Results
The Cells are alive on the Monolayer Disks with the labelling system.
To detect the cells whether or not survive after using celltrackers, as mentioned above again, the Porcine EEC were labeled with GFT and PCF were labeled with Texas Red. From the Fig. 2, the cells are observed by Lecia microscope with 10x and 20x lens after 72 hours. Thus, different types of the cells are alive after using celltrackers. Then, the cells are transferred to the Monolayers (Disks), using the ECM medium for the Porcine EEC and Porcine Fibroblasts were maintained in Fibroblast Growth Media 3. After culturing for 72 hours, the Monolayers were fixed and the images were taken by Yokogawa CellVoyager CV8000 under the 4x objective in the Fig 3. The images were from different z-depth such as -50, 100, 350 and 500 μm, and cells are widely distributed in the Monolayers.
The mixed cells survived on the multiple layers
To investigate that whether or not the PCF and Porcine EEC survive in the multiple layers (Disks), we mixed the cells and transferred them to the multiplex layers. After fixing and washing the Disks, we still utilized the Yokogawa CellVoyager CV8000 to monitor the cells. From the Fig 4, we found that the Porcine fibroblast- Texas Red and EEC- GFP were mixed and survived after 48 hours in the Disks. Also, from the images, the cell numbers of PCF and Porcine EEC were different at the different z-depth of 100 and 200 μm. From the images, we confirmed that the PCF and Porcine EECs were alive after bioprinting and mixed together with conditioning mediums.
The mixed cells on the Disks were alive after bioreactor treatment.
To further detect the mixed cells on the Disks whether can be potential facilitated for DSS project, we utilized the cone and plate bioreactors since it can imitate the heart fluids as Fig.1 & Fig. 6 showed the schematic and model diagrams. In addition, we designed two groups to conducted for experimental comparing, one group is control group without bioreactor treatment, and the other one is the experimental group within bioreactor treatment for 24 hours. During the bioreactor treatment, we also used the 1:1 ratio conditioning medium as mentioned above. In addition, the Disks were all fixed by 4% PFA and sectioned for Yokogawa CellVoyager CV8000 to observe the cells. From the Fig 5, the Disks under bioreactor treatment seems to be widely spread or distributed comparing to the 0-hour control group. Also, Disks after bioreactor treatment had less cells comparing to control groups, especially the PCF labeled with Texas-Red. Additionally, cells are still alive and stayed on the Disks after bioreactor treatment.