Poly-N-isopropylacrylamide

The muscular thin film chips were made with a multi-step fabrication process on round or rectangular glass cover slips. The circular chips were made with 25 mm cover slips to fit into standard assay setups (e.g. heating stages). The substrates were cleaned by sonicating for 60 min in 95% ethanol and air dried. For round cover slips, two pieces of protective film (regular Scotch tape) were attached on the edges approximately 1 cm apart (Fig. 1a(i)). Next, poly(N-isopropylacrylamide), (PIPAAm, Polysciences, Inc., Warrington, PA) was dissolved in 99.4% 1-butanol at 10%wt (w/v). An excess of the PIPAAm solution (~1 μL mm⁻²) was deposited onto the portions of glass not covered with the protective film and spin coated onto the surface at 6000 RPM for 1 min (Fig. 1a(ii)). The top protective film was peeled off without disturbing the layer of PIPAAm (Fig. 1a(iii)). Next, Sylgard 184 (Dow Corning, Midland, MI) polydimethylsiloxane (PDMS) elastomer was mixed at a 10 : 1 base to curing agent ratio and cured at room temperature for 3 to 6 h prior to further spin coating. PDMS properties for different curing times were described in detail in previous publications.^{29 (link)} PDMS was spin coated over the whole glass section (Fig. 1a(iv)) with a 2.5 min ramp protocol and 4000 RPM as the maximum rotation speed. PDMS-coated glass was cured at 65 °C for 8 h. For round cover slips, every seventh sample was saved to measure the thickness of the PDMS thin film.
For batch production of multiple chips, we attached protective film (Static Cling Film, McMaster-Carr, Robbinsville, NJ) on both the top and the bottom of a large glass section (7 cm × 11.5 cm) and used a razor blade to cut strips out of the top film (10 × 6 mm) that were separated by 4 mm horizontally and by 6 mm vertically (Fig. 1b(i)). Next, we spin coated PIPAAm onto the surface, and then peeled off the top film, leaving small patches of PIPAAm (Fig. 1b(ii)). The PDMS was spin coated over the glass section (Fig. 1b(iii)) and, after curing, we cut the glass, using a diamond glass cutter, into 42 rectangular cover slips (14 × 12 mm) to fit into differently sized multi-well plates (Fig. 1b(iv)). Two samples from each glass section were saved for thickness measurements. The thickness of the PDMS thin film was measured using a profilometer and ranged 10–30 μm (Dektak 6M, Veeco Instruments Inc., Plainview, NY).
Fibronectin (FN, Sigma, St. Louis, MO or BD Biosciences, Sparks, MD), an extracellular matrix protein, was microcontact printed onto the surface of the substrate to provide guidance cues for self-assembly of the chemically disassociated myocytes.^{29 (link),31 ,33 (link),34 (link),38} PDMS stamps with “brick wall” patterns (each brick was 20 μm wide and 100 μm long, and each short edge terminated with 5 μm long “saw-tooth”) were sterilized by sonicating for 30 min in 50% ethanol and air dried in a biohood under sterile conditions. The patterned surface of the dry stamps was covered in 250 μL of 50 μg mL⁻¹ FN and incubated for 60 min. The surface of the cover slips was sterilized and functionalized by exposing them for 8 min to UV ozone (Model No. 342, Jetlight Company, Inc., Phoenix, AZ). The stamps were dried with compressed air and used to transfer the FN pattern to the cover slips. The cover slips were then transferred to multi-welled plates and covered with 1% Pluronics F127 (BASF Group, Parsippany, NJ) in DI water for five minutes and washed three times with Phosphate Buffered Saline (PBS). Alternatively, for isotropic tissues, the cover slips were incubated in 25 μg mL⁻¹ FN for 15 min and washed three times with PBS. Following previously developed procedures all cover slips were stored dry at 4 °C for no more than three days prior to myocyte seeding.^{29 (link),30 (link)}