*2.8. Development of a Bioreactor System for Tissue-Engineered Vascular Graft (TEVG) Culture*

The TEVG approach was performed with a custom-made bioreactor setup. The culture chamber consists of a 250-mL glass bottle (Schott Duran, Wertheim, Germany) and encloses a removable custom-designed graft frame that holds the vascular graft. A computer-aided design (CAD) model for the graft frame was created in Solidworks (Dassault Systèmes, Vélizy-Villacoublay, France) and milled out of polyether ether ketone (PEEK; ADS Kunststofftechnik, Ahaus, Germany) using a 2.5-axis flatbed milling setup (Isel, Eichenzell, Germany) with computer numerical control (CNC). The constructed parts were subjected to the aforementioned cytotoxicity test to ensure no toxic leachables are released into the medium under culture (Figure S2b). The modular design of the culture chamber allows for a toolless assembly of the bioreactor system under a sterile bench.

The graft frame—once inserted into the culture chamber—is connected to medium reservoirs and a bubble trap with flexible silicone tubing. Sterile gas exchange is facilitated by sterile filters connected to the medium reservoirs. The entire setup is driven by a multichannel roller pump (Ismatec) (Figure 2).

The flow rates *Q* for dynamic culture were determined with a derived formulation of the Hagen–Poiseuille equation for laminar flow in straight circular pipes with internal radius *r*:

$$
\pi = \frac{4\mu Q}{\pi r^3} \tag{2}
$$

where μ denotes the dynamic viscosity. This gave an analytical approximation of the achieved wall shear stress (τ) within the cultured vascular graft. To validate this approximation and the assumption of a laminar regime within the vascular graft, in silico simulations were used to assess the local fluid dynamics within the vascular graft and graft frame interior. Briefly, the CAD model of the graft frame was meshed and exported to a computational fluid dynamics (CFD) solver (ANSYS Fluent). Dynamic culture with a wide range of flow rates was simulated under steady-state flow and Newtonian rheological conditions, after which the calculated wall shear stress on the interior graft wall was analyzed and compared to the aforementioned analytical solution (Figures S4 and S5).
