Materials and Methods
Gelatin from bovine skin powder (9000-70-8), Calcium Chloride pellets
(10043-52-4), and sodium alginate (9005-38-3) obtained from Sigma
Aldrich. Commercial Cellink BIOX Printer (Model: S-10001-001; S/N:
202041). ECM media (Sciencell, 1001), Fibroblast growth media 3
(PromoCell Inc. C-23025). 0.05% Trypsin EDTA (Gibco™ 25300054).
Research Design
4 different types of disks were fabricated. The 1stgroup contained only EECs in all 3 layers, adding \(3x10^{5}\) EECs per
layer. The 2nd group contained only Porcine
Fibroblasts in all 3 layers, \(3x10^{5}\) Porcine Fibroblasts per layer.
The 3rd group of disks were comprised of 2 layers of
Porcine Fibroblasts and 1 layer of EECs as the top layer, with same
ratio of cells per layer as the first 2 groups. Lastly, the
4th group of disks contained 3 layers of contained
Porcine Fibroblasts and EECs dispersed throughout all 3 layers,\(3x10^{5}\) EECs and \(3x10^{5}\) Porcine Fibroblasts in each layer for
a total of \(6x10^{5}\) cells per layer. Cells were cultured in the disk
for 48 hours and then each group subjected to shear stress in the
cone-and-plate bioreactor at 6 dynes/cm2 for each group for 24 hours.
Before and after inducing shear stress, confocal images were collected
of each layer of each disk.
Cell culture
Porcine Cardiac Fibroblasts and Endocardial Endothelial Cells were
cultured each cell type on an individual monolayer of 0.1% gelatin,
seeding \(5x10^{5}\) cells. EECs and Porcine Fibroblasts were maintained
in ECM media and Fibroblast Growth Media 3, respectively. Once cells
reach confluency and before trypsinization, each cell type was labelled
using CellTrackers. To be more specific, the Porcine EECs were labelled
by GFP (ThermoFisher, Cat #Q25041MP), and Porcine Cardiac Fibroblasts
were labelled by Texas Red (ThermoFisher, Cat # Q25021MP). We then
detached them using 0.05% Trypsin EDTA. We then cultured the
Celltracker-labelled cells as previously mentioned.
Optimization of Bioink with Cells –
In previous studies [35], we established that 8% alginate produced
the best results of reproducibility, ease of crosslinking, and
appropriate stiffness. We established the appropriate seeding density of
cells in alginate hydrogel by using a 3-way luer lock to combine one
syringe containing 1mL of 8% alginate with a separate syringe
containing 200uL of 1x10^6 cell suspension. After printing the disks,
confocal images were collected and analyzed to determine that 1 million
cells in 200uL of media is optimal cell concentration to print the
disks. Then, printed under sterile conditions using cellink.
Disk Fabrication
From the Fig 1., the disks were developed through a 4-step process.
Firstly, we created an STL file using bioprinter that formulates a set
of instructions on how to build a disk with 3 dimensions through
programmed printhead movements in the x, y, and z planes [35]. The
disks were 55 mm in diameter. The file was then transferred to the 3D
printer, where it is sliced into layers and printed as a grid pattern.
In order to ensure the disk can withstand the cone-and-plate bioreactor,
we utilized the printers’ customizable features to modify the number of
layers of the disk, inserting 2 additional layers, 0.1mm each.
3g of gelatin was combined with 30mL of 0.2% CaCl2, sodium alginate
crosslinking agent. After 10 minutes, the gelatin mixture was
centrifuged and plated to fill the entire volume of the Nunclon ™ dish.
Next, the cartidridge containing 1mL of alginate with 200uL of cell
suspension is attached to a stainless steel dispense tip (Nordson EFD,
Cat #7018345), which is calibrated to a position 10mm deep into the
gelatin support bath. Once printing is complete, the dish is placed in
the incubator for 2 hours, to allow the gelatin to completely melt.
Lastly, the gelatin is aspirated and 20mL of media with 1% CaCl2 is
added to supply nutrients to the cells imbedded in the disk and allow
the disk to achieve maximum stiffness over 24 hours. The disk is then
stored in the incubator.