Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes

Abstract

Source: Xu, Z., Orkwis, et al. Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification. J. Vis. Exp. (2020).

The video describes the preparation of a micropatterned substrate to study cell phenotype specification. A protein-coated micropatterned PDMS stamp is used to transfer the micropattern on a PDMS-coated coverslip. The coverslip is then treated with a surfactant to hinder cell adhesion at the unwanted surfaces. Cells are then added to settle in the pattern and incubated for further analysis.

Protocol

1. Tunable cell culture substrate preparation and characterization

1. Substrate preparation​
   1. Mix the Polydimethylsiloxane (PDMS) base elastomer and curing agents using a pipette tip vigorously at a ratio between 10:1 and 60:1 until bubbles are homogeneously dispersed within the mixture. Remove bubbles using vacuum desiccation until bubbles are dissipated.
      ​NOTE: During PDMS polymerization, the curing agent crosslinks with the base elastomer to provide the final polymer's desired mechanical properties. Crosslink ratios can be adjusted to alter PDMS stiffness.
   2. Place a drop (~0.2 mL) of desiccated PDMS mixture on a square or circular coverslip (e.g., 22 mm x 22 mm) and rotate the coverslip on a spin coater at 2500 rpm for 30 s.
   3. Incubate the coverslip in either an oven at 60 °C for 1-2 h or at room temperature overnight for PDMS to solidify.
   4. Treat the coverslip using UV-Ozone cleaner for 7 min (UV wavelength: 185 nm and 254 nm) to increase the surface hydrophilicity. Place it into a sterilized 6-well plate.
   5. Before using it for cell culture, incubate substrates in 70% ethanol for at least 30 minutes.
      ​CAUTION: UV-Ozone cleaner can generate Ozone that is harmful to humans. Work in a chemical fume hood or with some form of ventilation.
2. Micropatterned substrate preparation
   1. Draw the desired geometry and cell adhesive areas (900 µm², 1,600 µm² and 2,500 µm²) using computer-aided design (CAD) software. Create a chrome photomask based on those patterns from a commercial supplier.
   2. In a clean room or dust-free environment, use standard photolithography techniques to fabricate silicon wafers. Critical parameters for this particular application are as follows: Photoresist: SU-8 2010; Spin profile to disperse the photoresist: 500 rpm for 10 s with an acceleration of 100 rpm/s, then 3500 rpm for 30 s with an acceleration of 300 rpm/s; Exposure energy of UV light: 130 mJ/cm2.
      ​NOTE: The height of patterns on the silicon wafers is approximately 10 µm following these parameters. Potential cracks around the edge outside the rectangular or triangular patterns can be seen using a light microscope after step 1.2.2. Baking the silicon wafer at 190 °C for 30 min helps to eliminate the cracks.
   3. Place the patterned silicon wafer inside a circular 150 mm diameter x 15 mm height Petri dish and pour de-gassed PDMS (mixing ratio 10:1) as prepared in step 1.1.1 onto the silicon wafer.
      ​NOTE: Ensure the thickness of PDMS is at least 5 mm for ease of handling during microcontact printing steps.
   4. Solidify PDMS on a silicon wafer in an oven at 60 °C overnight. Allow PDMS to cool to room temperature. Precisely cut 30 mm x 30 mm square stamps containing the correct patterns from the silicon wafer using a surgical scalpel. Do not damage the silicon wafer.
      ​NOTE: Silicon wafers can be reused many times at this point to produce more stamps following cleaning with isopropanol.
   5. Sterilize PDMS stamps and the tunable coverslips (prepared in steps 1.1.1 to 1.1.3) by immersing them into 70% ethanol for 30 min.
   6. To confirm the efficacy of micropattern by PDMS stamps after microcontact printing, dry the surface of PDMS stamps using a filtered air stream and pipette 50 µg/mL Bovine serum albumin (BSA) (Texas Red conjugated) solution to cover the entire patterned side of the PDMS stamp.
   7. Incubate PDMS stamps with BSA solution for 1 h at room temperature to allow for protein adsorption.
   8. Dry the surface of tunable coverslips using a filtered air stream and increase surface hydrophilicity as described in step 1.1.5.
   9. Air dry the PDMS stamps to remove the remaining BSA solution.
      ​NOTE: Take care that the BSA solution is completely removed from the stamp because any remaining solution will cause stamps to slide on the coverslip during microcontact printing.
   10. Bring the patterned side of the stamp into conformal contact with the tunable coverslip for BSA adsorption on the coverslip surface. Gently press the stamp against the coverslip for 5 min.
       NOTE: Do not apply excessive force on the stamp since it will bend and cause non-specific contact between the stamp and the coverslip. The appropriate amount of force applied to the stamp is essential for successful microcontact printing.
   11. Examine the micropattern using a fluorescence microscope with a FITC (Fluorescein isothiocyanate) filter.
   12. To print cell adhesive areas rather than fluorescent patterns, substitute laminin for BSA protein and repeat steps 1.2.5 to 1.2.10.
   13. Remove stamps from coverslips and transfer coverslips into a sterilized 6-well plate. Add 2 mL of 0.2% w/v Pluronic F-127 solution into each well to cover the surface of the coverslip and incubate for 1 h at room temperature.
       ​NOTE: Pluronic F-127 can be adsorbed to the PDMS surface, increasing the hydrophobicity of the PDMS surface to block cells from adhesion.
   14. Aspirate Pluronic F-127 solution and wash 5x with Phosphate buffer saline (PBS) and 1x with the cell culture medium before seeding cells. A typical seeding density for SCs is 1,000 cells/cm².
   15. 45 min following cell seeding, remove the cell culture medium and wash coverslips with PBS 2x to prevent multiple SCs from adhering to the same pattern. Maintain cells in the desired cell culture environment for 48 h before quantification.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin from Bovine Serum (BSA), Texas Red conjugate	Thermo Fisher Scientific	A23017	BSA staining to show micropatterns
Dulbecco's Modified Eagle Medium	Thermo Fisher Scientific	11965092	Cell culture medium
Fetal Bovine Serum	Thermo Fisher Scientific	16000044	Cell culture medium supplemental
Fibronectin	Thermo Fisher Scientific	33010-018	Protein used to coat coverslips
Fluorescence microscope	Nikon	Eclipse Ti2	Fluorescence microscope
Laminin	Thermo Fisher Scientific	23017015	Protein used to coat coverslips
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122	Cell culture medium supplemental
Pluronic F-127	Sigma Aldrich	P-2443	Block non-specific protein binding
SYLGARD 184 PDMS base and curing agent	Sigma Aldrich	761036	Tunable polymer used to coat coverslips
UV-Ozone cleaner	Novascan		Increase hydrophicility of PDMS