## 4 Discussion  
The operating protocol involving the decellularization agents, such as SDS, DNAse, and deionized water, was adopted and modified according to the studies by [PERSON] et al. and by [PERSON] et al. [10; 11]. Many of the methods described require repeated steps involving changing decellularization agents or water after a certain period to maintain a chemical concentration gradient. Also, to improve the method and ensure homogenous decellularization, agitation in a shaker is used. These steps can be time-consuming. To ease these steps and ensure uninterrupted automation of the overall process, a novel system was built. This system implements a cyclic-based process of changing agents and washing water based on the desired setting. This approach alters the manual changing of them. Also, a special cultivation chamber was used where the tissue is fixed, and the chamber allows the flowing of used liquids to flow over the tissue, improving homogeneity. As demonstrated in Figure 6, the native tissue has visible cell nuclei, whereas in the fully decellularized tissues there were no visible cell nuclei or DNA residues. The cell cultivation, both in the dynamic cultivation system and in static conditions, also demonstrated that the tissue decellularized by the method described here is suitable for recolonization with cells. The cells adhering to the surface of the decellularized tissue were able to penetrate the tissue. However, under static conditions, the cells formed clusters instead of a homogeneous spread on the surface of the tissue when observed under dynamic conditions, as illustrated in Figure 9.  
Figure 12: Paraffin-embedded 20 \(\upmu\)m sections of ovine recellularized pericardium stained with hematoxylin-eosin (HE) and DAPI. Cells were seeded from both sides of the decellularized tissue prior to dynamic stimulation. Cultivation was held for 5 days in a dynamic culture system. It can, therefore, be summarized that this protocol, implemented into our decellularization system, provided a decellularized porcine pericardium matrix that is non-toxic, sterile, and ready for further recellularization and use as an experimental cardiovascular patch. The whole process can be completed within 24 h. The decellularization chamber designed for fixing the planar tissue can prepare tissues up to 12 \(\times\) 8 cm\({}^{2}\) in size in a single run. As was mentioned above, the porcine pericardium was harvested from a local slaughterhouse and was a cheap and easily obtainable source of tissue for validating the method. The ovine pericardium was also decellularized and was further tested to form a xenogeneic substrate with autologous cells (PrASCs) for further implantation into a porcine in vivo model to verify low immunogenicity of decellularized matrices reported in the literature [8; 9; 26]. Cell seeding is an important component of vascular tissue engineering that decreases graft thrombogenicity and promotes graft patency, regardless of the cell type used. A possible mechanism is the recruitment of autologous cells in a paracrine manner and accelerated regeneration of the seeded matrices into a neovessel. A method of static seeding passively introduces cells onto a scaffold. The cell maturation and penetration into the scaffold is low. As has been described above, the process of recellularization in static conditions does not give satisfactory results. As is illustrated in Figures 7 and 8, only 1/5 (50 \(\upmu\)m) of the decellularized tissue was penetrated with cells after 10 days of static cultivation. Similar results have also been obtained in other studies, in which several weeks of cultivation were needed to overgrow various matrices with cells and to achieve cell maturation [15]. The speed of the overall recellularization process is increased when perfusion bioreactors are used to provide mechanical stimulation of the cells according to studies by [PERSON] et al. [27]. Also, many studies have investigated the correlation between pressure strain, cell proliferation, and stem cell differentiation towards SMCs.