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.