Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

This protocol provides a cell culture platform where the properties of extracellular matrix such as stiffness, protein composition, and cell morphology can be precisely controlled in a cost-effective and facile manner. The method is cost-effective, and does not require special training or facilities. It is also compatible with multiple cellular quantification methods such as immunofluorescent staining and western blot.

This technique provides a mechanistic understanding of Schwann cell biology. With additional insight into biomaterial design for nervous system regeneration, it can be applied to any anchorage dependent cells. The most challenging portion of this protocol, is microcontact printing.

Prior to beginning of the protocol, microcontact printing on substrate using a florescent to tag the protein to visually verify the final print to the pattern. Begin by vigorously mixing the PDMS base elastomer and curing agents with a pipette tip until bubbles are homogeneously dispersed within the mixture. Then remove the bubbles with vacuum desiccation.

Place a drop of the desiccated PDMS mixture on a square or circular coverslip, and rotate the coverslip on a spin coater at 2, 500 rpm for 30 seconds. Incubate the coverslip in either an oven at 60 degrees Celsius, for one to two hours, or at room temperature overnight. To prepare a micro patterned substrate, place a patterned silicon wafer inside a circular 150 millimeter diameter by 15 millimeter height Petri dish and pour de-gassed PDMS onto the wafer.

Solidified the PDMS in an oven at 60 degrees Celsius overnight. Allow the PDMS to cool to room temperature. Then use a surgical scalpel to cut stamps of 30 by 30 millimeters squares containing the correct patterns taking care to not damage the silicon wafer.

Sterilize the PDMS stamps and tuneable cover slips by immersing them in 70%ethanol for 30 minutes. To confirm the efficacy of the micropattern, dry the surface of the PDMS stamps using a filtered air stream and pipette 15 micrograms per milliliter BSA solution to cover the entire patterned side of the stamp. Incubate the stamps with the BSA solution for one hour at room temperature to allow for protein adsorption, then air dry the stamps to remove the BSA solution.

Use the filtered air stream to dry the surface of the tuneable cover slips. Bring the patterned side of the stamp into conformal contact with the tuneable coverslip to allow for BSA adsorption on the coverslip surface, and gently press the stamp against the coverslip for five minutes. Examine the micropattern using a fluorescence microscope with a FITC filter.

To print cell adhesive areas rather than fluorescent patterns substitute laminin for BSA protein. Remove the stamps from the cover slips and transfer the cover slips into a sterilized six-well plate. Add two milliliters of 0.2%Pluronic F-127 solution into each well to cover the surface of the cover slip and incubate the plate for one hour at room temperature.

Aspirate the F-127 solution and wash the cover slips five times with PBS, once with the cell culture medium, then seed the Schwann cells at a seeding density of 1000 cells per centimeter squared. 45 minutes after cell seeding, remove the cell culture medium and wash the cover slips twice with PBS to prevent multiple cells from adhering to the same pattern. Maintain cells in the desired cell culture environment for 48 hours before quantification.

To create line-patterned cell culture substrates for examining aligned cells, make the stamps according to the manuscript directions, and cut them to the desired dimensions. Prepare two Petri dishes coated with a tuneable PDMS surface as previously described, and perform microcontact printing to print line-patterned cell adhesive areas on one of the dishes. After removing the stamps, fill the Petri dish with four milliliters of 0.2%Pluronic F-127 solution and incubate it for an hour.

Rinse each side of the PDMS stamp three times with 70%ethanol and dry it with air. Rotate the PDMS stamps and print the unpatterned cell adhesive area on the second Petri dish as previously described. After incubating the dishes with the F-127 solution, remove the solution and wash the dishes three times with PBS and once with fresh cell culture medium.

Then seed cells on the dishes, and maintain them at the desired conditions for 48 hours before preparing lysates. To prepare lysates, wash the cells with ice cold PBS for two minutes, then add 80 microliters of RIPA solution, prepared according to the manuscript directions, onto each cell adhesive area, and incubate the cells on an ice block for 25 minutes. Scrape the Schwann cells with the cell scraper for five minutes and collect the lysate into a labeled 1.5 milliliter microcentrifuge tube.

Centrifuge the lysate for 15 minutes at 12 thousands times G and four degrees Celsius. Collect the supernatant and transfer it to a clean microcentrifuge tube. Laminin coated substrates resulted in a higher proliferation rate when compared to collagen and fibronectin coated substrates.

Schwann cells on substrates with laminin coating and differing moduli showed that relatively softer substrates decreased cell proliferation rates. The cells were also analyzed for protein expression. Transcriptional factor c-Jun expression was upregulated as substrates became softer.

However, it was significantly downregulated on the softest substrates. On stiff substrates, collagen coated substrates resulted in the highest c-Jun expression, as substrates became softer, laminin showed the highest levels of c-Jun. The cells were then stained with rhodamine-phalloidin to explore the role of cell spreading and area in c-Jun expression.

When cell adhesive lines were created on the cell culture substrates, the nuclear aspect ratio of cells seeded on the pattern substrate increased compared to that of cells on unpatterned substrates. Expression of both c-Jun and p75 neurotrophin receptor were upregulated in dense cells with a smaller spreading area. Line-patterned cells also resulted in a higher expression of both c-Jun and p75 NTR when compared to nonpatterned cells.

Microcontact printed cell adhesive geometries were created to precisely control cell spreading area and elongation while eliminating cell to cell interactions. Increasing the cellular aspect ratio caused nuclear elongation to increase. The expression of c-Jun was upregulated by both cellular elongation and cell spreading inhibition.

Immunofluorescence staining for both nuclei and actin confirmed that cell spreading area and elongation were precisely controlled through this micropatterning method. This technique paved a way to using our tunable substrates as a method to mechanistically analyze Schwann cell biology and the signaling in the context of regeneration and cancer.