Cell Culture and Collagen I-Matrigel Hydrogel Encapsulation

Rat myoblasts and mouse embryonic fibroblasts were purchased from ATCC [H9c2(2-1), ATCC; CRL-1446; MEF (CF-1) SCRC-1040] and cultured in Dulbecco’s Modified Eagle’s Medium (ATCC; 30-2002) supplemented with 10% Fetal Bovine Serum (FBS) and 2% non-essential amino acids (Thermo Fisher Scientific; 11140050) in 5% CO₂ at 37°C. H9c2 and MEFs were dissociated once they reached ∼85% confluency using 0.25% trypsin/EDTA (Gibco; 25200-056). Each engineered tissue construct in the device contained approximately 2.8 × 10⁶ H9c2 and 1.4 × 10⁶ MEF resuspended in collagen I via the following method (Fig. ​(Fig.1a).1a). First, 100 μL of 10× PBS and 20 μL 1 N NaOH were added to a small centrifuge tube, vortexed, and then placed on ice. While on the ice, 860 μL of collagen I (Corning; 354236) was added to the PBS-NaOH mixture, vortexed, and quickly returned to the ice. Then 100 μL of Matrigel® (Corning; 354230) was added to the mixture to create a collagen I-Matrigel hydrogel. Following trypsinization and combination of H9c2 and MEF, the cells were centrifuged for 30 s to form a pellet and the supernatant was aspirated. A volume of 1,080 μL of collagen I-Matrigel hydrogel was then added to the cells and pipetted thoroughly to homogenize cell distribution throughout the gel. Afterwards, 250 μL of this suspension was pipetted into each dumbbell chamber. The device was placed into the incubator for 45 min for gelation, and 3 mL of complete DMEM media was then added to the device’s inner reservoir and returned to the incubator overnight. The next day, the agarose mold was carefully removed, and the media was replenished.

Overview of 3D cardiac tissue chips. a Engineered constructs, used for all experimental conditions, containing rat myoblasts (H9c2) and mouse embryonic fibroblasts (MEF) within a collagen I-Matrigel hydrogel. Three tissue constructs per PDMS cardiac tissue chip were done. Static experiments received no external hemodynamic stimuli. b The stretch experiments were performed with the PDMS cardiac tissue chip sandwiched between an open-top polycarbonate piece that allows for gas exchange and maintenance of constant atmospheric pressure in the media chamber, and a bottom polycarbonate piece with an open cavity and cylindrical obstacle connected to a programmable pneumatic pump. c For the pressure experiments, the cardiac tissue chip was positioned between 2 non-deformable polycarbonate pieces, and “systolic” pressure was created by connecting inlet and outlet ports on the top polycarbonate piece to a programmable pneumatic pump.