Continuous flow perfusion bioreactor system

For dynamic cell culture tests, electrospun vascular grafts were cultured in a custom-made perfusion bioreactor system. The bioreactor harboring the graft was assembled by combining two 15 mL Falcon tubes and silicone tubes. All parts were washed and Gamma-sterilized before assembling the bioreactor. Tubular scaffolds were inserted into the bioreactor under sterile conditions and mounted to respective connectors using ligature suture. Following, a sterile silicone tube was used to seal the bioreactor. Thereby, an extra luminal compartment was generated. The bioreactor system enabled distinct extra and intra luminal flow conditions.

At the in- and out-let of the bioreactor, a three-way valves were attached to enable controlled filling of the graft with cell suspension and to facilitate intra and extra luminal perfusion. hmvECs were harvested and 2 x 10⁶ cells were transferred in 1 mL Vasculife^® into the lumen of 4-cm-long scaffolds. Moreover, the extra luminal compartment was filled with cell culture medium. To allow cell attachment, the seeded scaffolds were cultured under static conditions without flow in an incubator. Every 30 min, the whole bioreactor was turned for 180° to enable gradual attaching of the hmvEC onto all sides of inner lumen of the graft. After 4 hours, the intra and extra luminal compartment of the bioreactor were perfused with a continuous flow of 4.9 mL/min. Therefore, cell culture medium was pumped from a medium reservoir through the bioreactor by using an ISMATEC^® pump (MCP-Z Process, Glattbrugg, Switzerland). The medium reservoir was equipped with a sterile filter to allow gas exchange. The bioreactor was operated in a tailored incubator system [17] that ensured controlled culture conditions of 37°C and 5% CO₂. During culture, 30 mL Vasculife^® medium was used and renewed once a week.