Discussion:
Preliminary experiments showed that labeled Porcine EEC and PCF cells remained viable after being labeled with a cell tracker and transferred to a single-layer disk. Observation of living cells with a Leica microscope confirmed the successful labeling and survival of different cell types. In addition, images of Yokogawa CellVoyager CV8000 at different z depths show that the cells are widely distributed within a single-layer disk, indicating their ability to adhere and proliferate on the disk surface. This finding is consistent with previous studies that have shown that monolayer cultures are suitable for supporting cell growth and maintaining viability [47].
The survival of mixed PCF and porcine EEC cells in multilayer disks was investigated with encouraging results. Yokogawa CellVoyager CV8000 images showed that Porcine fibroblasts - Texas Red and EEC-GFP cells co-existed and were viable inside the disks after 48 hours. Importantly, the different cell numbers observed at different Z-depths indicate a pattern of differential potential distribution within the multilayer. This finding is consistent with research highlighting the importance of spatial organization and cell-cell interactions in mixed cell cultures [48]. Future research should focus on characterizing the spatial arrangement and intercellular communication between different cell types within the composite layer.
The simulation of cardiac fluid using cone and plate bioreactors has provided valuable insights into the mixed cell populations under conditioning medium. There were significant differences in cell distribution between the control group and the experimental group after 24 hours of bioreactor treatment. Yokogawa CellVoyager CV8000 images showed that disks exposed to bioreactor processing showed a wider distribution of cells compared to controls. This observation suggests that the dynamic fluid environment promotes the dispersion of cells within the disk. In addition, a reduction in the number of cells in the petri dish after bioreactor treatment, particularly PCF labeled with Texas red, may indicate potential cell detachment or altered cell survival ratio under the shear stress conditions. Future research could explore the specific mechanisms of cell detachment under bioreactor conditions and potential differences in cell viability and function.
In this study, the bioreactor provided the shear stress was used as a model to study the DSS. The present study investigated the co-cultural system under bioreactor treatment. The results showed that the mixed cell population successfully survived, distributed, and reacted on a monolayer disk, highlighting the behavior of these cells under bioreactor treatment. The findings of this study provide valuable insights into cell behavior in DSS.
The use of bioreactors in co-culture systems can significantly affect cell distribution from the results. The decrease in the number of cells observed in the bioreactor treated samples suggests that shear stress caused by the flow dynamics of the bioreactor may lead to cell detachment. This finding is consistent with previous research and highlights the importance of optimizing bioreactor conditions to maintain cell adhesion and prevent cell loss during culture. Understanding the effects of shear stress on cell behavior is critical to successfully implementing a co-culture system in DSS.
Also, the successful survival and distribution of a mixed cell population in a co-culture system including single layer and multiple layers, which demonstrates the potential for a more holistic approach in DSS applications. By combining multiple cell types, such as endothelial cells, and fibroblasts, it is possible to develop more efficient tissue structures for DSS repair or replacement [49].
The co-culture system is also designed to address the limitations of single-cell type therapy. By integrating multiple cell types that are critical to the formation and function of target tissues, researchers can create tissue structures that more closely resemble natural tissues [50]. For instance, the addition of endothelial cells helps promote blood vessel formation, smooth muscle cells support mechanical properties, and fibroblasts contribute to extracellular matrix production and tissue stability in DSS [51].
By providing a more comprehensive and versatile approach, co-culture systems have the potential to create organizational structures that better mimic the complexity and function of native tissues [52]. This approach allows researchers to study cell-cell interactions, signaling pathways, and tissue development in a more physiologically relevant way. Ultimately, it provides more efficient and functional tissue structures to improve the treatment for DSS patients.