## 2 Materials and Methods
### Cell Seeding and Dynamic Cultivation  
Before the cells were seeded, the pH and the temperature in the cultivation system and in the chamber were equilibrated. In this phase, the culture medium was perfused through the cultivation chamber. All bubbles were also removed. This phase took about 2 h.  
Then the cell suspension was prepared. The cells were seeded at a density of 90,000 cells/cm\({}^{2}\). This density was experimentally adjusted to form a homogeneous, confluent layer on the top of the decellularized tissue. As was mentioned above, 35 \(\times\) 35 mm\({}^{2}\) squares of the tissue were fixed in the chamber. The total area for cell growth was 30 \(\times\) 30 mm\({}^{2}\); the borders were used for fixing the tissue in the holder. Thus, there were 810,000 cells on one side of the tissue, which were suspended in 500 \(\upmu\)L of the culture medium. This cell suspension was then injected through a sterile septum into the appropriate side of the cultivation chamber. Then, the chamber was aligned horizontally, allowing the cells to adhere. During this phase, there was no perfusion and no dynamic stimulation. The cell adhesion phase took 60 min. After that, mild perfusion (flow 2 mL/min) with the culture medium was started. The perfusion lasted for 2 h. After that, the stimulation was set to 15.9/10.6 kPa (120/80 mmHg) high/low pressure with a frequency of 1 Hz (60 pulses per minute). Every 2 h, the stimulation was changed to the perfusion mode for 10 min in order to refill the reservoirs of the chamber with a fresh culture medium. This cultivation was performed for 5 days and for 10 days.  
The first goal of this study was to test how well the bioreactor promotes the ingrowth of the cells through the decellularized tissue, so only one side of the tissue was seeded with cells and analyzed. The second goal was to prepare fully recellularized cardiovascular patches. When these patches were being prepared, the cells were seeded from both sides. This procedure involved the described seeding on one side. Then, the cells were left for 60 min, allowing their adhesion. After this phase, the chamber was turned and then the same seeding procedure, but from the second side, was performed, also followed by 60 min of cell adhesion.  
The cultivation medium consists of DMEM/F12 with a 1:1 mixture, supplemented by 10% of fetal bovine serum, 50 \(\upmu\)g/mL of ascorbic acid, 2.5 ng/mL of TGF-\(\beta\) (Transformation growth factor \(\beta\)), 2.5 ng/mL of BMP4 (Bone morphological protein 4), and 1% of ABAM antibiotics. This composition was set on the basis of our previous study [17], in order to promote both the growth and the differentiation of the ASCs towards smooth muscle cells. This culture medium was replaced every two days. During replacement, the cultivation chamber and the perfusion circuit were still fully flooded, and only the remaining medium was refilled into a syringe from the working reservoir. Then, the syringe was replaced by a new syringe containing fresh culture medium. These syringe replacements were carried out in a biohazard box to ensure sterility.  
A comparative control under static cultivation conditions was performed. In order to ensure the homogeneity of the experimental setup, the second cultivation system with cultivation chambers was used. The preparation included the same preparation steps as above, until the beginning of the 2 mL/min perfusion. Instead of perfusion followed by mechanical stimulation, the system with chambers was disassembled, and the tissues were inserted into standard six-well plates. The same culture medium was used and was replaced every two days.