## 3 Results
### Preparation of Recellularized Cardiovascular Patches  
As was mentioned in the Material and Methods section of this study, we tried to demonstrate the efficiency of dynamic cultivation in stimulating cell ingrowth inside the decellularized tissue. For this experiment, only one side of the pericardium was seeded with cells, and the depth of the cell penetration into the tissue was evaluated. As described above, cells were penetrating nearly the whole tissue after 10 days of dynamic cultivation. However, the density was decreasing with depth. After 5 days of dynamic cultivation, cells were able to penetrate at half of the depth of the decellularized tissue.  
To achieve more homogeneous cell colonization for preparing implantable cardiovascular patches, both sides of the decellularized tissue were seeded with cells prior to pressure stimulation. The design of the cultivation chamber was also optimized when the two covers of the chamber implemented Luer-Locks (LLs) in order to use a sterile septum for cell seeding. In this case, ovine decellularized pericardium was also used. The protocol for the ovine pericardium was the same as for the porcine tissue, except that the tissue was sourced from experimental sheep. We had only a limited amount of pericardial tissue because we had access to just a small number of sheep that had originally been used for other studies. The overall method for testing and validation was, therefore, based on the porcine pericardium. However, in further studies, ovine pericardium will be used for implantation into pigs in order to simulate a xenogeneic tissue that will be repopulated with autologous porcine cells.  
Also, the total dynamic cultivation time was reduced to 5 days when both sides of tissue were seeded prior to dynamic cultivation. This method offers a considerable advantage when preparing autologous cell-seeded grafts due to the reduced time needed for recellularizing the decellularized tissue. The difference between single-side and both-sides seeding after 5 days of dynamic cultivation is illustrated in Figure 10.  
As is demonstrated in Figures 11 and 12 on 20 \(\upmu\)m paraffin-embedded sections stained with hematoxylin and eosin HE and DAPI, the PrASCs were homogeneously distributed inside the recellularized porcine tissue samples and ovine tissue samples. There was no difference between the ovine and porcine tissues in terms of cell colonization.  
Figure 9: Immunofluorescence staining of the cultivated decellularized pericardium (red—F-actin, green—H1-calponin, blue—DAPI) in static and dynamic conditions for 10 days. For overall growth images, a Carl Zeiss 880 NLO confocal microscope was used with 10x objective and extended field of view. For differentiation images, a Leica DMi8 with 20x objective was used.  
Figure 11: Paraffin-embedded 20 \(\upmu\)m sections of porcine 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.  
Figure 10: Comparison between single- and both-sides cell seeding procedures. The cultivation time in both procedures was 5 days in dynamic conditions.