## 2 Materials and Methods
### Decellularization System and pericardium Decellularization  
To automate the overall process of decellularization, a custom-made decellularization system was built. This solution consists of two peristaltic pumps with attached pinch valves. This system is controlled by an embedded controller compactRIO (National Instruments, Austin, TX, USA) with custom LabVIEW (also National Instruments) software to set appropriate parameters of the decellularization and to monitor the overall process. The first pump mixes and prepares decellularization agents, such as SDS (Sodium dodecyl sulfate) or DNAse, with dH\({}_{2}\)O (deionized water) and fills the decellularization chamber. The second pump maintains the recirculation of the agents in the decellularization chamber. This approach promotes the overall decellularization of large tissues.  
The process of decellularization works in cycles. First, the decellularization agent or water is filled into the chamber. Then, the agent/water is recirculated for a defined period. After that, the agent/water is flushed into waste. Finally, a new agent or water is filled again. These steps are repeated depending on the tissue properties or experimental setup. This multicycle approach maintains a high chemical gradient promoting the efficiency of agents and washing. The system is installed in a heated chamber that holds the temperature to 37 \({}^{\circ}\)C. Higher temperature promotes the activity of detergents. Also, a special chamber was designed for fixing the planar pericardial tissue. The flow in this chamber is optimized to be homogeneously spread over both sides of the tissue. This helps to decellularize the tissue homogenously and to avoid the persistence of non-decellularized areas. The tissue in this chamber is fixed on a silicone holder using surgical silk. The overall view of the decellularization system and the fixing chamber is in Figure 1.  
The frozen pericardium was thawed in a 37 \({}^{\circ}\)C water bath. Then, the tissue was installed in the decellularization chamber using a 4--0 suture. The 0.5% SDS solution was used as a decellularization agent. This solution was used in 6 recirculation cycles lasting 10 min each. Then the SDS was washed out using 5 recirculation cycles of dH2O lasting 5 min each. After that, a 60 min-lasting DNAse cycle was performed. The DNAse was dissolved in a DNAse buffer to the concentration of 40 \(\upmu\)g/mL. This treatment was crucial to remove the remaining residues of DNA spilt over the tissue as a result of the previous SDS treatment. Then, the overall process was finished with a thorough washing of the tissue, consisting of a hundred 10-min cycles of dH2O. The agent concentration, the cycle repetitions, and the exposure times were optimized to neutralize the potentially toxic effects of the agents and to minimize the mechanical degradation of the tissue described earlier [19]. The decellularized tissues were then sterilized in a 70% ethanol solution for 1 h, and then they were rinsed and stored in sterile PBS at 4 \({}^{\circ}\)C. To evaluate the effectiveness of the decellularization process, 10 \(\upmu\)m-thin cryosections from randomly selected parts of the tissues were prepared. These sections were stained using DAPI to visualize the cell nuclei or the remaining DNA.  
Cytotoxicity and sterility tests were also performed. The cultivation of cells in the leachate and directly on the decellularized tissue were done to verify the potential cytotoxic effects. The leachates were prepared in a laboratory. Decellularized and sterile tissue was incubated in DMEM culture media with 10% FBS for 72 h. Then, this culture media was added to cells, and proliferation was observed. The isolated porcine adipose tissue-derived stromal cells were used for these cytotoxic tests. The sterility tests were performed according to Ph. eur. 2.6.1 and ISO 11737-2 [20] in thioglycollate broth and tryptic soy broth.