Cation The PDMS functions fabricated by soft-lithography have been bonded around the surface with the MEA substrates to define the cell patterns and also the bridges between clusters of cardiac cells. The clusters were patterned into circles with a diameter of 800 connected by the bridges with a dimension of 500 300 . The SU-8 3050 photoresist (Kayaku Sophisticated Components, Inc., Westborough, MA, USA) was spin-coated on a silicon wafer at 800 rpm for 30 s to attain a thickness of 140 . Soon after baking at 65 C for 15 min and 95 C for 60 min, the wafer was exposed to UV light at a dose of 250 mJ/cm2 through a adverse photomask of the desired pattern. Then the wafer was baked again at 65 C for 10 min and 95 C for 30 min to additional cure the SU-8. The wafer was immersed in SU-8 developer (Kayaku Sophisticated Supplies, Inc., Westborough, MA, USA) to eliminate the uncured photoresist and cleaned with isopropanol andMicromachines 2021, 12,4 ofDI water. Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (TFOCS, Fisher Triacsin C MedChemExpressOthers https://www.medchemexpress.com/triacsin-c.html �Ż�Triacsin C Triacsin C Purity & Documentation|Triacsin C Description|Triacsin C manufacturer|Triacsin C Autophagy} Scientific, Waltham, MA, USA) was vacuum-coated on the surface of the silicon wafer mold for the straightforward release of PDMS. Then common PDMS replica molding was conducted to fabricate PDMS attributes. PDMS pre-polymer (SYLGARD184 silicone elastomer, Dow Corning, Midland, MI, USA) and curing agent (SYLGARD184 silicone elastomer curing agent, Dow Corning, Midland, MI, USA) were mixed at a weight ratio of ten:1 and after that poured onto the SU-8 mold. Immediately after degassing and baking, the PDMS options were manually removed in the mold and bonded with MEA substrate utilizing the plasma cleaner to receive the surface topographic features. The SU-8 blockers having a dimension of 300 320 were fabricated by exactly the same process of photolithography. The width of blockers (320) is slightly wider than the bridge width (300) as a way to blockade the bridge region. two.3. Cell Patterning and Culture Rat CMs have been isolated from two-day-old Sprague Dawley rat hearts following a previously established protocol [19] in compliance together with the IACUC suggestions and under an approved protocol from the University of Notre Dame. The differentiation induction of iCMs followed our previously established protocols [20]. Briefly, DiPS 1016 SevA human induced pluripotent stem cells (hiPSCs) derived from human skin fibroblasts (Passage quantity 400) have been seeded on a Geltrex-coated (1 TL-895 Protein Tyrosine Kinase/RTK Invitrogen, Carlsbad, CA, USA) tissue culture flask applying mTeSR (StemCell Technologies, Vancouver, BC, Canada) supplemented with 1 penicillin (VWR, Radnor, PA, USA). At 80 confluence, hiPSCs were detached and reseeded into the culture cell-plate and incubated in mTeSR1 media supplemented with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (five , StemCell Technologies, Vancouver, BC, Canada). The media have been changed each day till the cells reached 95 confluence. Then, hiPSCs were treated with RPMI Medium 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with B27 without the need of insulin (two , Invitrogen, Carlsbad, CA, USA), beta-mercaptoethanol (final concentration of 0.1 mM, Promega, Madison, WI, USA) and penicillin (1) (CM (-)) using the addition of Wnt activator, CHIR99021 (CHIR) (12 , Stemgent, Cambridge, MA, USA). Twenty-four hours later, media had been changed to CM (-) with no any CHIR. On day 4, iCMs had been treated with CM (-) media supplemented using the Wnt inhibitor IWP-4 (5 , Stemgent, Cambridge, MA, USA), media were changed back to CM (-) two days later. On day 9, iCMs have been cultured with RPMI Medium 1640 supp.
