The overall goal of the following experiment is to create cell microenvironments to study biological questions relating to individual and group cell behaviors that take place at micro and nanoscale dimensions. This is achieved by first designing templates that ultimately will guide cell placement. Next, these designs are then translated into pattern surfaces via photolithography, which allows for the creation of plasma shielding molds via soft lithography.
Finally, these shielding molds are arranged as a tripod in a Petri dish to allow for selective exposure of the underlying surfaces to plasma, which will leave a cell sensitive chemical pattern on the areas under each mold. Results can be obtained that show how a cell's behavior depends upon its microenvironment and its interaction with neighboring cells. This method can help answer key questions about the effects of microenvironments on cell behavior, such as how do interactions amongst groups of cells affect higher order phenomena?
And how do subcellular nanoscale cues affect functions such as health migration and differentiation? First, use super glue to attach a pattern slide to a stack of blank slides. Next, create a container of aluminum foil slightly larger than the stack of slides, and large enough to hold 30 grams of poly dimethyl suboxane or PDMS.
Put the stack of slides in the container and then bore PDMS over the stack of slides to create an initial mold. Then Degas PDMS by placing the container inside a vacuum chamber. Break any bubbles present in the PDMS by quickly removing and replacing a finger over the intake valve tubing several times to briefly allow air to flow into the chamber.
The valve is closed for five minutes of uninterrupted degassing. Then allow the PDMS to cure in the clean flow hood at room temperature for one to two days. Once cured, the PDMS is peeled off of the master pattern, forming a mold.
Next, pour a thin layer of six grams of two part epoxy that has just been mixed for one minute into the master mold. Degas the epoxy immediately using many bubble breaking cycles before the epoxy sets. After the Degas epoxy has been allowed to sit undisturbed for an hour, it will be partially cured.
Add more epoxy to the mold without degassing to produce a thicker structure that will be easier to handle in later steps. Then allow the epoxy to cure for one to two days. Once the epoxy mold is cured, remove the epoxy from the PDMS master mold and then wrap tape around the epoxy mold to form a container.
A working mold can then be created by pouring PDMS onto the epoxy mold, degassing the PDMS and curing the new mold for one to two days. After the working mold is cured, peel the mold off of the epoxy and store both inside Petri dishes in a sealed plastic container inside the flow hood to maintain cleanliness. To prepare a mold for surface patterning, cut small sections of the working mold with a razor blade and place the sections into a Petri dish.
The locations of these mold sections are labeled on the backside of the Petri dish with a permanent marker by making an outline around where the mold sections are sitting form a tripod from the sections and wait the tripod to ensure conformal contact between the working mold and the Petri dish surface. Look at the bottom of the dish to confirm good placement as evidenced by the presence of channels that can be seen by the naked eye or with a small magnifying lens. Place the assembly into a plasma chamber.
Initiate plasma treatment after the plasma treatment. Remove the mold and weight arrangement and place the Petri dish inside a biosafety cabinet under UV light for 10 minutes for sterilization. Store the dish there until it is seeded with cells.
Dilute the experimental cells of choice in their standard culture media to achieve the desired confluence. When placed onto the patterned Petri dish surface, pour three milliliters of the cell solution into the Petri dish. Incubate the patterned Petri dish at 37 degrees Celsius, 5%carbon dioxide, 100%humidity for several hours to several days to allow the cells to assemble onto the pattern Created by the plasma cell.
Patterning is checked with phase contrast microscopy. When culturing neuroblastoma cells, add 10 micromolar of retinoic acid daily until cell differentiation into a neuron like state is observed. Replace the standard cell culture media every three to four days.
A typical result of implementing the plasma lithography technique is the formation of a pattern of cells that resembles some arbitrary or natural structure. An example of this can be seen here in this figure with neurons that have been differentiated with retinoic acid on a pattern of lines. And here in this figure where lines and networks of neurons have been created, cell types other than neurons can also be used as seen in these next two figures, this figure shows human umbilical vein endothelial cells, and this figure shows C two C 12 skeletal muscle cells forming grids as seen.Here.
Materials such as polylysine can also be patterned to facilitate attachment of certain cell types and for other uses Following this procedure, incorporation of additional inputs such as microfluidics, cell probing or genetic manipulation of a specific proteins can be performed in order to answer additional questions, such as how do combinations of stimuli affect higher order and individual cell behavior?
