Introduction: 2-dimensional cultures have traditionally been used as in vitro models to simulate the effects of diseases and drug testing. However, the 2D environment does not accurately simulate the extracellular matrix (ECM) inside the human body. 3D bioprinting shows potential to more closely mimic the ECM and advance drug testing and novel therapies. Hydrogels are the most common bioink because of their biocompatibility and their ability to acquire a similar structure to the ECM. Printing parameters directly influence the precision and accuracy of the bioink disposition and directly impact cell viability as they determine the shear forces within the bioink during the printing process. This study aims to optimize the printing parameters (print speed, extrusion speed, nozzle temperature) and other variables (alginate viscosity and printing tip diameter) through seeing their effects on the pore factor of the unloaded 3D grid structure. This study aims to provide the basis for the establishment of more accurate in vitro organoid models.
Materials and
Methods: Hydrogels were made out of 5% alginate and 5% gelatin in phosphate buffered saline (PBS). Before printing, hydrogels were warmed in a 37 °C incubator and then cooled to room temperature. A 10mm x 10mm x 1mm grid structure was chosen and kept constant. Pre-extrusion was done to ensure unclogged syringes and more uniform prints. Pictures of prints (not crosslinked) were taken with a light microscope and pores were analyzed with ImageJ software. Ideally, the pore factor is 0.95-1 for optimal cell viability. If pore factor < 0.95, then the structure is too soft. If pore factor >1, then the structure is too stiff. This procedure is repeated for multiple different print speeds, extrusion speeds, nozzle temperature, printing tip diameter, and alginate viscosity.
Results, Conclusions, and Discussions: Initially, all the medium viscosity (773 kDa) prints (with 0.27 mm tip diameter) were too stiff, regardless of print speed and extrusion speed combinations. After switching to low viscosity (24 kDa), the prints were still too stiff, so nozzle temperature was turned on to try to soften the prints and so the prints don’t get progressively stiffer throughout the day (controlling another variable). After turning the nozzle temperature on, the prints became too soft and uneven. A wider syringe tip was used (0.4 mm diameter) to see if it would produce more consistent prints. The 0.4 mm tip did end up resulting in more consistent prints, and after testing more print speed, extrusion speed, and nozzle temperature combinations, a one such combination consistently resulted in an ideal pore structure. Low viscosity (24 kDa) is preferred compared to medium viscosity, which was consistently too stiff. Nozzle temperature started getting turned on to soften prints and reduce the impact of time elapsed since warming the hydrogel. Ideal temperature approximately 23-24 °C. The wider tip (0.4 mm diameter) is preferred because the lower shear force is more consistent and easier to work around. Most preferred print speed and extrusion speed combination is 5 mm/s print speed and 0.6 mm/s extrusion speed. These settings most consistently yielded a pore factor between 0.95 and 1.
Acknowledgements (Optional): Special thanks to Dr. Jamel Ali for the opportunity to work in his lab and for his support and education. I would also like to thank Annie Scutte and Tyler Gregory for their guidance and training. I am very thankful for the Maglab Externship program run by Mr. Carlos Villa. The National High Magnetic Field Laboratory is supported by the National Science Foundation through NSF/DMR-2128556 and the State of Florida.
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