Construction of cardiac bioreactor

The scaffolds were cut into disks with a diameter of 10 mm and a thickness of 2 mm. Neonatal rat cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats following our established protocol approved by the Institute’s Committee on Animal Care.³³ The cells were maintained in DMEM supplemented with 10 vol % FBS. Each scaffold was placed in a well of a 12-well plate and seeded with 4 × 10⁶ cells by dropping a suspension of cells in 30 μL of medium. The medium containing the cardiomyocytes immediately wetted the scaffold via capillary action ensuring a uniform distribution of the cells inside the scaffold. Every 30 min, 10 μL of medium was introduced around the scaffold to maintain the moisture and prevent the cells from dehydration. The cells were allowed to attach to the matrix of the scaffold for 1 h, followed by addition of 1 mL of medium to cover the scaffold. The medium was changed every day during the first 3 days of culture.

The bioreactors were designed and fabricated according to our recently developed protocol.³⁴ A laser cutter was used to cut off a poly(methyl methacrylate) (PMMA) positive mold with an elliptical shape and a straight tail. The mold was taped to the bottom of a 10-mm petri dish, and filled with polydimethylsiloxane (PDMS) prepolymer mixture (10:1 ratio). After curing of PDMS at 80°C for 24 h, the elastomer was peeled off from the PMMA mold to achieve the negative chamber/channel complex. Two such PDMS structures were placed against each other to construct the bioreactor, where the inlet/outlet were punched for connection with external metal connectors and Teflon tubing. To prevent potential leakage, the PDMS device was further clamped in between a pair of PMMA slides held together by four screw/bolt sets. The scaffold was secured at the bottom of the chamber of a bioreactor by fixation using a custom-designed holder constructed from a ring of PMMA board with four metal legs. The bioreactors were filled with media and the tubing from the inlet/outlet was connected into a closed loop. A peristaltic pump was used to drive the liquid at a flow rate of 200 μL/h.

The viability of the cardiomyocytes inside the scaffolds under both static and dynamic conditions was evaluated at days 3, 7, and 14 post seeding using the Live/Dead viability kit (ThermoFisher, USA) following the manufacturer’s instructions.