Pluronics f 127

The surfaces of the wells and posts were coated with 1% Pluronics F127 (Thermo Fisher Scientific) to prevent cell adhesion to the PDMS and walls of the plate. 2% Poly(N-isopropylacrylamide) (NIPAM) gel was then cast as a temporary spacer at the bottom of each well to ensure gel formation at the top of the posts. Cell-populated fibrin-based tissue constructs were created by mixing two solutions (60 μl each) in the wells on a heating plate. In one solution, fibrinogen was diluted in sterile Dulbecco’s phosphate buffered saline (DPBS, Cellgro), while in the other solution, cells (4 million cells/ml) were suspended in tissue culture medium mixed with thrombin dissolved in 40 μM calcium chloride. The final concentration of fibrinogen (3.3mg/ml) and thrombin (1 U/ml) were selected based on previous studies (Quinlan et al., 2011 (link)). The casted constructs were incubated for 1 hour before being fed with tissue culture medium (DMEM supplemented with10% FBS, 1,000 KIU/ml Aprotinin (Thermo Fisher Scientific), 250 μg/ml L-Ascorbic acid 2-phosphate salt hydrate (Sigma), and 5 μg/ml human insulin (Sigma)). After compacting overnight, the samples were placed at 4 °C for 15 minutes to liquefy the NIPAM spacer which was then removed via pipette. The media were changed daily throughout the entire duration of the experiment.