(B-77) Standardized Parameters for 3D Bioprinting with Gelma

The prospect of tissue engineered therapies is quickly expanding as better methodology and techniques are discovered. Amongst these techniques is 3D bioprinting which employs the fundamental principles of additive manufacturing to tissue scaffolds for the creation of complex 3D cellular structures, bringing unbridled potential. Injuries that once required autologous or allogeneic transplants can be treated without the risk of site morbidity from the patient’s own cells. The complex geometry of bioprinted scaffolds allows for unique culturing techniques that induce differentiation, or closely mimic the natural environment of the body on a larger scale than organ-on-a-chip technology that allows for an easier harvest and analysis of cells. This can be especially useful for exosome therapies to see how they impact the growth and development of cellular components. However, there is a steep learning curve for the necessary skills. To properly operate a bioprinter, you need to understand the instrument’s limitations and variables, the properties of the gel used as the scaffold base, and a fundamental understanding of G Code. The purpose of this study is to identify the function of each parameter available in the Bio X6 Cellink, and optimize them in a manner that creates reliable, successful, prints.

The materials used for the bioprinting were mouse fibroblast cells, 6mL Gelma, and 1.2 cell media mixer. The instrumentation used during the process are the Cellink BioX6 and a standard confocal microscope. When cells were used, the printing solution was a 1:5 cell to Gelma ratio using the provided cell mixer by Cellink. The Gelma prepared was a PhotoGel 50% DOM and a 10% LAP mix. The GCode was generated to print out 8 layers of 15 x 15 mm structures as shown in Figure 1 that possessed a 10% infill. Temperature, pressure, nozzle size, print bed temperature, crosslinking wavelength, and layer height were parameters that were tested and optimized in prior prints. These specifications can be seen in Table 1. The structure was duplicated in three separate wells. The print was run until completion or failure was obvious. They were left to incubate in an incubator at 37 C for one week.
For the analysis of the structures, PBS and live/dead viability assay. Once complete, the completed prints were washed with PBS three times. A Live/Dead assay stain was performed to assess the viability of the bioprinted samples. The scaffolds were left to sit in the assay staining for fifteen minutes. Two of the three prints were then observed under a confocal microscope, and a z-scan was performed for spatial depth.

The process of bioprinting the 3D structures was successfully replicated in two trials, and the relevant parameters were determined. The most common reason prints failed was due to the temperature of the print head. If the temperature inside the BioX6 chamber deviated too far from 26 C, the Gelma inside the cartridge would begin to behave differently. This means it would either solidify and not be extruded or it would become runny, compromising its integrity. To circumvent this limitation, the Gelma was kept in a water bath at 37 C. Once it was time to print, the Gelma was transferred to a cartridge and the temperature of the chamber was monitored until it reached 26 C. Doing this meant the print could be completed before the temperature would alter the behavior of the printing material. 

Print head temperature was correlated to the consistency of the Gelma, the gauge size was correlated to the ease of extrusion, and the print speed to the fidelity of the print. Pressure seemed to correlate with how much gel was extruded, but the values were too varied across trials to create a consistent trend to follow and decided on an as-needed basis. The first print made under these parameters resulted in its dissolution when incubating. Believing the issue to be structural integrity, the infill was reduced and the amount of layers increased. The print completed with no issues, and was able to be incubated. The resulting print had a 99.74% cell survival rate in Well One. 

A successful bioprinting of tissue constructs requires an optimization of the parameters related to the bioprinter and the bioink composition. This includes the temperature and handling of the printing material. In addition to sacrificial printing as used in this study, coaxial printing will be also explored to bioprint vascularized tissue constructs with embedded channels for perfusion of nutrients.