate the chips in serum for three to 12 hours at 37 degrees Celsius. Now remove the chips from their activation solution. Wash once for 10 seconds in Hank's balanced salt solution.
Then place the chips in a culture well and plate the chosen cell type as a suspension in its usual growth media. Optimum cell plating density depends both on cell type and geometric pattern of paraline C on chip. A density of five times 10 to the fourth cells per milliliter is a sensible starting point.
Imaging is tailored to the underlying motivation for cell patterning, but live cell behavior can easily be assessed using a dissecting microscope and a digital camera with a suitable relay lens. Here is the live cell imaging of HEC 2 93 cells cultured on paraline C silicon dioxide after three days. In vitro, the ships had been incubated for three hours in fetal bovine serum, and the cells were plated in suspension at a concentration of five times 10 to the fourth cells per milliliter.
Paraline C promotes cell adhesion whilst bare silicon dioxide repels cells. This figure shows the immunofluorescence images of primary human glioma derived stem-like cells grown on various patterns of paraline C on silicon dioxide. Here the schematic illustrates the reticular paraline design, the fluorescence micrograph of fixed cells stained for glial fibrillary acidic protein, and the light micrograph of live cells on the same chip.
Here is a fluorescent image illustrating GFAP stained cells on a different paraline design and the reflectance image of the node in spoke paraline design. And this figure shows the live cell imaging of three T three L one cells cultured on paraline C silicon dioxide. After four days in vitro, the chips had been activated for three hours in fetal bovine serum, and the cells were plated in suspension at a concentration of five times 10 of the fourth cells per milliliter.
In this instance, the platform does not enable patterning with cells becoming equally confluent on Lene C and silicon dioxide regions. When performing this protocol, it's important to remember that serum activation must occur immediately after piranha treatment. Failure to do so results in undermining the patterning process.
Don't forget that working with piranha acid can be extremely dangerous. Prepare and use piranha acid in a chemical fume hood and make sure that you wear appropriate goggles, lab coats and gloves.
