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.